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Inactivation of Cronobacter malonaticus cells and inhibition of its biofilm formation exposed to hydrogen peroxide stress

  • Author Footnotes
    2 These authors contributed to the manuscript equally.
    Yingwang Ye
    Correspondence
    Corresponding authors
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    2 These authors contributed to the manuscript equally.
    Affiliations
    School of Food Science and Engineering, Hefei University of Technology, Hefei 230009, 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
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  • Author Footnotes
    2 These authors contributed to the manuscript equally.
    Maofeng Zhang
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    2 These authors contributed to the manuscript equally.
    Affiliations
    School of Food Science and Engineering, Hefei University of Technology, Hefei 230009, China
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  • Author Footnotes
    2 These authors contributed to the manuscript equally.
    Rui Jiao
    Footnotes
    2 These authors contributed to the manuscript equally.
    Affiliations
    School of Food Science and Engineering, Hefei University of Technology, Hefei 230009, China
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  • Na Ling
    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
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  • Xiyan Zhang
    Affiliations
    School of Food Science and Engineering, Hefei University of Technology, Hefei 230009, China
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  • Liaowang Tong
    Affiliations
    School of Food Science and Engineering, Hefei University of Technology, Hefei 230009, China
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  • Haiyang Zeng
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    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
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  • Jumei Zhang
    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
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  • Qingping Wu
    Correspondence
    Corresponding authors
    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
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  • Author Footnotes
    2 These authors contributed to the manuscript equally.
Open ArchivePublished:October 25, 2017DOI:https://doi.org/10.3168/jds.2017-13463

      ABSTRACT

      Presence of Cronobacter malonaticus in powdered infant formula (PIF) poses a high risk to infant and public health. Cronobacter malonaticus has been widely distributed in food and food processing environments, and the true origin of C. malonaticus in PIF is poorly understood. Control and prevention of C. malonaticus is necessary for achieving microbial safety of PIF. However, little information about decontamination of C. malonaticus is available. In this study, effects of hydrogen peroxide on inactivation and morphological changes of C. malonaticus cells were determined. Furthermore, inhibitory effects of H2O2 on biofilm formation in C. malonaticus were also performed. Results indicated that H2O2 could completely inactivate C. malonaticus in sterile water with 0.06% H2O2 for 25 min, 0.08% H2O2 for 15 min, and 0.10% for 10 min, respectively, whereas the survival rates of C. malonaticus in tryptic soy broth medium significantly increased with the same treatment time and concentration of H2O2. In addition, morphological changes of C. malonaticus cells, including cell shrinkage, disruption of cells, cell intercession, and leakage of intercellular material in sterile water after H2O2 treatment, were more predominant than those in tryptic soy broth. Finally, significant reduction in biofilm formation by H2O2 was found using crystal violet staining, scanning electron microscopy, and confocal laser scanning microscopy detection compared with control samples. This is the first report to determine the effects of H2O2 on C. malonaticus cells and biofilm formation. The findings provided valuable information for practical application of H2O2 for decontamination of C. malonaticus in dairy processing.

