Emergent methods for inactivation of Cronobacter sakazakii in foods: A systematic review and meta-analysis.

Cronobacter sakazakii is a potentially pathogenic bacterium that is resistant to osmotic stress and low aw, and capable of persisting in a desiccated state in powdered infant milks. It is widespread in the environment and present in various products. Despite the low incidence of cases, its high mortality rates of 40 to 80 % amongst neonates make it a microorganism of public health interest. This current study performed a comparative assessment between current reduction methods applied for C. sakazakii in various food matrices, indicating tendencies and relevant parameters for process optimization. A systematic review and meta-analysis were conducted, qualitatively identifying the main methods of inactivation and control, and quantitatively evaluating the effect of treatment factors on the reduction response. Hierarchical clustering dendrograms led to conclusions on the efficiency of each treatment. Review of recent research trend identified a focus on the potential use of alternative treatments, with most studies related to non-thermal methods and dairy products. Using random-effects meta-analysis, a summary effect-size of 4-log was estimated; however, thermal methods and treatments on dairy matrices displayed wider dispersions - of τ2 = 8.1, compared with τ2 = 4.5 for vegetal matrices and τ2 = 4.0 for biofilms. Meta-analytical models indicated that factors such as chemical concentration, energy applied, and treatment time had a more significant impact on reduction than the increase in temperature. Non-thermal treatments, synergically associated with heat, and treatments on dairy matrices were found to be the most efficient.

Lack of correlation between growth rate and sequence type among Cronobacter sakazakii.

Species identification and growth rates for a collection of Cronobacter strains from clinical and non-clinical sources have been previously reported. However, advancements in DNA sequencing-based identification methods now allow for more accurate identification. Here we report the sequence types (STs) for 24 strains of Cronobacter sakazakii and examine any possible correlation between sequence type and growth rate, which could influence risk through greater pathogen multiplication and reach of infectious doses during time between formula preparation and feeding. The most common clonal complexes (CCs) identified were C. sakazakii CC1 and CC4. CC1 strains belonged to ST1 (n = 8) and ST391 (n = 1), while CC4 included ST4 (n = 4), ST255 (n = 1) and ST295 (n = 1). Three strains were found to belong to CC100 and two were found to belong to ST64. The remaining STs identified were represented by single strains. CC4 strains have a slightly not significant tendency for faster growth rates at 25 °C; however, the small sample size suggests that more strains need to be analysed to determine if this is a true result. In conclusion, the growth rates of C. sakazakii strains do not appear to be strongly correlated to ST.

Evaluation of the Membrane Damage Mechanism of Chlorogenic Acid against Yersinia enterocolitica and Enterobacter sakazakii and Its Application in the Preservation of Raw Pork and Skim Milk.

Plant-derived antimicrobial agents have adequate antimicrobial effects on food-borne pathogens, which can be used as food preservatives. The purpose of this study was to evaluate the antibacterial mechanism of chlorogenic acid (CA) against Yersinia enterocolitica and Enterobacter sakazakii. The minimum inhibitory concentration (MIC) of CA was determined by employing the broth microdilution method. Then, the cell function and morphological changes of Y. enterocolitica and E. sakazakii treated with CA were characterized. Finally, the growth inhibition models of Y. enterocolitica in raw pork and E. sakazakii in skim milk were constructed through the response surface methodology. The results demonstrated that CA has a satisfactory inhibitory effect against Y. enterocolitica and E. sakazakii with a MIC of 2.5 mg/mL. In addition, CA inhibited the growth of Y. enterocolitica and E. sakazakii via cell membrane damage, such as depolarization of the cell membrane, reduction in intracellular adenosine triphosphate (ATP) and pH levels, and destruction of cell morphology. Moreover, CA reduced two log cycles of Y. enterocolitica in raw pork and E. sakazakii in skim milk at a certain temperature. According to the corresponding findings, CA has the potential to be developed as an effective preservative to control Y. enterocolitica and E. sakazakii-associated foodborne diseases.

Quantitative detection of trace VBNC Cronobacter sakazakii by immunomagnetic separation in combination with PMAxx-ddPCR in dairy products.

