A total of 7 Cronobacter strains were isolated from 703 fecal samples collected in Jinan from June 13 to December 30, 2011, with the positive rate of Cronobacter spp. being 1.0% (95% confidence interval 0.6%-1.4%). Three Cronobacter sakazakii stains were isolated from 157 fecal samples of healthy neonates (95% confidence interval 0.4%-5.5%). This number was slightly higher than that isolated from 273 fecal samples of healthy adults, in which 1 strain of C. sakazakii and 1 strain of Cronobacter malonaticus were isolated, and that from 173 fecal samples of adults with acute diarrhea, in which 1 strain of C. sakazakii and 1 strain of C. malonaticus were isolated, but the differences were not statistically significant (P>0.05). The Cronobacter isolates were all from different genetic sources. It should be noted that Cronobacter carriage may cause infection under certain conditions, especially in neonates.
Bacterial Typing Techniques ; Cronobacter ; Cronobacter sakazakii ; Food Microbiology ; Humans ; Infant, Newborn

Cronobacter sakazakii (C. sakazakii), formerly Enterobacter sakazakii, is an emerging pathogen associated with the ingestion of contaminated reconstituted formula that causes serious illnesses such as bacteremia, septicemia, necrotizing enterocolitis, meningitis and death in low-birth-weight preterm neonatal infants. The objective of this study was to develop an animal model for human neonatal C. sakazakii infections. We acquired timed-pregnant ICR mice and allowed them to give birth naturally. On postnatal day 3.5, each pup was administered orally a total dose of approximately 107 CFU C. sakazakii strain 3439. Mice were observed twice daily for morbidity and mortality. At postnatal day 10.5, the remaining pups were euthanized, and brain, liver, and cecum were excised and analyzed for the presence of C. sakazakii. C. sakazakii was isolated from cecum and other tissues in inoculated mice. In the tissues of C. sakazakii infected mice, meningitis and gliosis were detected in brain. In this study, we confirmed the neonatal ICR mice may be used a very effective animal model for human neonatal C. sakazakii infections.
Animals ; Bacteremia ; Brain ; Cecum ; Cronobacter ; Cronobacter sakazakii ; Eating ; Enterocolitis, Necrotizing ; Gliosis ; Humans ; Infant ; Liver ; Meningitis ; Mice ; Mice, Inbred ICR ; Models, Animal ; Parturition ; Sepsis ; Sprains and Strains

BACKGROUND/AIMS: Cronobacter sakazakii, an emergent pathogen is considered as a major concern to infants and neonates fed on reconstituted powdered infant milk formula. In conjunction with many other factors, biofilm forming capacity adds to its pathogenic potential. In view of the facts that infants are at highest risk to C. sakazakii infections, and emerging antibiotic resistance among pathogens, it is imperative to evaluate probiotic cultures for their efficacy against C. sakazakii. Therefore, pure probiotic strains were isolated from commercial probiotic products and tested for their antimicrobial and anti-biofilm activities against C. sakazakii. METHODS: A total of 6 probiotic strains were tested for their antibiotic susceptibility followed by antimicrobial activity using cell-free supernatant (CFS) against C. sakazakii. The inhibitory activity of CFS against biofilm formation by C. sakazakii was determined using standard crystal violet assay and microscopic observations. RESULTS: All the probiotic strains were sensitive to ampicillin, tetracycline, vancomycin and carbenicillin whereas most of the strains were resistant to erythromycin and novobiocin. Four of the 6 probiotic derived CFS possessed antimicrobial activity against C. sakazakii at a level of 40 μL. A higher biofilm inhibitory activity (>80%) was observed at initial stages of biofilm formation with weaker activity during longer incubation upto 48 hours (50%–60%). CONCLUSIONS: The study indicated the efficacy of isolated commercial probiotics strains as potential inhibitor of biofilm formation by C. sakazakii and could be further explored for novel bioactive molecules to limit the emerging infections of C. sakazakii.
Ampicillin ; Biofilms ; Carbenicillin ; Cronobacter sakazakii ; Cronobacter ; Drug Resistance, Microbial ; Erythromycin ; Gentian Violet ; Humans ; Infant ; Infant, Newborn ; Milk ; Novobiocin ; Probiotics ; Tetracycline ; Vancomycin

Enterobacter sakazakii previously known as a 'yellow-pigmented Enterobacter cloacae', was designated as a unique species in 1980. This reclassification was based on differences from E. cloacae in DNA relatedness, pigment production and biochemical reactions. In 1961, Urmenyi and Franklin reported the first two known cases of neonatal meningitis and septicemia caused by E. sakazakii, although at that time it was still considered as an E. cloacae strain. Since 1961, a number of cases of neonatal infection caused by E. sakazakii have been reported worldwide. Usually newborn with E. sakazakii meningitis have a poor prognosis, many of them dying within days of infection. The reported case-fatality rate vary from 40-80%. We diagnosed and experienced a case of epidural abscess caused by E. sakazakii in a 1 month old male patient, who was asymptomatic with brain mass detected on brain ultrasound examination. We presented this case with a brief review of literatures.
Brain ; Cloaca ; Cronobacter sakazakii* ; DNA ; Enterobacter* ; Epidural Abscess* ; Humans ; Infant, Newborn* ; Male ; Meningitis ; Prognosis ; Sepsis ; Ultrasonography

OBJECTIVETo analyze the ribotyping fingerprint of Enterobacter sakazakii (E. sakazakii) isolated from food and its typing power.