      Key words

      INTRODUCTION

      Cronobacter species are considered to be occasional food-borne pathogens involved in neonatal meningitis, necrotizing enterocolitis, and sepsis (
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      Bloodstream infections in neonatal intensive care unit patients: Results of a multicenter study.
      ;
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      Citrobacter freundii invades and replicates in human brain microvascular endothelial cells.
      ;
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      NO and oxyradical metabolism in new cell lines of rat brain capillary endothelial cells forming the blood-brain barrier.
      ), and also isolated from various samples including food-processing plants, food, water, soil, and clinical samples (
      • Gurtler J.B.
      • Kornacki J.L.
      • Beuchat L.R.
      Enterobacter sakazakii: A coliform of increased concern to infant health.
      ;
      • Ye Y.W.
      • Li H.
      • Wu Q.P.
      • Zhang J.M.
      • Lu Y.D.
      The Cronobacter sp. in milk and dairy products: Detection and typing.
      ). Genomic analysis indicated that Cronobacter sakazakii, Cronobacter malonaticus, and Cronobacter turicensis were associated with severe infections (
      • Kucerova E.
      • Clifon S.W.
      • Xia X.Q.
      • Long F.
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      • Fulton L.
      • Fronick C.
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      • Kyung K.
      • Warren W.
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      • Wollam A.
      • Shah N.
      • Bhonagiri V.
      • Nash W.E.
      • Hallwworth-Pepin K.
      • Wilson R.K.
      • McClelland M.
      • Forsythe S.J.
      Genome sequence of Cronobacter sakazakii BAA-894 and comparative genomic hybridization analysis with other Cronobacter species.
      ). The true origin of C. malonaticus in powdered infant formula is poorly understood, although Cronobacter (formerly known as Enterobacter sakazakii) strains in powdered infant formula might originate from food-processing environments, as described by
      • Mullane N.R.
      • Whyte P.
      • Wall P.G.
      • Quinn T.
      • Fanning S.
      Application of pulsed-field gel electrophoresis to characterize and trace the prevalence of Enterobacter sakazakii in an infant formula processing facility.
      .
      Cross-contamination or persistent contamination of food-borne pathogens in food samples have been determined to be from bacterial biofilms on surfaces of food engineering plants and equipment (
      • Srey S.
      • Jahid I.K.
      • Ha S.-D.
      Biofilm formation in food industries: A food safety concern.
      ;
      • Cappitelli F.
      • Polo A.
      • Villa F.
      Biofilm formation in food processing environments is still poorly understood and controlled.
      ). In recent years, biofilm-forming abilities on surfaces in Cronobacter strains have been studied (
      • Lee Y.-D.
      • Park J.-H.
      • Chang H.
      Detection, antibiotic susceptibility and biofilm formation of Cronobacter spp. from various foods in Korea.
      ;
      • Jung J.-H.
      • Choi N.-Y.
      • Lee S.-Y.
      Biofilm formation and exopolysaccharide (EPS) production by Cronobacter sakazakii depending on environmental conditions.
      ;
      • Ye Y.W.
      • Ling N.
      • Jiao R.
      • Wu Q.P.
      • Han Y.J.
      • Gao J.N.
      Effects of culture conditions on the biofilm formation of Cronobacter sakazakii strains and distribution of genes involved in biofilm formation.
      ), and a capsule biofilm was considered to contribute to survival of Cronobacter strains under environmental stresses (
      • Iversen C.
      • Forsythe S.
      Risk profile of Enterobacter sakazakii, an emergent pathogen associated with infant milk formula.
      ). Therefore, controlling food-borne C. malonaticus strains and biofilm formation is extremely important to newborn and public health.
      Hydrogen peroxide is a well-studied sanitizer for inactivate food-borne pathogens (
      • Guan W.
      • Fan X.
      • Yan R.
      Effect of combination of ultraviolet light and hydrogen peroxide on inactivation of Escherichia coli O157:H7, native microbial loads, and quality of button mushrooms.
      ;
      • Back K.H.
      • Ha J.W.
      • Kang D.H.
      Effect of hydrogen peroxide vapor treatment for inactivating Salmonella Typhimurium, Escherichia coli O157:H7 and Listeria monocytogenes on organic fresh lettuce.
      ) with bacteriostatic and bactericidal activity (
      • Olmez H.
      • Kretzschmar U.
      Potential alternative disinfection methods for organic fresh-cut industry for minimizing water consumption and environmental impact.
      ). In addition, hydrogen peroxide is regarded as a safe disinfecting agent due to no harmful residue after treatment, and inactivation of H2O2 solutions on Escherichia coli O157, Listeria monocytogenes, and Salmonella have been studied (
      • Sapers G.M.
      • Miller R.L.
      • Jantschke M.
      • Mattrazzo A.M.
      Factors limiting the efficacy of hydrogen peroxide washes for decontamination of apples containing Escherichia coli..
      ;
      • Huang Y.
      • Chen H.
      Effect of organic acids, hydrogen peroxide and mild heat on inactivation of Escherichia coli O157: H7 on baby spinach.
      ;
      • Choi N.Y.
      • Baek S.Y.
      • Yoon J.H.
      • Choi M.R.
      • Kang D.H.
      • Lee S.Y.
      Efficacy of aerosolized hydrogen peroxide-based sanitizer on the reduction of pathogenic bacteria on a stainless steel surface.
      ;
      • Guan W.
      • Fan X.
      • Yan R.
      Effect of combination of ultraviolet light and hydrogen peroxide on inactivation of Escherichia coli O157:H7, native microbial loads, and quality of button mushrooms.
      ;
      • Back K.H.
      • Ha J.W.
      • Kang D.H.
      Effect of hydrogen peroxide vapor treatment for inactivating Salmonella Typhimurium, Escherichia coli O157:H7 and Listeria monocytogenes on organic fresh lettuce.
      ;
      • Jiang Y.
      • Sokorai K.
      • Pyrgiotakis G.
      • Demokritou P.
      • Li X.H.
      • Mukhopadhyay S.
      • Jin T.
      • Fan X.T.
      Cold plasma-activated hydrogen peroxide aerosol inactivates Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria innocua and maintains quality of grape tomato, spinach and cantaloupe.
      ). However, little work has focused on the effects of hydrogen peroxide on inactivation C. malonaticus cells and inhibition of biofilm formation.
      In the present study, the effects of hydrogen peroxide on the survival and morphological injury of C. malonaticus cells were determined using plate counting method and scanning electron microscopy. Furthermore, inhibitory roles of hydrogen peroxide on biofilms formation in C. malonaticus were also performed using crystal violet staining (CVS), scanning electron microscopy, and confocal laser scanning microscopy (CLSM).