One immunomagnetic separation (IMS) assay based on immunomagnetic beads (IMBs) has been evaluated as a potential pretreatment tool for the separation and enrichment of target bacteria. In this study, we successfully immobilized antibodies onto magnetic bead surfaces to form IMBs through biotin and a streptavidin (SA) system to capture viable but nonculturable (VBNC) Cronobacter sakazakii (C. sakazakii) from dairy products. Various parameters that affected the capture efficiency (CE) of IMS, including the number of antibodies, IMBs dose, incubation time, magnetic separation time, and immunoreaction temperature, were systematically investigated. We further determined the optimal enrichment conditions for different dairy substrates to ensure maximum enrichment of target pathogens in the system. An IMS technique combining improved propidium monoazide (PMAxx) and droplet digital PCR (ddPCR) was established to detect the pathogenic VBNC C. sakazakii. The IMS-PMAxx-ddPCR method after IMBs enrichment showed higher accuracy when the VBNC C. sakazakii was under 1 Log10 copies/g. The detection limit for this method in a background of powdered infant formula (PIF) was 5.6 copies/g. In summary, the developed IMS-PMAxx-ddPCR method has great potential for the analysis and detection of VBNC bacteria in food.

Impact of pmrA on Cronobacter sakazakii planktonic and biofilm cells: A comprehensive transcriptomic study.

Cronobacter sakazakii is an emerging opportunistic foodborne pathogen causing rare but severe infections in neonates. Furthermore, the formation of biofilm allows C. sakazakii to persist in different environments. We have demonstrated that the mutator phenotype ascribed to deficiency of the pmrA gene results in more biomass in the first 24 h but less during the post maturation stage (7-14 d) compared with BAA 894. The present study aimed to investigate the regulatory mechanism modulating biofilm formation due to pmrA mutation. The transcriptomic analyses of BAA 894 and s-3 were performed by RNA-sequencing on planktonic and biofilm cells collected at different time points. According to the results, when comparing biofilm to planktonic cells, expression of genes encoding outer membrane proteins, lysozyme, etc. were up-regulated, with LysR family transcriptional regulators, periplasmic proteins, etc. down-regulated. During biofilm formation, cellulose synthase operon genes, flagella-related genes, etc. played essential roles in different stages. Remarkably, pmrA varies the expression of a number of genes related to motility, biofilm formation, and antimicrobial resistance, including srfB, virK, mviM encoding virulence factor, flgF, fliN, etc. encoding flagellar assembly, and marA, ramA, etc. encoding AraC family transcriptional regulators in C. sakazakii. This study provides valuable insights into transcriptional regulation of C. sakazakii pmrA mutant during biofilm formation.

Lactobacillus gastricus BTM 7 prevents intestinal colonization by biofilm forming Cronobacter sakazakii in Caenorhabditis elegans model host.

The study reports protective role of potential probiotic cultures against infection by biofilm forming Cronobacter sakazakii in Caenorhabditis elegans model system. Among the fifteen indigenous potential probiotics, the cell free supernatant of Lactobacillus gastricus BTM7 possessed highest antimicrobial action and biofilm inhibition against C. sakazakii. The competitive exclusion assays revealed that preconditioning with probiotics resulted in increased mean life span of the nematode to 12-13 days as compared to 5-6 days when the pathogen was administered alone. Enhanced expression of the marker genes (pmk-1, daf-16 and skn-1) was observed during the administration of probiotic cultures. The highest expression of pmk-1 (2.5 folds) was observed with administration of L. gastricus BTM7. The principal component analysis on selected variables revealed that L. gastricus BTM7 has the potential to limit the infection of C. sakazakii in C. elegans and enhance the expression of key genes involved in extending life span of the worm.

Survival strategy of Cronobacter sakazakii against ampicillin pressure: Induction of the viable but nonculturable state.

