Xiaojuan Yang

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.

Prevalence and Characterization of Monophasic Salmonella Serovar 1 ,4,[5],12:i:- of Food Origin in China

Salmonella enterica subsp. enterica serovar 1,4,[5],12:i:- is a monophasic variant of Salmonella Typhimurium, which has recently been recognized as an emerging cause of infection worldwide. This bacterium has also ranked among the four most frequent serovars causing human salmonellosis in China. However, there are no reports on its contamination in Chinese food. Serotyping, polymerase chain reaction, antibiotic resistance, virulotyping, and multilocus sequence typing (MLST) assays were used to investigate the prevalence of this serological variant in food products in China, and to determine phenotypic and genotypic difference of monophasic isolates and Salmonella Typhimurium isolated over the same period. Salmonella 1,4,[5],12:i:- was prevalent in various food sources, including beef, pork, chicken, and pigeon. The study also confirmed the high prevalence (53.8%) of resistance to ampicillin, streptomycin, sulfonamides, and tetracycline in Salmonella 1,4,[5],12:i:-, which was higher than that in Salmonella Typhimurium. Moreover, Salmonella 1,4,[5],12:i:- isolates in our study were different from Salmonella Typhimurium isolates by the absence of three plasmid-borne genes (spvC, pefA, and rck) and the presence of gipA in all isolates. All Salmonella 1,4,[5],12:i:- isolates demonstrated MLST pattern ST34. Genomic deletions within the fljBA operon and surrounding genes were only found in Salmonella 1,4,[5],12:i:- isolates, with all isolates containing a deletion of fljB. However, hin and iroB were identified in all Salmonella 1,4,[5],12:i:- isolates. Three different deletion profiles were observed and two of them were different from the reported Salmonella 1,4,[5],12:i:- clones from Spain, America, and Italy, which provided some new evidence on the independent evolution of the multiple successful monophasic clones from Salmonella Typhimurium ancestors. This study is the first report of Salmonella 1,4,[5],12:i:- in food products from China. The data are more comprehensive and representative, providing valuable information for epidemiological studies, risk management, and public health strategies.

Prevalence of Staphylococcus aureus and Methicillin-Resistant Staphylococcus aureus in Retail Ready-to-Eat Foods in China.

Staphylococcus aureus, particularly methicillin-resistant S.aureus (MRSA), is a life-threatening pathogen in humans, and its presence in food is a public health concern. MRSA has been identified in foods in China, but little information is available regarding MRSA in ready-to-eat (RTE) foods. We aimed to investigate the prevalence of S. aureus and MRSA in Chinese retail RTE foods. All isolated S. aureus were tested for antimicrobial susceptibility, and MRSA isolates were further characterized by multilocus sequence typing (MLST) and staphylococcal cassette chromosome mec (SCCmec) typing. Of the 550 RTE foods collected from 2011 to 2014, 69 (12.5%) were positive for S. aureus. Contamination levels were mostly in the range of 0.3–10 most probable number (MPN)/g, with five samples exceeding 10 MPN/g. Of the 69 S. aureus isolates, seven were identified as MRSA by cefoxitin disc diffusion test. Six isolates were mecA-positive, while no mecC-positive isolates were identified. In total, 75.8% (47/62) of the methicillin-susceptible S. aureus isolates and all of the MRSA isolates were resistant to three or more antibiotics. Amongst the MRSA isolates, four were identified as community-acquired strains (ST59-MRSA-IVa (n = 2), ST338-MRSA-V, ST1-MRSA-V), while one was a livestock-associated strain (ST9, harboring an unreported SCCmec type 2C2). One novel sequence type was identified (ST3239), the SCCmec gene of which could not be typed. Overall, our findings showed that Chinese retail RTE foods are likely vehicles for transmission of multidrug-resistant S. aureus and MRSA lineages. This is a serious public health risk and highlights the need to implement good hygiene practices.