      MATERIALS AND METHODS

      Strains and Preparation of Hydrogen Peroxide

      Cronobacter malonaticus GDMCC-11 is isolated from aquatic food samples and comes from Guangdong Microbiology Culture Center (GDMCC). Hydrogen peroxide (30%, Sinopharm Chemical Reagent Co. Ltd., Shanghai, China) were added into sterile tryptic soy broth (TSB; Huankai, Guangzhou, China) for preparation of 0 (control, vol/vol), 0.02, 0.04, 0.06, 0.08, and 0.10% H2O2.

      Inactivity of C. malonaticus Cells by Hydrogen Peroxide

      Cronobacter malonaticus was inoculated into TSB for incubation at 37°C for 14 to 16 h. The C. sakazakii with 106 cfu/mL was then transferred into sterile water and TSB containing 0 (control, vol/vol), 0.02 0.04%, 0.06, 0.08, and 0.10% H2O2 for incubation of 10, 20, and 30 min. Finally, the survival of C. malonaticus was counted using plating method and each experiment was done in triplicate.

      Morphological Changes of C. malonaticus by H2O2 using Scanning Electron Microscopy

      To determine the morphological changes of H2O2 treatment, 2 set of experiments were designed. In one set of experiments, C. malonaticus cells were inoculated into sterile TSB at 37°C, and then the enrichment culture (100 µL, optical density (OD) at 600 nm = 0.5) was transferred into 5 mL of sterile TSB broth or sterile water containing 0, 0.02, 0.04, 0.06, 0.08, and 0.10% H2O2 for 15 min of treatment at 37°C. In another set of experiments, the enrichment culture (100 µL, OD at 600 nm = 0.5) was transferred into 5 mL of sterile TSB broth or sterile water containing 0.6% H2O2 for treatment of about 5, 10, 15, 20, 25, and 30 min. The cultures were then harvested, and pellets cells were used for gradient dehydration with ethanol described by
      • Wang C.Y.
      • Hsu C.P.
      • Huang H.W.
      • Yang B.B.
      The relationship between inactivation and morphological damage of Salmonella enterica treated by high hydrostatic pressure.
      with little modification. Finally, the treated cells by H2O2 were subject to scanning electron microscopy analysis (S8020, Hitachi, Tokyo, Japan).