In a viable but nonculturable (VBNC) state, bacteria are no longer culturable on standard laboratory media, but still, remain a pathogenic potential and present possible health risks. In this study, we investigated ampicillin's ability, which is commonly used in dairy cattle disease treatment, to induce Cronobacter sakazakii into the VBNC state. After treatment with ampicillin, the counts of culturable cells decreased from 108 CFU/mL to an undetected level 7-30 days post-treatment. Meanwhile, viable cells were still approximately 104-105 cells/mL, and could be resuscitated under appropriate conditions. Fluorescence microscopy showed that VBNC cell maintained apparent cellular integrity, but that the morphology of VBNC cells differed visibly from that of normal cells. Moreover, the respiratory chain activity of VBNC cells were confirmed by flow cytometry (FCM) analysis, suggesting that cells in a VBNC state were physiologically active. Finally, transcriptomics analysis and real-time PCR (qPCR) validation were used to explore the underlying mechanisms of VBNC cell formation. Over-expression of relA, lon, ppx, and ppk in the toxin-antitoxin (TA) trigger system contributed to VBNC cell formation. In the TA trigger system, RelA and exopolyphosphatases/guanosine pentaphosphate phosphohydrolases (PPX/GPPA) synthesize ppGpp, which activates polyphosphate kinase (PPK), the cellular enzyme that accumulates plyphosphate (PolyP). PolyP combines with and stimulates Lon to degrade the antitoxins, thereby activating the toxins that induce a VBNC state. The results of our research will facilitate a better understanding of the survival strategies that bacteria develop to deal with ampicillin pressure and the health risks associated with VBNC Cronobacter sakazakii induced by antibiotics.

Amaranthus tricolor crude extract inhibits Cronobacter sakazakii isolated from powdered infant formula.

The purpose of this study was to elucidate the antibacterial activity and possible mechanism of action of Amaranthus tricolor crude extract (ATCE) against Cronobacter sakazakii isolated from powdered infant formula (PIF). The antibacterial activity of ATCE was assessed by measuring the diameter of inhibition zone (DIZ), minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC). The possible mechanism of action of ATCE was revealed by analyzing the effects of ATCE on growth curves and changes in cell membrane potential, intracellular pH, content of bacterial protein and genomic DNA, and cell morphology. Finally, ATCE was applied to the disinfection of C. sakazakii in biofilm on stainless steel tube. The results showed that the DIZ, MIC, and MBC of ATCE against C. sakazakii strains were from 14.35 ± 0.67 to 14.84 ± 0.67 mm, 20 mg/mL, and 40 mg/mL, respectively. Treatment with ATCE ended the logarithmic growth phase of C. sakazakii, and led to depolarization of the cell membranes, reducing intracellular pH and bacterial protein and genomic DNA contents, and resulting in cytoplasmic leakage and deformation. In addition, ATCE effectively inactivated C. sakazakii in biofilm, reducing viable bacteria by approximately 6.5 log cfu/mL bacterial count after treatment with 1 MIC (1 MIC = 20 mg/mL) of ATCE for 20 min at 25°C. Our findings showed that ATCE inactivated C. sakazakii strains isolated from PIF and has potential as a natural disinfectant to reduce the contamination of PIF by C. sakazakii.

Multi-locus sequence typing and antimicrobial susceptibility profile of Cronobacter sakazakii and Cronobacter malonaticus isolated from corn-based farinaceous foods commercialized in Brazil.

The aim of this study was to evaluate the microbiological quality of 45 samples of corn-based farinaceous foods commercialized in Brazil. The bacteriological analysis performed were: detection of Salmonella and Cronobacter, and enumeration of faecal coliforms and Bacillus cereus. The Cronobacter isolates were phenotypically characterized by Vitek 2.0 and the antibiotic susceptibility profile. Molecular characterization was accomplished by real-time PCR targeting dnaG gene and MLST. No sample presented contamination by Salmonella or B. cereus (<102 UFC/g). Faecal coliforms were detected in two (4.4%) samples but in low concentration (≤23.0 MPN/g), and 20 samples (44.4%) contained Cronobacter. Twenty-nine unique Cronobacter isolates were identified as C. sakazakii (n = 18), C. malonaticus (n = 2); that presented 11 different fusA alleles, including new fusA 183. MLST analysis revealed 17 sequence types (STs), six of which were newly identified (ST687-690, 693, and 694). Resistance or intermediary resistance were found to ceftazidime (15.0%), aztreonam (15.0%), nalidixic acid (15.0%), nitrofurantoin (15.0%), cefepime (10.0%), gentamicin (5.0%), and tetracycline (5.0%). The presence of Cronobacter in corn-based farinaceous foods could be a significant risk to infants as these products are used as alternatives to commercially available infant formula. Strategies to manage the risk of Cronobacter infections due to the consumption of these alternative feeds need to be developed by the regulatory agencies.