METHODSTwo standard strains and twenty-eight isolates of E.sakazakii were analyzed by the DuPont Riboprinter(TM) microbial characterization system. The relevant database was established and the fingerprint patterns were analyzed with BioNumerics software.

RESULTSThis system grouped two standard strains and twenty-eight E.sakazakii isolates into 26 ribotypes, and four ribotypes included two strains respectively, the other twenty-two strains showed different ribotypes. The lowest similarity was 31.58%. The number of bands by ribotyping was approximately ten and the molecular weight of these bands ranged from 1 to 50 kb. By the clustering program in BioNumerics, these isolates could be grouped into four clusters.

CONCLUSIONThe automatic ribotyping method is convenient and fast in E.sakazakii typing.

Objective: To assess the risk of foodborne diseases caused by Cronobacter sakazakii in infant formula powder from retail to feeding and provide formulate suggestions for safe feeding of infants at home. Methods: This study used the special monitoring and risk monitoring data of infant formula powder in Heilongjiang Province and combined data at home and abroad. The @RISK software was used to evaluate the disease risk caused by Cronobacter sakazakii in the process of infant formula powder from retail to feeding. Results: According to the results of this quantitative risk assessment, the risk of foodborne diseases caused by Cronobacter sakazakii at the current consumption pattern in Heilongjiang Province was 5.158×10^-5 persons/million (40.0 ℃ and 50.0 ℃), 1.072×10^-7 persons/million (60.0 ℃), 5.544×10^-14 persons/million (70.0 ℃). When the feeding time of infant formula powder was adjusted to 0-2 h and 2-3 h respectively, the above prediction results did not change. When it was adjusted to 3-4 h, the risk increased. If it was adjusted to 4-24 h, the number of Cronobacter sakazakii increased by 14-24 orders of magnitude at room temperature. If the initial pollution concentration (after flushing) was adjusted to 1 MPN/ml, the average disease risk per meal was 805.7 persons/million (40.0 ℃ and 50.0 ℃), 1.7 persons/million (60.0 ℃) and 9.1 × 10^-7 persons/million (70.0 ℃). The results of sensitivity analysis showed that the water temperature (70.0 ℃), initial pollution concentration, room storage time and temperature were important factors of risk. Conclusion: Controlling the contamination level of Cronobacter sakazakii in infant formula powder, controlling the feeding time within 3 h, storing in refrigerator and mixing with water with temperature not lower than 70.0 ℃ are effective measures to prevent infants from eating infant formula powder infected by Cronobacter sakazakii.
Infant ; Humans ; Cronobacter sakazakii ; Infant Formula ; Food Microbiology ; Powders ; Risk Assessment ; Foodborne Diseases

We used a proteomic approach to identify IbpA in Cronobacter sakazakii (C. sakazaki), which is related to heat tolerance in this strain. The abundance of IbpA in C. sakazakii strains strongly increased after heat shock. C. sakazakii CMCC 45402 ibpA deletion mutants were successfully constructed. The C. sakazakii CMCC 45402 ΔibpA and wild-type strains could not be distinguished based on colony morphology on LB agar plates or biochemical assays. The growth of the C. sakazakii CMCC 45402 ΔibpA mutant in heat shock conditions was indistinguishable from that of the isogenic wild-type, but showed greater heat resistance than E. coli O157:H7 strain CMCC 44828. This study suggests that the absence of a single ibpA gene has no obvious effect on the phenotype or heat resistance of the strain C. sakazakii CMCC 45402.
Bacterial Proteins ; genetics ; metabolism ; Cronobacter sakazakii ; genetics ; physiology ; Gene Expression Regulation, Bacterial ; physiology ; Genotype ; Heat-Shock Proteins ; genetics ; metabolism ; Hot Temperature ; Stress, Physiological