      Effects of H2O2 on Biofilm Formation in C. malonaticus

      For CVS detection, C. malonaticus was inoculated into 5 mL of TSB and grown for 12 to 14 h at 37°C with constant shaking. Fifty microliters of culture (OD at 600 nm = 0.5) were inoculated into 96-well polystyrene plates containing 250 µL of sterile Luria-Bertani broth with 0, 0.02, 0.04, 0.06, 0.08, and 0.10% H2O2, 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.
      For biofilm formation detection by scanning electron microscopy, C. malonaticus was inoculated in sterile TSB broth at 37°C for 12 to 14 h, and 0.05 mL of the cultures were transferred to 24-wells plates (Baiyan, Shanghai, China) containing 5 mL of fresh TSB with 0 (control), 0.02, 0.04, and 0.06% H2O2. To test biofilm formation on glass, glass coverslips (Jingan, Shanghai, China) were immersed in TSB broth and then inoculated with C. malonaticus. The coverslips were incubated inside 24-well plates at 37°C for 24, 48, and 72 h, after which time bacterial biofilm formation was observed. During this biofilm-formation period, at 24 h-intervals old culture medium was replaced with fresh TSB. The glass coverslips with different incubation times were rinsed in PBS and fixed with 2.5% glutaraldehyde overnight at 4°C. Postfixation 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.
      To better visualize the biofilm architecture, C. malonaticus was inoculated in TSB at 37°C for overnight and 0.05 mL of the cultures were transferred to 24-well plates (Baiyan) containing 5 mL of sterile TSB with 0 (control), 0.02, 0.04, and 0.06% H2O2. To test biofilm formation on glass, glass coverslips (Jingan) were immersed in TSB broth and then inoculated with C. malonaticus. The coverslips were incubated inside 24-well plates at 37°C for 24, 48, and 72 h, after which time bacterial biofilm formation on glass slips was stained with LIVE/DEAD BacLight bacterial viability Kit (Invitrogen, Carlsbad, CA) and were subsequently observed by CLSM (Zeiss, Berlin, Germany).