Effect of hydrolysis and microwave treatment on the antibacterial activity of native bovine milk lactoferrin against Cronobacter sakazakii.

Bovine lactoferrin (bLF) is an iron-binding glycoprotein used in functional and therapeutic products due to its biological properties, the most important being its antimicrobial activity. In this study, hydrolysates of bovine lactoferrin (bLFH) obtained with pepsin, chymosin and microbial rennet were assayed against Cronobacter sakazakii (104 CFU/mL) in different media: phosphate buffered saline (PBS), bovine skim milk and whey, and reconstituted powdered infant formula (PIFM). The results obtained have shown that hydrolysis of bLF enhances its antibacterial activity against C. sakazakii. The three types of bLFH dissolved in PBS reduced C. sakazakii growth from a concentration of 0.1 mg/mL and inhibited it completely above 0.5 mg/mL, after 4 and 8 h of incubation at 37 °C. The three bLFH (1 and 2 mg/mL) did not show any antibacterial activity in skim milk, whey and reconstituted PIFM after 8 h of incubation at 37 °C. However, C. sakazakii growth was completely inhibited in whey when pepsin and chymosin bLFH (2 mg/mL) were combined with undigested bLF (2 mg/mL), after 8 h of incubation at 37 °C. On the other hand, the combination of any of the three hydrolysates with bLF showed very low activity in skim milk and practically no activity in reconstituted PIFM. Furthermore, the effect of temperature after reconstitution (4, 23 and 37 °C), on the antibacterial activity of bLF (2.5 and 5 mg/mL) in reconstituted PIFM contaminated with C. sakazakii (10-102 CFU/mL) was also investigated. bLF at 5 mg/mL significantly reduced (p < .05) the proliferation of C. sakazakii in reconstituted PIFM at 37 °C until 2 h. C. sakazakii did not grow at 4 °C for 6 days in reconstituted PIFM with or without bLF. The effect of microwave heating (450, 550 and 650 W for 5, 10 and 15 s) on the antibacterial activity and stability of bLF (2.5 mg/mL) in reconstituted PIFM contaminated with C. sakazakii (10-102 CFU/mL) was also studied. The antibacterial activity of bLF was maintained after treatments at 450 and 550 W for 5 s, which kept 94 and 89% of bLF immunoreactivity, respectively. Moreover, microwave treatments of reconstituted PIFM with or without bLF, at 650 W for 5 s, and at 450, 550 and 650 W for 10 and 15 s, completely inactivated C. sakazakii.

Inhibition of Cronobacter sakazakii in reconstituted infant formula using triglycerol monolaurate and its effect on the sensory properties of infant formula.

Cronobacter sakazakii (C. sakazakii) is an opportunistic foodborne pathogen in infant formula. This study was designed to explore the inhibitory effect of TGML on C. sakazakii in reconstituted infant formula (RIF). Firstly, the growth curve of C. sakazakii in RIF treated by TGML and the effect of different temperatures (4, 10, 21, 30 and 37 °C), pH values (5, 6, 7, 8 and 9) and ionic strengths (25, 50, 100, 200, 400 and 800 mM) on its activity were assessed. The results showed that the inhibitory effect of TGML on C. sakazakii was dose-dependent, and 1, 2 and 5 µg/mL TGML delayed the visible growth of pathogen by 4, 12 and 24 h, respectively. Storage temperature above or below room temperature enhanced the bioactivity of TGML. And a decrease in pH also increased the antibacterial effect of TGML. However, the effect of ionic strength on its activity was not obvious. Subsequently, the antibacterial effect of TGML in physiological gastric acid and simulated gastric juice in vitro was further explored. We found that only 5 µg/mL TGML could inhibit the growth of pathogen below the infectious dose (10,000 CFU in total) in simulated gastric juice during the whole gastric emptying period (3.5-21 h), weaker than its antibacterial effect in physiological gastric acid and room temperature culture. Finally, the effect of TGML and the above environmental factors on the color and aroma of infant milk was evaluated by a 12-person panel. The results revealed that TGML did not affect the sensory flavor of milk, and the color and odor scores of infant milk under different environmental conditions did not show any significant differences. Therefore, it is concluded that TGML has a good inhibitory effect on C. sakazakii in RIF and a high sensory acceptability for consumers. Adjusting the temperature or lowering the pH enhances its bacteriostatic activity. However, the presence of infant gastric juice can impair the bioactivity of TGML. Overall, this study will provide some new ideas for controlling and eliminating the potential risk of C. sakazakii infection during infant feeding.

