Random Mutagenesis Applied to Reveal Factors Involved in Oxidative Tolerance and Biofilm Formation in Foodborne Cronobacter malonaticus.

Cronobacter species are linked with life-treating diseases in neonates and show strong tolerances to environmental stress. However, the information about factors involved in oxidative tolerance in Cronobacter remains elusive. Here, factors involved in oxidative tolerance in C. malonaticus were identified using a transposon mutagenesis. Eight mutants were successfully screened based on a comparison of the growth of strains from mutant library (n = 215) and wild type (WT) strain under 1.0 mM H2O2. Mutating sites including thioredoxin 2, glutaredoxin 3, pantothenate kinase, serine/threonine protein kinase, pyruvate kinase, phospholipase A, ferrous iron transport protein A, and alanine racemase 2 were successfully identified by arbitrary PCR and sequencing alignment. Furthermore, the comparison about quantity and structure of biofilms formation among eight mutants and WT was determined using crystal violet staining (CVS), scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM). Results showed that the biofilms of eight mutants significantly decreased within 48 h compared to that of WT, suggesting that mutating genes play important roles in biofilm formation under oxidative stress. The findings provide valuable information for deeply understanding molecular mechanism about oxidative tolerance of C. malonaticus.

Exploration of factors in response to low acid tolerance using random mutagenesis in Cronobacter malonaticus.

Cronobacter species are associated with rare but severe infections in newborns, and their tolerance to environmental stress such as acid stress has been described. However, the factors involved in low acid tolerance in Cronobacter are poorly understood. Here, a transposon mutagenesis approach was used to explore the factors involved in acid tolerance in C. malonaticus. Eight mutants from mutant library (n = 215) were successfully screened through a comparison of growth with wild type (WT) strain under acid stress (pH 4.0). Eight mutating sites including glucosyltransferase MdoH, extracellular serine protease, sulfate transporter, phosphate transporter permease subunit PstC, lysine transporter, nitrogen regulation protein NR (II), D-alanine-D-alanine ligase, glucan biosynthesis protein G were successfully identified by arbitrary polymerase chain reaction and sequencing. The biomass of biofilm of eight mutants were significantly reduced using crystal violet staining (CVS) compared with that of WT. furthermore, the more compact biofilms of WT was observed than those of eight mutants through scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM). Disassembly of biofilms appeared among mutants and WT strain from 48 h to 72 h through the increasing of dead cells and reduction of viable cells and exopolysaccharide. The study reveals the molecular basis involved in acid tolerance of C. malonaticus and a possible relationship between biofilm formation and acid tolerance, which provides valuable information for survival of C. malonaticus under acid stress.

Short communication: Roles of outer membrane protein W on survival, cellular morphology, and biofilm formation of Cronobacter sakazakii in response to oxidative stress.

Cronobacter species are a group of opportunistic food-borne pathogens that cause rare but severe infections in neonates. Tolerance to environmental stress in Cronobacter is known; however, factors involved in oxidative stress are undefined. In this study, Cronobacter sakazakii survival, cellular morphology, and biofilm formation in response to oxidative stress were evaluated between the wild type (WT) and an outer membrane protein W (OmpW) mutant. The survival rates of ΔOmpW strain after treatment with 1.0 and 1.5 mM hydrogen peroxide were significantly reduced compared with those of WT. Morphological changes, including cell membrane damage and cell fragmentation, in ΔOmpW were more predominant than those in WT. By crystal violet staining, we also observed increased biomass in ΔOmpW biofilms as compared with WT following treatment with 0.5 and 1.0 mM H2O2. Biofilms using scanning electron microscopy and confocal laser scanning microscopy further confirmed the structural changes of biofilms between WT and ΔOmpW in response to oxidative stress. The current findings show that OmpW contributed to survival of planktonic cells under oxidative stress and the deletion of OmpW facilitated the biofilm formation in C. sakazakii to adapt to oxidative stress.

Genes involved in tolerance to osmotic stress by random mutagenesis in Cronobacter malonaticus.

Cronobacter malonaticus is one of the opportunistic food-borne pathogens in powdered infant formula and has unusual abilities to survive under environmental stresses such as osmotic conditions. However, the genes involved in osmotic stress have received little attention in C. malonaticus. Here, genes involved in osmotic stress were determined in C. malonaticus using a transposon mutagenesis approach. According to the growth of mutants (n = 215) under 5.0% NaCl concentration, the survival of 5 mutants under osmotic stress was significantly decreased compared with that of the wild type strain. Five mutating sites, including potassium efflux protein KefA, inner membrane protein YqjF, peptidylprolyl isomerase, Cys-tRNA(Pro)/Cys-tRNA(Cys) deacylase, and oligogalacturonate lyase were successfully identified. In addition, the biofilm formation of 5 mutants was determined using crystal violet staining, scanning electron microscopy, and confocal laser scanning microscopy, and the biofilms of 5 mutants significantly decreased within 72 h compared with that of wild type strain. This is the first report to determine the genes involved in osmotic tolerance in C. malonaticus. The findings provided valuable information for deep understanding of the mechanism of survival of C. malonaticus under osmotic stress, and a possible relationship between biofilm formation and tolerance to osmotic stress was also demonstrated in C. malonaticus.

Roles of outer membrane protein W (OmpW) on survival, morphology, and biofilm formation under NaCl stresses in Cronobacter sakazakii.

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.

Short communication: Roles of outer membrane protein W (OmpW) on survival and biofilm formation of Cronobacter sakazakii under neomycin sulfate stress.

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.

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

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.