      RESULTS AND DISCUSSION

      As one of the important species within the genus of Cronobacter, the control and prevention of C. malonaticus due to cross- or persistent-contamination is important for achieving microbial safety of powdered infant formula. In our study, with increasing amounts of H2O2 in sterile water, the survival rates of C. malonaticus cells significantly decreased (P < 0.01), as shown in Figure 1A. In addition, survival rates of C. malonaticus cells in sterile water with 0.02 and 0.04% H2O2 after 15 min of treatment tended to stabilize, and C. malonaticus cells were completely killed in sterile water with 0.06, 0.08, and 0.10% H2O2 for 25, 15, and 10 min, respectively. In Figure 1B, C. malonaticus in TSB medium was completely inactivated after treatment of 0.08% H2O2 for 25 min and 0.10% H2O2 for 20 min. In addition, H2O2 treatment at 0.06% could not inactivate C. malonaticus cells within 30 min, suggesting the TSB medium could significantly contribute (P < 0.01) to the increasing survival rates of C. malonaticus cells under the same concentration of H2O2 and treatment time. Consequently, nutrients played a protective role on C. malonaticus cells under treatment of H2O2, and organic contents in processing environments should be taken into consideration during control and prevention of C. malonaticus.
      Figure thumbnail gr1
      Figure 1Inactivation of Cronobacter malonaticus cells exposed to hydrogen peroxide with different concentrations in sterile water (A) and tryptic soy broth (TSB) medium (B). Survival rates: means ± SD.
      Aerosolized hydrogen peroxide-based sanitizer were used to reduce the levels of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella Typhimurium on stainless steel surfaces, and the 3 pathogens were not detected after 60 min with 0.25% H2O2 (
      • Choi N.Y.
      • Baek S.Y.
      • Yoon J.H.
      • Choi M.R.
      • Kang D.H.
      • Lee S.Y.
      Efficacy of aerosolized hydrogen peroxide-based sanitizer on the reduction of pathogenic bacteria on a stainless steel surface.
      ).
      • Abadias M.
      • Alegre I.
      • Usall J.
      • Torres R.
      • Vinas I.
      Evaluation of alternative sanitizers to chlorine disinfection for reducing foodborne pathogens in fresh cut apple.
      found that a hydrogen peroxide-based sanitizer at a concentration of 20 mL/L for 5 min caused about 4 log reductions of E. coli O157:H7 and Listeria spp.
      • Yeon J.H.
      • Kim I.J.
      • Park K.H.
      • Park B.K.
      • Park H.K.
      • Park D.W.
      • Kim Y.S.
      • Kim H.I.
      • Jeon D.H.
      • Lee Y.J.
      • Ha S.D.
      Treatment and effect of sanitizers and disinfections in animal food manufacturing plant.
      demonstrated that an aerosolized hydrogen peroxide-based sanitizer (1,100 mg/L) resulted in reductions of 3.5, 3.4, 3.0, and 2.8 log cfu/100 cm2 of E. coli, Staphylococcus aureus, Salmonella Typhimurium, and L. monocytogenes in or on stainless steel, respectively. The aerosolized hydrogen peroxide-based sanitizer at 22.0% for 1 h was effective and resulted in an approximately 5 to 9 log reduction in Salmonella Typhimurium (
      • Lee S.Y.
      • Jung J.H.
      • Jin H.H.
      • Kim Y.H.
      • Oh S.W.
      Inhibitory effect of aerosolized commercial sanitizers against foodborne pathogens.
      ). Vaporized 10% hydrogen peroxide treatment for 10 min decreased levels of Salmonella Typhimurium, E. coli O157:H7, and L. monocytogenes on lettuce by 3.12, 3.15, and 2.95 log10 cfu/g, respectively (
      • Back K.H.
      • Ha J.W.
      • Kang D.H.
      Effect of hydrogen peroxide vapor treatment for inactivating Salmonella Typhimurium, Escherichia coli O157:H7 and Listeria monocytogenes on organic fresh lettuce.
      ). Treatment of lettuce with 2% H2O2 at 50°C for 2 min decreased Escherichia coli O157:H7 by 4 log (
      • Lin C.M.
      • Moon S.S.
      • Doyle M.P.
      • McWatters K.M.
      Inactivation of Escherichia coli O157:H7, Salmonella enterica serotype Enteritidis, and Listeria monocytogenes on lettuce by hydrogen peroxide and lactic acid and by hydrogen peroxide with mild heat.
      ); however, the effects of these sanitizers on inactivation of E. coli O157:H7 inoculated onto baby spinach are very limited (
      • Lee S.Y.
      • Baek S.Y.
      Effect of chemical sanitizer combined with modified atmosphere packaging on inhibiting Escherichia coli O157:H7 in commercial spinach.