The antimicrobial activity of coenzyme Q0 against planktonic and biofilm forms of Cronobacter sakazakii.

Coenzyme Q0 (CoQ0) has demonstrated antitumor, anti-inflammatory, and anti-angiogenic activities. Cronobacter sakazakii is an opportunistic foodborne pathogen associated with high mortality in neonates. In this study, the antimicrobial activity and possible antimicrobial mechanism of CoQ0 against C. sakazakii were investigated. Moreover, the inactivation effect of CoQ0 on C. sakazakii in biofilms was also evaluated. The minimum inhibitory concentration (MIC) of CoQ0 against C. sakazakii strains ranged from 0.1 to 0.2 mg/mL. Treatment caused cell membrane dysfunction, as evidenced by cell membrane hyperpolarization, decreased intracellular ATP concentration and cell membrane integrity, and changes in cellular morphology. CoQ0 combined with mild heat treatment (45, 50, or 55 °C) decreased the number of viable non-desiccated and desiccated C. sakazakii cells in a time- and dose-dependent manner in reconstituted infant milk. Furthermore, CoQ0 showed effective inactivation activity against C. sakazakii in biofilms on stainless steel, reducing the number of viable cells and damaging the structure of the biofilm. These findings suggest that CoQ0 has a strong inactivate effect on C. sakazakii and could be used in food production environments to effectively control C. sakazakii and reduce the number of illnesses associated with it.

Effect of low doses of biocides on the antimicrobial resistance and the biofilms of Cronobacter sakazakii and Yersinia enterocolitica.

The susceptibility of Cronobacter sakazakii ATCC 29544 (CS) and Yersinia enterocolitica ATCC 9610 (YE) to sodium hypochlorite (10% of active chlorine; SHY), peracetic acid (39% solution of peracetic acid in acetic acid; PAA) and benzalkonium chloride (BZK) was tested. Minimum inhibitory concentration (MIC) values (planktonic cells; microdilution broth method) of 3,800 ppm (SHY), 1,200 ppm (PAA) and 15 ppm (BZK) for CS, and 2,500 ppm (SHY), 1,275 ppm (PAA) and 20 ppm (BZK) for YE, were found. In some instances, an increase in growth rate was observed in presence of sub-MICs (0.25MIC, 0.50MIC or 0.75MIC) of biocides relative to the samples without biocides. The cultures exhibited an acquired tolerance to biocides and an increase in antibiotic resistance after exposure to sub-MICs of such disinfectants. Strains were able to form strong biofilms on polystyrene after 48 hours (confocal laser scanning microscopy), with average biovolumes in the observation field (14,161 µm2) of 242,201.0 ± 86,570.9 µm3 (CS) and 190,184.5 ± 40,860.3 µm3 (YE). Treatment of biofilms for 10 minutes with disinfectants at 1MIC or 2MIC reduced the biovolume of live cells. PAA (YE) and BZK (CS and YE) at 1MIC did not alter the percentage of dead cells relative to non-exposed biofilms, and their effect of countering biofilm was due principally to the detachment of cells. These results suggest that doses of PAA and BZK close to MICs might lead to the dissemination of live bacteria from biofilms with consequent hazards for public health.