BACKGROUND: There have been few studies about the kinds of species causing surgical site infections and their resistance pattern in Korea. An increase of extended-spectrum beta-lactamase (ESBL) producing strains is a worldwide problem. However, there is not enough data on the prevalence of ESBL-producing strains in Korea and the true extent of this problem seems to be under-recognized. METHODS: Minimal inhibitory concentrations of gram-negative bacilli isolated from surgical site infections were tested using the standard agar dilution method according to the National Committee for Clinical Laboratory Standards. To identify and characterize beta-lactamases, we performed conjugation test, isoelectric focusing, Southern hybridization, and polymerase chain reaction. RESULTS: A total of 54 strains of gram-negative enteric bacilli were identified:two strains of Acinetobacter spp., one of Citrobacter freundii, nine of Enterobacter cloacae, one of Enterobacter sakazakii, one of Escherichia coli, two of Klebsiella pneumoniae, one of Morganella morganii, one of Proteus vulgaris, 23 of Pseudomonas aeruginosa, four of Xanthomonas maltophila, and nine of Serratia marcescens. Three strains produced ESBL. CONCLUSION: Various species of gram-negative organisms isolated from surgical site infections showed complex antibiograms to various beta-lactams, even to the new generation of antibiotics. A large proportion of these strains showed conjugally transferable, plasmid-mediated, beta-lactam resistance. Some strains were ESBL-producing. This evidence suggests that there has been a molecular evolution of beta-lactamase genes to a great extent in Korea, possibly due to indiscriminate use of antibiotics.
Acinetobacter ; Agar ; Anti-Bacterial Agents ; beta-Lactam Resistance* ; beta-Lactamases ; beta-Lactams ; Citrobacter freundii ; Cronobacter sakazakii ; Drug Resistance ; Enterobacter cloacae ; Escherichia coli ; Evolution, Molecular ; Isoelectric Focusing ; Klebsiella pneumoniae ; Korea ; Microbial Sensitivity Tests ; Morganella morganii ; Polymerase Chain Reaction ; Prevalence ; Proteus vulgaris ; Pseudomonas aeruginosa ; Serratia marcescens ; Xanthomonas

BACKGROUND: There have been few studies about the kinds of species causing surgical site infections and their resistance pattern in Korea. An increase of extended-spectrum beta-lactamase (ESBL) producing strains is a worldwide problem. However, there is not enough data on the prevalence of ESBL-producing strains in Korea and the true extent of this problem seems to be under-recognized. METHODS: Minimal inhibitory concentrations of gram-negative bacilli isolated from surgical site infections were tested using the standard agar dilution method according to the National Committee for Clinical Laboratory Standards. To identify and characterize beta-lactamases, we performed conjugation test, isoelectric focusing, Southern hybridization, and polymerase chain reaction. RESULTS: A total of 54 strains of gram-negative enteric bacilli were identified:two strains of Acinetobacter spp., one of Citrobacter freundii, nine of Enterobacter cloacae, one of Enterobacter sakazakii, one of Escherichia coli, two of Klebsiella pneumoniae, one of Morganella morganii, one of Proteus vulgaris, 23 of Pseudomonas aeruginosa, four of Xanthomonas maltophila, and nine of Serratia marcescens. Three strains produced ESBL. CONCLUSION: Various species of gram-negative organisms isolated from surgical site infections showed complex antibiograms to various beta-lactams, even to the new generation of antibiotics. A large proportion of these strains showed conjugally transferable, plasmid-mediated, beta-lactam resistance. Some strains were ESBL-producing. This evidence suggests that there has been a molecular evolution of beta-lactamase genes to a great extent in Korea, possibly due to indiscriminate use of antibiotics.
Acinetobacter ; Agar ; Anti-Bacterial Agents ; beta-Lactam Resistance* ; beta-Lactamases ; beta-Lactams ; Citrobacter freundii ; Cronobacter sakazakii ; Drug Resistance ; Enterobacter cloacae ; Escherichia coli ; Evolution, Molecular ; Isoelectric Focusing ; Klebsiella pneumoniae ; Korea ; Microbial Sensitivity Tests ; Morganella morganii ; Polymerase Chain Reaction ; Prevalence ; Proteus vulgaris ; Pseudomonas aeruginosa ; Serratia marcescens ; Xanthomonas

PURPOSE: We aim to fabricate a thermoplastic poly(methylmethacrylate) (PMMA) Lab-on-a-Chip device to perform continuous- flow polymerase chain reactions (PCRs) for rapid molecular detection of foodborne pathogen bacteria. METHODS: A miniaturized plastic device was fabricated by utilizing PMMA substrates mediated by poly(dimethylsiloxane) interfacial coating, enabling bonding under mild conditions, and thus avoiding the deformation or collapse of microchannels. Surface characterizations were carried out and bond strength was measured. The feasibility of the Lab-on-a-Chip device for performing on-chip PCR utilizing a lab-made, portable dual heater was evaluated. The results were compared with those obtained using a commercially available thermal cycler. RESULTS: A PMMA Lab-on-a-Chip device was designed and fabricated for conducting PCR using foodborne pathogens as sample targets. A robust bond was established between the PMMA substrates, which is essential for performing miniaturized PCR on plastic. The feasibility of on-chip PCR was evaluated using Escherichia coli O157:H7 and Cronobacter condimenti, two worldwide foodborne pathogens, and the target amplicons were successfully amplified within 25 minutes. CONCLUSIONS: In this study, we present a novel design of a low-cost and high-throughput thermoplastic PMMA Lab-on-a-Chip device for conducting microscale PCR, and we enable rapid molecular diagnoses of two important foodborne pathogens in minute resolution using this device. In this regard, the introduced highly portable system design has the potential to enable PCR investigations of many diseases quickly and accurately.
Bacteria ; Cronobacter ; Diagnosis* ; Escherichia coli ; Lab-On-A-Chip Devices* ; Plastics* ; Polymerase Chain Reaction* ; Polymethyl Methacrylate