      ). Treatment with 3% H2O2 and a combination of H2O2 and UV resulted in 0.77 and 0.87 log cfu/g reductions of E. coli O157:H7 inoculated on mushrooms (
      • Guan W.
      • Fan X.
      • Yan R.
      Effect of combination of ultraviolet light and hydrogen peroxide on inactivation of Escherichia coli O157:H7, native microbial loads, and quality of button mushrooms.
      ).
      • Jiang Y.
      • Sokorai K.
      • Pyrgiotakis G.
      • Demokritou P.
      • Li X.H.
      • Mukhopadhyay S.
      • Jin T.
      • Fan X.T.
      Cold plasma-activated hydrogen peroxide aerosol inactivates Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria innocua and maintains quality of grape tomato, spinach and cantaloupe.
      demonstrated that efficacy of aerosolized hydrogen peroxide depended on type of inoculated bacteria, location of bacteria, and type of produce items, and aerosolized hydrogen peroxide could potentially be used to sanitize fresh fruits and vegetables.
      From Figure 2, morphological changes of C. malonaticus cells were also observed after treatment of H2O2 in sterile water. After treatment with 0.02 and 0.04% H2O2 for 15 min, slight or weak cell shrinkage of C. malonaticus was observed, and significant injuries of C. malonaticus, including cell shrinkage, disruption of cell, and leakage of intracellular material, were observed under treatment with 0.06, 0.08, and 0.10% H2O2. After treatment with H2O2 in TSB medium, injuries of C. malonaticus cells were slightly reduced compared with those in sterile water showed in Figure 3. Furthermore, with extension of treatment time by 0.06% H2O2 in sterile water (Figure 4) and TSB medium (Figure 5), injuries of C. malonaticus, such as cell shrinkage, cell disruption, and cell intercession, clearly increased. In addition, morphological injuries of cells were more predominant than those in TSB medium, which further confirmed protective roles of medium on C. malonaticus cells under H2O2 stress. In E. coli, exposure to low or high H2O2 concentrations resulted in morphological changes including cell filamentation, cell volume, and cell membrane damage, followed by loss of intercellular material (
      • Brandi G.
      • Fiorani M.
      • Pierotti C.
      • Albano A.
      • Cattabeni F.
      • Cantoni O.
      Morphological changes in Escherichia coli cells exposed to low or high concentrations of hydrogen peroxide.
      ). In contrast, obvious filamentation of C. malonaticus cells was not observed using scanning electron microscopy detection in our study.
      Figure thumbnail gr2
      Figure 2Morphological changes of Cronobacter malonaticus cells exposed to hydrogen peroxide with different concentrations in sterile water for 15 min. A = Control group (0% H2O2); B = 0.02% H2O2; C = 0.04% H2O2; D = 0.06% H2O2; E = 0.08% H2O2; F = 0.10% H2O2.
      Figure thumbnail gr3
      Figure 3Morphological changes of Cronobacter malonaticus cells exposed to hydrogen peroxide with different concentrations in tryptic soy broth (TSB) medium for 15 min. A = Control group (0% H2O2); B = 0.02% H2O2; C = 0.04% H2O2; D = 0.06% H2O2; E = 0.08% H2O2; F = 0.10% H2O2.
      Figure thumbnail gr4
      Figure 4Morphological changes of Cronobacter malonaticus cells exposed to hydrogen peroxide with 0.06% H2O2 in sterile water. A = 5 min; B = 10 min; C = 15 min; D = 20 min; E = 25 min; F = 30 min.
      Figure thumbnail gr5
      Figure 5Morphological changes of Cronobacter malonaticus cells exposed to hydrogen peroxide with 0.06% H2O2 in tryptic soy broth (TSB) medium. A = 5 min; B = 10 min; C = 15 min; D = 20 min; E = 25 min; F = 30 min.
      A hydrogen peroxide-based sanitizer at concentrations of 3 and 5% for 5 min led to reduction of the viable cells of >5 log cfu/mL on biofilms of Staphylococcus epidermidis (
      • Presterl E.
      • Suchomel M.
      • Eder M.
      • Reichmann S.
      • Lassnigg A.
      • Graninger W.
      • Rotter M.
      Effects of alcohols, povidone-iodine and hydrogen peroxide on biofilms of Staphylococcus epidermidis..
      ). The attachment of food-borne pathogens to biotic and abiotic surfaces, followed by biofilm formation, might be the critical factor for persistent-contamination of microorganism in food samples (
      • Choi N.Y.
      • Baek S.Y.
      • Yoon J.H.
      • Choi M.R.