Effects of intense pulsed light on Cronobacter sakazakii and Salmonella surrogate Enterococcus faecium inoculated in different powdered foods.

Cronobacter sakazakii and Salmonella spp. are foodborne pathogens associated with low moisture foods. An intense pulsed light (IPL) system is being developed as an alternative novel method to pasteurize powdered food. The aim of the study is to investigate the microorganism inactivation in different powdered foods and a variety of related variables using a vibratory-assisted IPL system. The results showed that C. sakazakii on non-fat dry milk (NFDM), wheat flour, and egg white powder were significantly inactivated by 5.27, 4.92, and 5.30 log10 CFU/g, respectively, after 3 or 4 passes of IPL treatments. For decontamination of E. faecium, 3-4 passes of IPL treatments reduced the E. faecium level on NFDM, wheat flour, and egg white by 3.67, 2.79, 2.74 log10 CFU/g, respectively. These results demonstrated that the enhanced microbiological inactivation can be achieved using this vibratory-assisted IPL system after multiple passes.

Principal component analysis of normalized full spectrum mass spectrometry data in multiMS-toolbox: An effective tool to identify important factors for classification of different metabolic patterns and bacterial strains.

RATIONALE: Explorative statistical analysis of mass spectrometry data is still a time-consuming step. We analyzed critical factors for application of principal component analysis (PCA) in mass spectrometry and focused on two whole spectrum based normalization techniques and their application in the analysis of registered peak data and, in comparison, in full spectrum data analysis. We used this technique to identify different metabolic patterns in the bacterial culture of Cronobacter sakazakii, an important foodborne pathogen. METHODS: Two software utilities, the ms-alone, a python-based utility for mass spectrometry data preprocessing and peak extraction, and the multiMS-toolbox, an R software tool for advanced peak registration and detailed explorative statistical analysis, were implemented. The bacterial culture of Cronobacter sakazakii was cultivated on Enterobacter sakazakii Isolation Agar, Blood Agar Base and Tryptone Soya Agar for 24 h and 48 h and applied by the smear method on an Autoflex speed MALDI-TOF mass spectrometer. RESULTS: For three tested cultivation media only two different metabolic patterns of Cronobacter sakazakii were identified using PCA applied on data normalized by two different normalization techniques. Results from matched peak data and subsequent detailed full spectrum analysis identified only two different metabolic patterns - a cultivation on Enterobacter sakazakii Isolation Agar showed significant differences to the cultivation on the other two tested media. The metabolic patterns for all tested cultivation media also proved the dependence on cultivation time. CONCLUSIONS: Both whole spectrum based normalization techniques together with the full spectrum PCA allow identification of important discriminative factors in experiments with several variable condition factors avoiding any problems with improper identification of peaks or emphasis on bellow threshold peak data. The amounts of processed data remain still manageable. Both implemented software utilities are available free of charge from http://uprt.vscht.cz/ms.

Gold nanoparticle-based enhanced lateral flow immunoassay for detection of Cronobacter sakazakii in powdered infant formula.

Cronobacter sakazakii is an opportunistic foodborne pathogen that can infect newborns through powdered infant formula (PIF). In this study, we developed a novel enhanced lateral flow immunoassay (LFA) with enhanced sensitivity for detection of C. sakazakii in PIF by the naked eye. The proposed strategy for signal enhancement of the traditional LFA used concentrated gold nanoparticles (AuNP) as the enhancer to conjugate with capture antibodies, which could increase the immobilized capture antibodies concentration at the detection zone to improve capture efficiency. Besides, the detection signal was further amplified by accumulated AuNP as the C. sakazakii labeled with AuNP probes was captured by antibodies conjugated with enhancer at the test line. We also studied the effect of different concentrations of capture antibodies and concentrated AuNP on detection performance, and found that 2.2 mg/mL of capture antibodies and 0.06 nM concentrated AuNP were the optimal combination that could avoid a false-positive signal and maximally amplify the detection signal of the enhanced LFA. Using this strategy, the detection sensitivity of the enhanced LFA was 103 cfu/mL and improved 100-fold compared with traditional LFA. The strip was highly specific to C. sakazakii, and the time for detection of C. sakazakii in PIF was shortened by 3 h. In summary, the enhanced LFA developed by the addition of concentrated AuNP as the enhancer can be used as a sensitive, rapid, visual qualitative and point-of-care test method for detecting target analytes.

