Xiaoke Xu

Prevalence, molecular characterization, and antibiotic susceptibility of Cronobacter spp. in Chinese ready-to-eat foods.

Cronobacter spp. are foodborne pathogens that cause rare but life-threatening diseases in neonates and infants; they can also cause disease in adults. Cronobacter spp. contamination of ready-to-eat (RTE) foods has been reported previously. However, to date, the prevalence and contamination levels of these bacteria in RTE foods in China have not yet been determined. Therefore, the aim of this study was to investigate the prevalence of Cronobacter spp. in RTE foods marketed in China. Two-hundred and eighty RTE food samples were collected from different producers and retailers and analyzed using quantitative methods. The isolates obtained were identified to the species level based on fusA sequences, and were subtyped using a PCR-based serotyping technique. Selected isolates were further characterized by multilocus sequence typing (MLST) and antimicrobial sensitivity determination. Of 280 samples tested, 52 (18.6%) were positive for Cronobacter spp. The contamination levels were less than 110 MPN/g for 78.8% (41/52) of the samples. The results of the O-antigen serotyping for 111 isolates showed that Cronobacter sakazakii serogroup O2 (28 isolates) was the most prevalent serotype. MLST analyses produced 41 sequence types (STs), including 20 novel STs. ST8 was the most prevalent ST (9 isolates) followed by ST4 (5 isolates). Antimicrobial sensitivity testing showed that 84.5% and 46.5% of the isolates were resistant to penicillin G and cephalothin, respectively; in contrast, all of the tested isolates were susceptible to cefotaxime, ciprofloxacin, tetracycline, and nalidixic acid. To the best of our knowledge, this is the first report on Cronobacter spp. prevalence in RTE foods in China, and the findings of our study nonetheless suggested that Cronobacter spp. contamination of Chinese RTE foods poses a potential risk for the consumer. Thus, the study highlights the significance of developing more effective control strategies during the manufacturing process.

Development of an immobilization and detection method of Enterobacter sakazakii from powdered infant formula

Enterobacter sakazakii is an emerging opportunistic pathogen that is associated with rare but life-threatening cases of meningitis, necrotizing enterocolitis, and sepsis in premature and full-term infants. In the present study, a procedure was developed for immobilization of E. sakazakii with zirconium hydroxide coupled with detection by a species-specific duplex PCR, based on 16s-23s rDNA internal transcribed spacer (ITS) and ompA gene. Specificity of duplex PCR was tested against two-type strains, six isolates of E. sakazakii and other eight non-E. sakazakii species. When pure culture of E. sakazakii was used for immobilization, total recovery rate ranged from 79.4% to 99.6% of input bacteria, and the detection limit of duplex PCR was 3x10(5)CFU/ml. Different levels of E. sakazakii were inoculated into 90ml reconstituted powdered infant formula (PIF), and detection limit of duplex PCR was 3x10(0)CFU/ml with 24-30h enrichment after immobilization. When the experiment was performed in the presence of 10(2)CFU/ml Salmonella typhimurium, the detection limit of duplex PCR was not affected after enrichment. Seven out of 13 commercial PIF were detected positive by duplex PCR after immobilization, while only three were positive by biological methods. This study demonstrates that the combination of immobilization method with duplex PCR is easy, rapid, and efficient, and may have applications for the detection of E. sakazakii in more PIF samples.

Occurrence and Characterization of Cronobacter spp. in Powdered Formula from Chinese Retail Markets

Cronobacter spp. (formerly known as Enterobacter sakazakii) are foodborne pathogens that cause rare but life-threatening diseases in neonates and infants through consumption of contaminated powdered infant formula. This study was conducted to investigate the occurrence of Cronobacter spp. in powdered formula in China and to further characterize Cronobacter isolates. Isolates were identified to the species level based on the fusA gene sequence, and strains of C. sakazakii were further subtyped by applying the polymerase chain reaction (PCR)-based serotyping method. A total of 23 strains of Cronobacter spp. isolated from 530 powdered formula samples were identified using conventional biochemical methods and duplex PCR. Cronobacter spp. were detected in 6.25%, 1.82%, 3.64%, 5.45%, and 2.50% of the general formula, infant formula (age <6 months), follow-up formula (6-12 months of age), growing-up formula (1-3 years of age), and childrens formula (3-6 years of age), respectively. The individual species were identified as C. sakazakii (22 isolates) and C. malonaticus (1 isolate). Among 22 C. sakazakii isolates, representatives of all but two O-antigen serotypes (serotypes O5 and O6) were recognized.