      • Kang D.H.
      • Lee S.Y.
      Efficacy of aerosolized hydrogen peroxide-based sanitizer on the reduction of pathogenic bacteria on a stainless steel surface.
      ). In our study, the inhibitory effects of H2O2 on biofilm formation in C. malonaticus were studied for the first time. The highest biomass in C. malonaticus was formed after 48 h of incubation at 37°C by CVS in Figure 6A. The biofilm formation was significantly decreased with increasing concentration of H2O2 in medium, and we noted significant inhibition of biofilm formation of C. malonaticus in TSB containing 0.02, 0.04, and 0.06% H2O2 compared with control samples by the CVS method. In addition, C. malonaticus could hardly form biofilms in the TSB containing 0.08 and 0.10% H2O2.
      Figure thumbnail gr6a
      Figure 6Biofilm formation of Cronobacter malonaticus cells exposed to hydrogen peroxide. A = Quantitative analysis (mean ± SD) of biofilm formation using crystal violet staining; OD = optical density. B = Detection of biofilm formation under different H2O2 concentrations using scanning electron microscopy. C = Detection of biofilm formation under different H2O2 concentrations using confocal laser scanning microscopy. Color version available online. Continued.
      Figure thumbnail gr6b
      Figure 6Biofilm formation of Cronobacter malonaticus cells exposed to hydrogen peroxide. A = Quantitative analysis (mean ± SD) of biofilm formation using crystal violet staining; OD = optical density. B = Detection of biofilm formation under different H2O2 concentrations using scanning electron microscopy. C = Detection of biofilm formation under different H2O2 concentrations using confocal laser scanning microscopy. Color version available online. Continued.
      Furthermore, biofilm formation of C. malonaticus in TSB medium with 0, 0.02, 0.04, and 0.06% H2O2 was detected using scanning electron microscopy in Figure 6B. We found that biofilm formation of C. malonaticus was significantly inhibited with increasing concentrations of H2O2, and incompact biofilms were observed in TSB medium with 0.06% H2O2. To date, little research has focused on control of biofilm formation in C. malonaticus. Our previous study indicated that d-Tryptophan significantly inhibits formation of mature biofilm of C. malonaticus and contributes to disassembly of existing biofilms (
      • Li H.
      • Ye Y.W.
      • Ling N.
      • Wu Q.P.
      • Zhang J.M.
      Inhibitory effects of d-tryptophan on biofilm development by the foodborne Cronobacter sakazakii..
      ). After 72 h of incubation time, the percentage of dead cells (red or yellow) increased in all samples using CLSM detection. From Figure 6C, compared with control group, the amount of viable cells and dead or injured cells increased. In addition, incompact structure of biofilm after treatment of H2O2 was also confirmed by CLSM; however, 0.06% H2O2 could not completely inhibit the biofilm formation and inactivate C. malonaticus cell in TSB medium, suggesting a combination of 2 or more sanitizers is required for removal of C. malonaticus strain and biofilms.
      In summary, treatment of hydrogen peroxide with 0.08 and 0.10% H2O2 in sterile water and TSB within 25 min could completely inactivate C. malonaticus cells. In addition, medium played a protective role for C. malonaticus cells under H2O2 stress. With the increasing of H2O2 concentrations and treatment time, injuries of C. malonaticus cells were more predominant compared with the control groups. Furthermore, significant inhibition of biofilm formation in C. malonaticus by hydrogen peroxide was observed compared with the control groups for the first time. In addition, inhibitory effects of hydrogen peroxide on biofilm formation were also confirmed through detection of structural changes by scanning electron microscopy and CLSM. To achieve microbial safety of powdered infant formula from contamination with C. malonaticus, a combination of 2 or more sanitizers is recommended.

      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), Science and Technology Planning Project of Guangdong Province China (2016A050502033) Project of Science and Technology of Guangzhou, Guangdong Province, China (201604020036), and State Key Laboratory of Applied Microbiology Southern China Open Foundations, Guangzhou, China (SKLAM004-2015).

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