Comparison of Chromogenic Selective Media for the Detection of Cronobacter spp. (Enterobacter sakazakii).

　The four types of chromogenic selective media that are commercially available in Japan were compared for establishing a Japanese standard method for detecting Cronobacter spp. based on ISO/TS 22964:2006. When assessed using 9 standard Cronobacter spp. strains and 29 non-Cronobacter strains, Enterobacter sakazakii isolation agar, ChromocultTM Enterobacter sakazakii agar, CHROMagarTM E. sakazakii, and XM-sakazakii agar demonstrated excellent inclusivity and exclusivity. Using the ISO/TS 22964:2006 method, the recovered numbers of 38 Cronobacter spp. strains, including 29 C. sakazakii isolates obtained from each medium, were equivalent, indicating that there was no significant difference (p > 0.05) among the four types of chromogenic selective media. Thus, we demonstrated that these four chromogenic selective media are suitable alternatives when using the standard method for detecting Cronobacter spp. in Japan, based on the ISO/TS 22964:2006.

Survival of Cronobacter in powdered infant formula and their variation in biofilm formation.

Cronobacter is a ubiquitous Gram-negative pathogen bacterium capable of surviving in low water activity environments, in particular powdered infant formula (PIF). Seven Cronobacter strains representing four different species (C. sakazakii, n = 4; C. malonaticus, n = 1; C. muytjensii, n = 1; C. turicensis, n = 1) were subjected to dry stress and stored in PIF at room temperature. The resulting survivor curves showed that Cronobacter sp. can survive for extended periods of at least 3 months with a significant, but moderate, variability regarding the level of resistance between species; however, no correlation was evident regarding the origin of strains. These results are evaluated with regard to other key characteristics, including genomic profiles and biofilm formation capacities of the strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Cronobacter can survive extended periods of at least 3 months in PIF, with moderately significant interspecific variability in desiccation resistance. Results are evaluated with regard to genomic profiles and biofilm formation capacities of the strains, and contribute to an improved understanding of the environmental persistence of Cronobacter in contaminated PIF, and subsequent risk to infant exposure.

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.

Acetoacetate and ethyl acetoacetate as novel inhibitors of bacterial biofilm.

Acetoacetate (AAA) was identified as a biofilm inhibitor in a previous study, where the effect of 190 carbon and nitrogen sources on biofilm amounts by Escherichia coli O157:H7 was determined. With this study, we tested the effect of AAA on growth and biofilm amounts of Cronobacter sakazakii, Serratia marcescens and Yersinia enterocolitica. AAA reduced growth and biofilm amounts of the three pathogens, albeit at rather high concentrations of 10 to 35 mg ml-1 . Acetoacetate at a concentration of 5 mg ml-1 reduced Y. enterocolitica mRNA transcripts of the flagellar master regulator operon flhD, the invasion gene inv, and the adhesion gene yadA. Transcription of the regulator of plasmid-encoded virulence genes virF, the plasmid-encoded virulence gene yopQ, and ymoA were largely unaffected by AAA. Importantly, AAA did not cause an increase in transcription of any of the tested virulence genes. As a more cost efficient homologue of AAA, the effect of ethyl acetoacetate (EAA) was tested. EAA reduced growth, biofilm amounts and live bacterial cell counts up to 3 logs. IC50 values ranged from 0·31 mg ml-1 to 5·6 mg ml-1 . In summary, both AAA and EAA inhibit biofilm, but EAA appears to be more effective. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial biofilms are communities of bacteria that form on surfaces and are extremely difficult to remove by conventional physical or chemical techniques, antibiotics or the human immune system. Despite advanced technologies, biofilm still contributes to 60 to 80% of human bacterial infections (NIH and CDC) and cause problems in many natural, environmental, bioindustrial or food processing settings. The discovery of novel substances that inhibit biofilm without increasing the virulence of the bacteria opens doors for countless applications where a reduction of biofilm is desired.