Classification and fatty acid composition analysis of Cronobacter spp. isolated from powdered infant formula in China

This study aimed to classify a collection of Enterobacter sakazakii (E. sakazakii) strains previously identified from powdered infant formula (PIF) to species level by recN gene sequencing and biochemical testing to determine the distribution of Cronobacter species in China and investigate the strain diversity by cellular fatty acid (CFA) analysis. Of 24 E. sakazakii isolates, 23 were identified as C. sakazakii and one was C. malonaticus. The 23 C. sakazakii isolates showed the same CFA profiles. The C. malonaticus isolate was discriminated from the C. sakazakii isolates by the significant difference in the amounts of C12:0, C14:0, and C17:0 cyclo acids. These results showed that C. sakazakii and C. malonaticus were the common Cronobacter species distributed in PIF in China and that the isolates of the two species exhibited different CFA profiles. These findings are of value for epidemiological investigations and provide an alternative method for confirming various Cronobacter spp.

Multilocus Sequence Typing and Virulence-Associated Gene Profile Analysis of Staphylococcus aureus Isolates From Retail Ready-to-Eat Food in China

The aim of this study was to characterize the subtypes and virulence profiles of 69 Staphylococcus aureus isolates obtained from retail ready-to-eat food in China. The isolates were analyzed using multilocus sequence typing (MLST) and polymerase chain reaction (PCR) analysis of important virulence factor genes, including the staphylococcal enterotoxin (SE) genes (sea, seb, sec, sed, see, seg, seh, sei, sej), the exfoliative toxin genes (eta and etb), the toxic shock syndrome toxin-1 gene (tst), and the Panton-Valentine leucocidin-encoding gene (pvl). The isolates encompassed 26 different sequence types (STs), including four new STs (ST3482, ST3484, ST3485, ST3504), clustered in three clonal complexes and 17 singletons. The most prevalent STs were ST1, ST6, and ST15, constituting 34.8% of all isolates. Most STs (15/26, 57.7%) detected have previously been associated with human infections. All 13 toxin genes examined were detected in the S. aureus isolates, with 84.1% of isolates containing toxin genes. The three most prevalent toxin genes were seb (36.2%), sea (33.3%), and seg (33.3%). The classical SE genes (sea–see), which contribute significantly to staphylococcal food poisoning (SFP), were detected in 72.5% of the S. aureus isolates. In addition, pvl, eta, etb, and tst were found in 11.6, 10.1, 10.1, and 7.2% of the S. aureus isolates, respectively. Strains ST6 carrying sea and ST1 harboring sec-seh enterotoxin profile, which are the two most common clones associated with SFP, were also frequently detected in the food samples in this study. This study indicates that these S. aureus isolates present in Chinese ready-to-eat food represents a potential public health risk. These data are valuable for epidemiological studies, risk management, and public health strategies.

Investigation on the effects of ϵ-poly-L-lysine on a producing strain Streptomyces ahygroscopicus GIM8, for better understanding its biosynthesis

ϵ‐Poly‐L‐lysine (ϵ‐PL) is an L‐lysine homopolymer with strong antimicrobial activity, which is generally produced by Streptomyces strains. ϵ‐PL is only produced under acidic conditions in liquid culture, and to improve the current understanding of ϵ‐PL biosynthesis, the present study was undertaken to investigate the effects of ϵ‐PL on its producer Streptomyces ahygroscopicus GIM8, under acidic and neutral conditions. The results indicated that a neutral pH favored ϵ‐PL adsorption onto the cells, whereas minimal adsorption occurred at pH 4.0, the maximum pH for ϵ‐PL production. At pH 7.0, small amounts of ϵ‐PL caused considerable ATP leakage from the cells, which showed increased membrane permeability. Conversely, ATP leakage was inhibited by ϵ‐PL at pH 4.0. Transmission electron microscopy investigation indicated that the cytoplasmic membrane was the primary site of ϵ‐PL activity at pH 7.0, and that cell shape was maintained. Metabolic activity profiles revealed that ϵ‐PL decreased cellular metabolic activity at a relatively low rate at pH 7.0. However, the toxic effect was significantly enhanced at pH 4.0. Based on these data, a mechanism for the effect of ϵ‐PL on ϵ‐PL‐producing cells under neutral and acidic conditions is proposed. Additionally, acidic conditions may potentially be required for ϵ‐PL biosynthesis in liquid culture because low pH can increase membrane permeability and prevent binding of ϵ‐PL onto cells, both of which favor the secretion of the ϵ‐PL produced by the cells into the broth. This research contributes to the current understanding of ϵ‐PL biosynthesis.