Genetic Diversity Of The Cronobacter Spp.Isolated From Powdered Infant Formula And Production Environments

Cronobacter spp.is one of the important pathogenic bacteria contaminating powdered infant formula(PIF),can cause severe neonatal infections,such as necrotizing enterocolitis,bacteremia and meningitis.The organisms is widely distributed and has a high genetic diversity,can cause the contamination of PIF by contaminated production environment.China is an important region of production and consumption of PIF,and has been facing the problem that Cronobacter contamination,which troubles the PIF industry development.Therefore,the study on genetic diversity of the Cronobacter spp.isolated from PIF and production environments is very significant,can provide theory to prevent and control Cronobacter spp.in PIF production process,and reduces the harm of Cronobacter to the infants.In this study,the research objects were the 70 Cronobacter strains isolated from PIF and production environments.70 Cronobacter strains were analyzed by multilocus sequence typing(MLST),PCR-based O-antigen serotyping,ompA and rpoB sequence analysis to reveal the population characteristic of Cronobacter spp.from PIF and production environments.The clear phylogenetic relationship of 70 Cronobacter strains was constructed using bioinformatics software to illuminate the genetic diversity of the Cronobacter spp.isolated from PIF and production environments systematically.On the basis of MLST of Cronobacter,the correlation analysis was performed with the variables of ST of this pathogen and environmental sources.The riskiest processing links and distributed regularity of Cronobacter strains in PIF production process were determined to reach the purpose of tracing this pathogenic bacteria.Ultimately,Cronobacter strains with the typical sequence types were selected,and were analyzed to excavate deeper bioinformatics information of Cronobacter spp.using the whole genome sequencing technology.The results were as follows:(1)The 70 Cronobacter strains clustered into 19STs.C.sakazakii was the dominant species isolated from PIF and production environments.The main C.sakazakii STs were ST4(18/70,27%),ST1(14/70,21%)and ST64(11/70,16%).The nucleotide diversity(?)ranged from 0.0084(atpD)to 0.0300(gyrB and ppsA)per individual genes,which suggested that the nucleotide diversity of atpD was the lowest,and the nucleotide diversity of gyrB and ppsA were higher than other housekeeping genes.The phylogenetic tree based on 3036 bp of spliced seven housekeeping genes showed that C.sakazakii and C.malonaticus belonged to two phylogenetic branches.In C.sakazakii strains,there a closer phylogenetic relationships between ST4 and ST258,ST64 and ST261;In C.malonaticus strains,there was a closer phylogenetic relationship between ST7 and ST201.(2)The 70 Cronobacter strains were divided into five serotypes,including C.sakazakii serotype O1,C.sakazakii serotype 02,C.sakazakii serotype 04,C.sakazakii serotype 07,and C.malonaticus serotype 02.Furthermore,C.sakazakii serotype 02(55%)and C.sakazakii serotype 01(29%)were the dominant serotypes isolated from PIF and production environments.(3)The ompA gene 70 Cronobacter strains had 10 alleles.Of the 10 alleles,ompA6(42/70,42%),ompA3(13/70,19%)and ompA5(5/70,7%)were the dominant alleles.ompA21,ompA22,ompA23,ompA24,ompA54 and ompA55 were the new alleles.The rpoB gene 70 Cronobacter strains were divided into 10 clusters,which showed that the gene could be used to type the Cronobacter strains with acceptable distinguishability.The rpoBl was the dominant cluster,the number of Cronobacter belong to which was twenty-four,accounting for 34.28%of the total.(4)In the manufacturing process of powdered infant formula,spray drying,fluidized bed drying,and packing were the main areas with Cronobacter contamination.Although the Cronobacter strains had be found in raw materials and equipment surface,the Cronobacter strains with the same ST were not found in the final product.It suggested that the sterilization and closed production mode could prevent and control the Cronobacter strains from raw materials and equipment surface effectively(5)The statistical result of ANI values showed that the ANI value of CMa3(C.malonaticus)was less than 95%compared with other genomes of strains.The ANI values among other strains was more than 97%,and should belong to the C.sakazakii.The ANI values of CE65(ST269),CE64(ST4)and CE16(ST259)were less than 98%compared with other genomes of strains,which indicated that there may be more variation information in them.(6)The pan gene of Cronobacter spp.may be the close pan gene.The gene family number of CE75(ST261,3035)was the most among the 12 Cronobacterg strains,and the gene family number of CE52(ST21,29)was the least.The new gene number of CMa3(ST258,386)was the most among the 12 Cronobacterg strains,and the new gene in CE62(ST64)and CE75(ST261)was not found.In addition,the function genes of Cronobacter spp.was distributed in the core gene,accessory and unique genes,which showed that these features were regulated by the above three kinds of genes.(7)The KEGG analysis of Cronobacter spp.includesd 6 major KEGG categories(40 KEGG sub categories),which contained 385 KEGG pathways,among them,116 KEGG pathways were associated with related functional genes,including one pathway related to cell growth and death,two pathways related to cell motility,three pathways related to membrane transport,one pathway related to signal transduction,three pathways related to folding sorting and degradation,five pathways related to signaling molecules and interaction,four pathways related to translation,eleven pathways related to drug resistance,eight pathways related to amino acid metabolism,thirteen pathways related to carbohydrate metabolism,thirteen pathways related to energy metabolism,eleven pathways related to metabolism of cofactors and vitamins,thirteen pathways related to metabolism of other amino acids,two pathways related to nucleotide metabolism,sixteen pathways related to other metabolism,five pathways related to development,and five pathways related to environmental adaptation.In addition,most of the 40 pathway were regulated by the above three kinds of genes.This study revealed the genetic diversity of the Cronobacter spp.isolated from powdered infant formula and production environments comprehensively,traced the contamination sources of Cronobacter strains in PIF,further,excavated the genetic characteristics of Cronobacter spp.using the whole genome sequencing technology,which could help to solve the scientific question effectively that Cronobacter spp.contamination in the manufacturing process of PIF,made the basic research to serve the production practice.Not only provided a new idea for preventing and controlling the Cronobacter spp.in the production of PIF effectively,but also it was of great significance to the theoretical research of microbial safety in the production of PIF and infant food.