Yuanhong Li

Genome shuffling of Bacillus amyloliquefaciens for improving antimicrobial lipopeptide production and an analysis of relative gene expression using FQ RT-PCR

Genome shuffling is an efficient approach for the rapid improvement of the yield of secondary metabolites. This study was undertaken to enhance the yield of surfactin produced by Bacillus amyloliquefaciens ES-2-4 using genome shuffling and to examine changes in SrfA expression of the improved phenotype at the transcriptional level. Six strains with subtle improvements in lipopeptide yield were obtained from populations generated by ultraviolet irradiation, nitrosoguanidine, and ion beam mutagenesis. These strains were then subjected to recursive protoplast fusion. A strain library that was likely to yield positive colonies was created by fusing the lethal protoplasts obtained from both ultraviolet irradiation and heat treatments. After two rounds of genome shuffling, a high-yield recombinant F2-38 strain that exhibited 3.5- and 10.3-fold increases in surfactin production in shake flask and fermenter respectively, was obtained. Comparative analysis of synthetase gene expression was conducted between the initial and shuffled strains using FQ (fluorescent quantitation) RT-PCR. Delta CT (threshold cycle) relative quantitation analysis revealed that surfactin synthetase gene (srfA) expression at the transcriptional level in the F2-38 strain was 15.7-fold greater than in the ES-2-4 wild-type. The shuffled strain has a potential application in food and pharmaceutical industries. At the same time, the analysis of improved phenotypes will provide more valuable data for inverse metabolic engineering.

Optimization of a medium for enhancing nicotine biodegradation by Ochrobactrum intermedium DN2

Aims:  To optimize a medium for nicotine degradation by Ochrobactrum intermedium DN2 in presence of yeast extract, glucose and Tween 80 using response surface methodology (RSM).

Use of rpoB gene sequence analysis for phylogenetic identification of Cronobacter species

Here, we report the RNA polymerase beta-subunit gene (rpoB) as a new molecular marker for the identification of the Cronobacter species. The results indicated that members of the Cronobacter genus are more easily discriminated by rpoB sequencing than 16S rRNA sequencing, and reliable identification could be achieved by rpoB gene sequence comparison.

Development and application of a sensitive, rapid, and reliable immunomagnetic separation-PCR detection method for Cronobacter spp.

Cronobacter spp. have been linked to clinical cases of infection in both adults and infants. Enrichment of Cronobacter spp. before detection has been necessary but is quite time consuming. Hence, we sought to develop an immunomagnetic separation (IMS) PCR method that could shorten the time of enrichment before the detection of Cronobacter spp. The polyclonal antibody used in this immunomagnetic separation was prepared based on the outer membrane protein A of Cronobacter sakazakii China Center of Industrial Culture Collection 21560 and had high specificity to the target. The primers used in the IMS-PCR method also showed high specificity. The detection limit of IMS-PCR for pure C. sakazakii culture was 5.2 × 102 cfu/mL. Cronobacter sakazakii in artificially contaminated powdered infant formula (PIF) was also detected at a detection limit of 5.2 × 102 cfu/mL. After 8 h of enrichment, the detection limit in PIF was lower than 5.2 × 101 cfu/mL. An interference test using Escherichia coli in artificially contaminated PIF showed that the IMS-PCR method developed in this study had a good ability to resist interference. Finally, the IMS-PCR method was applied to the detection of Cronobacter in food samples and was shown to be reliable. Thus, this newly developed IMS-PCR detection method was quite sensitive, rapid, and reliable and could be applied to the detection of Cronobacter in foods.

Characterization of a single-chain variable fragment specific to Cronobacter spp. from hybridoma based on outer membrane protein A.

Monoclonal antibody and polyclonal antibody specific to Cronobacter spp. had been reported in previous studies. However, the preparation of single-chain variable fragment (scFv) was faster and convenient. Hence, the aim of this study was to construct a scFv using outer membrane protein A (OmpA) of C. sakazakii as antigen. The protein sequences of OmpA of Cronobacter spp. were analyzed first. The results showed protein OmpA with length of 347 amino acids was conserved in Cronobacter genus (94.83%-100% of protein identity) and was greater than that observed for the other genera tested (8.28-91.64% of protein identity). Then, purified protein OmpA expressed in E. coli was used to prepare hybridoma and to construct scFv further. The scFv was named scFvH81 and analyzed by bioinformatics. The model of scFvH81 built by homologous modeling had a good quality (residues in disallowed regions: 3%) and showed that scFvH81 had a standard pocket-like site. Purified scFvH81 was prepared by denaturation and renaturation of inclusion body and it showed a good specificity and its affinity of Ka=2.39×10(6)M(-1). Therefore, it could be used in the detection and the pathogenesis study of Cronobacter spp.

Genetic Diversity, Antimicrobial Susceptibility, and Biofilm Formation of Cronobacter spp. Recovered from Spices and Cereals

Cronobacter species are important food-borne opportunistic pathogens which have been implicated in the cause of necrotizing enterocolitis, sepsis, and meningitis in neonates and infants. However, these bacteria are routinely found in foodstuffs, clinical specimens, and environmental samples. This study investigated the genetic diversity, antimicrobial susceptibility, and biofilm formation of Cronobacter isolates (n = 40) recovered from spices and cereals in China during 2014–2015. Based on the fusA sequencing analysis, we found that the majority (23/40, 57.5%) of Cronobacter isolates in spices and cereals were C. sakazakii, while the remaining strains were C. dublinensis (6/40, 15.0%), C. malonaticus (5/40, 12.5%), C. turicensis (4/40, 10.0%), and C. universalis (2/40, 5.0%). Multilocus sequence typing (MLST) analysis produced 30 sequence types (STs) among the 40 Cronobacter isolates, with 5 STs (ST4, ST13, ST50, ST129, and ST158) related to neonatal meningitis. The pattern of the overall ST distribution was diverse; in particular, it was revealed that ST148 was the predominant ST, presenting 12.5% within the whole population. MLST assigned 12 isolates to 7 different clonal complexes (CCs), 4, 13, 16, 17, 72, 129, and 143, respectively. The results of O-antigen serotyping indicated that C. sakazakii serotype O1 and O2 were the most two prevalent serotypes. The antimicrobial susceptibility testing showed that the 40 Cronobacter isolates were susceptible to most of the antibiotics tested except for ceftriaxone, meropenem, and aztreona. Of the 40 Cronobacter strains tested, 13 (32.5%) were assessed as weak bioflim producers, one (2.5%) was a moderate biofilm producer, one (2.5%) was strong biofilm producer, and the others (62.5%) were non-biofilm producers. MLST and O-antigen serotyping have indicated that Cronobacter strains recovered from spices and cereals were genetically diverse. Isolates of clinical origin, particularly the C. sakazakii ST4 neonatal meningitic pathovar, have been identified from spices and cereals. Moreover, antimicrobial resistance of Cronobacter strains was observed, which may imply a potential public health risk. Therefore, the surveillance of Cronobacter spp. in spices and cereals should be strengthened to improve epidemiological understandings of Cronobacter infections.