Identification of potential virulence factors of Cronobacter sakazakii isolates by comparative proteomic analysis.

Cronobacter is a group of important foodborne pathogens associated with neonatal meningitis, septicemia, and necrotizing enterocolitis. Among Cronobacter species, Cronobacter sakazakii is the most common species in terms of isolation frequency. However, the molecular basis involved in virulence differences among C. sakazakii isolates is still unknown. In this study, based on the determination of virulence differences of C. sakazakii G362 (virulent isolate) and L3101 (attenuated isolate) through intraperitoneal injection, histopathologic analysis (small intestine, kidney, and liver) further confirmed virulence differences. Thereafter, the potential virulence factors were determined using two-dimensional electrophoresis (2-DE) coupled with MALDI/TOP/TOF mass spectrometry. Among a total of 36 protein spots showing differential expression (fold change>1.2), we identified 31 different proteins, of which the expression abundance of 22 was increased in G362. These up-regulated proteins in G362 mainly contained DNA starvation/stationary phase protection protein Dps, OmpA, LuxS, ATP-dependent Clp protease ClpC, and ABC transporter substrate-binding proteins, which might be involved in virulence of C. sakazakii. This is the first report to determine the potential virulence factors of C. sakazakii isolates at the proteomic levels.

Prevalence and population analysis of Vibrio parahaemolyticus in aquatic products from South China markets

Vibrio parahaemolyticus is a common foodborne pathogen in aquatic products. To investigate the prevalence of Vibrio parahaemolyticus in aquatic products in South China, 224 samples were collected from markets in four provinces (11 cities) from May 2013 to January 2014. One hundred and fifty isolates were isolated from 98 samples. All isolates were analyzed for the presence of thermostable direct haemolysin (TDH) and TDH-related haemolysin (TRH) by PCR, antibiotic susceptibility analysis by disk diffusion method, serotyping by multiplex PCR and molecular typing by enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) typing. Although all 150 isolates were negative for tdh, 61 strains were trh positive (40.67%). Antimicrobial susceptibility results indicated that most strains were resistant to streptomycin (88.67%), cefazolin (66.00%) and ampicillin (62.67%). All strains were susceptible to chloramphenicol. Forty percent of all isolates were O2 type. The 150 isolates were grouped into three clusters by ERIC-PCR typing. The results demonstrated the presence of V. parahaemolyticus in aquatic products from the retail market and this methodology can be used for microbiological risk assessment in China.

Prevalence, Molecular Characterization, and Antibiotic Susceptibility of Vibrio parahaemolyticus from Ready-to-Eat Foods in China

Vibrio parahaemolyticus is the leading cause of foodborne outbreaks, particularly outbreaks associated with consumption of fish and shellfish, and represents a major threat to human health worldwide. This bacterium harbors two main virulence factors: the thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH). Additionally, various serotypes have been identified. The extensive use of antibiotics is a contributing factor to the increasing incidence of antimicrobial-resistant V. parahaemolyticus. In the current study, we aimed to determine the incidence and features of V. parahaemolyticus in ready-to-eat (RTE) foods in China. We found 39 V. parahaemolyticus strains on Chinese RTE foods through investigation of 511 RTE foods samples from 24 cities in China. All isolates were analyzed for the presence of tdh and trh gene by PCR, serotyping was performed using multiplex PCR, antibiotic susceptibility analysis was carried out using the disk diffusion method, and molecular typing was performed using enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) typing and multilocus sequence typing (MLST). The results showed that none of the isolates were positive for tdh and trh. Most of the isolates (33.3%) were serotype O2. Antimicrobial susceptibility results indicated that most strains were resistant to streptomycin (89.7%), cefazolin (51.3%), and ampicillin (51.3%). The isolates were grouped into five clusters by ERIC-PCR and four clusters by MLST. We updated 10 novel loci and 33 sequence types (STs) in the MLST database. Thus, our findings demonstrated the presence of V. parahaemolyticus in Chinese RTE foods, provided insights into the dissemination of antibiotic-resistant strains, and improved our knowledge of methods of microbiological risk assessment in RTE foods.