Construction And Application Of A Phage Recognition Element-based Specific Detection Method For Cronobacter Sakazakii

Cronobacter sakazakii is a Gram-negative food-borne pathogen belonging to the family of Enterobacteriaceae.It is widely distributed in ready-to-eat,animal-derived foods,and lowmoisture foods,especially in powdered infant formula（PIF）.Infection with C.sakazakii can cause meningitis,septicemia,and necrotizing tiny intestinal colitis in infants,and the fatality rate is high.Currently,the detection of C.sakazakii in PIF depends on the selective culturebased method,while this method is complicated,inefficient,and always time-consuming.Immunological assays,such as enzyme-linked immunosorbent（ELISA）and immunofluorescence assay,also have defects such as high cost,low precision,complicated pretreatment,difficulty in automation,and inability to distinguish live/dead bacteria.Therefore,it is necessary to develop highly efficient,specific,sensitive,and rapid assays to identify C.sakazakii in foods.Phages can capture and infect host bacteria by their tail and thus can act as biorecognition elements for the specific detection of hosts.Combining phages with other detection techniques or signal amplification technologies,more sensitive and specific assays can be developed to meet the demand for rapid,accurate,and efficient bacterial detection in food safety.In addition,phages can be used as biocontrol agents to control foodborne pathogens.Therefore,the screening of C.sakazakii phages with high lytic activity and the establishment of a rapid detection method using phages as biometric components to achieve rapid and accurate detection of C.sakazakii in PIF are of practical significance for the safe production,regulation of PIF,and the health protection of infants.This study isolated four phages with high lytic activity against C.sakazakii,and their biological and genome characteristics were clarified.Application for the detection and biocontrol of C.sakazakii in PIF,and a rapid detection method combining quantitative fluorescence PCR and colorimetric method with phages as biometric elements were successfully established.The results of the study are as follows.（1）Isolation,identification,and biological characterization of C.sakazakii phages.Four C.sakazakii phages,EspYZU12,EspYZU13,EspYZU14,and EspYZU15,were isolated from wastewater samples collected in Yangzhou.The transmission electron microscopy results showed that the four phages had an equal head（67-82 nm in diameter）and tail（102-121 nm in length）,as well as a contractable tail sheath.The four phages were classified into Caudovirales and Myoviridae based on morphological characteristics.EspYZU13 exhibited lysis performance against 9 out of 18 tested strains.All four phages were non-lytic,and the frequency of insensitive mutations of C.sakazakii（CICC 22919 and CICC 21569）to the four phages ranged from 3.2 × 10-6-6.5 × 10-5,with the lowest frequency of resistant mutations in phage EspYZU 12.Phage EspYZU 12 and EspYZU 15 showed good infection when the optimal infection MOI was 0.001,while phages EspYZU 13 and EspYZU14 showed the best infection when the MOI was 0.1.The one-step growth curve showed that the incubation period of these four phages ranged from 5 to 20 min,and the lysis amount was 10 to 250 PFU/host cell,among which the incubation period of phage EspYZU 12,EspYZU13,and EspYZU14 was shorter and the lysis amount of phage EspYZU 12 was the highest.The four phages showed high stability in the range of 50℃-60℃ and pH 4.0-10.0.In addition,all four phages showed different abilities against C.sakazakii at 37℃,and the maximum inhibition rate against the host was 46%-84%at MOI of 0.01-100.（2）Whole genome sequencing and bioinformatics analysis of C.sakazakii phages.The whole genome of four C.sakazakii phages was sequenced,and the comparative analysis of genome and protein function annotation was performed.The known functional genes of the four strains included structural proteins（e.g.,head protein,tail protein,coat protein,etc.）,phage cleavage genes（e.g.,lytic enzyme,perforin）,DNA packaging genes,DNA manipulation genes（e.g.,DNA polymerase genes）,and DNA manipulation genes.All four phages were free of virulence and lysogenic genes,indicating their high application safety.Phylogenetic tree analysis showed that they belonged to two different genetic groups and had a significant genetic evolutionary distance between them.The results of the comparative genomics analysis are as follows:Salmonella phage GEC_vB_MG,accession number:MW006477.1;Salmonella phage PVP-SE1,accession number:NC₀16071.1 showed that the genomic sequences of the phages EspYZU 12,EspYZU 14,and EspYZU 15 had a total of 131 homologous genes with the above two phages,while the phage EspYZU 13 has only 11 homologous genes with C.sakazakii phage vB_CskP_GAP227（accession number:KC107834）.（3）Establishment of a method for detecting C.sakazakii in PIF by phage amplification in combination with quantitative real-time PCR（PAA-qPCR）.An amplification technique using C.sakazakii phage EspYZU12 as a biological recognition element combined with quantitative real-time PCR（PAA-qPCR）was developed to detect C.sakazakii indirectly.The phage EspYZU12（103 PFU/mL）was co-cultured with C.sakazakii standard gradient suspension（101-105 CFU/mL）for 4 h.The linear relationship between the number of viable C.sakazakii and the number of proliferating phages was established by qPCR,and the number of viable C.sakazakii in the samples was calculated based on the number of proliferating phages.Based on this strategy,PAA-qPCR could detect C.sakazakii in the range of 3.2 × 101-3.2 × 105 CFU/mL with a detection limit of 6.58 × 102 CFU/mL.In addition,PAA-qPCR could be used for the specific detection of C.sakazakii in PIF,and the accuracy factor（Af）,deviation factor（Bf）,and root mean square error（RMSE）were 1.029,1.026,and 1.808,respectively,indicating that the method is highly reliable.The above results showed that PAA-qPCR could rapidly detect live C.sakazakii in PIF.（4）Preparation of phage-directed immobilized palladium-based nanozyme and its detection of C.sakazakii.The specific detection of C.sakazakii was achieved by electrostatically immobilizing the phage EspYZU15 on the surface of a palladium-based nanozyme.EspYZU15@Pd exhibited excellent peroxidase-like activity and catalyzed the color development reaction of 3,3’,5,51tetramethylbenzidine（TMB）in the presence of H2O2.Based on the specific recognition and capture ability of EspYZU15@Pd to the host,C.sakazakii could specifically and efficiently inhibit the peroxidase-like activity of EspYZU15@Pd and suppress the above colorimetric reaction.Based on this strategy,EspYZU15@Pd could be used for the colorimetric detection of C.sakazakii in the range of 3.2 × 102-3.2 × 107 CFU/mL with the detection limit of 3.6 ×10 CFU/mL.The sample recoveries of EspYZU15@Pd for the colorimetric detection of C.sakazakii in PIF ranged from 95.8%to 104.9%with a relative standard deviation of less than 4.6%,indicating that the method is highly reliable.The detection results were consistent with the plate count method,indicating that the method has good accuracy.Therefore,the colorimetric detection method based on EspYZU15@Pd in this chapter can be used to specifically detect C.sakazakii in PIF.（5）Preparation of phage magnetic nanospheres and their detection of C.sakazakii.Covalent immobilization of the phage EspYZU13 on the surface of magnetic nanospheres was used to achieve magnetic enrichment and sensitive detection of C.sakazakii.The phage EspYZU13 was immobilized on the surface of carboxylated magnetic Fe3O4 nanomicrospheres by amidation.The phage EspYZU13@Fe3O4 nanomicrospheres were prepared to achieve magnetic enrichment of C.sakazakii in complex environments（low concentration and multi-component environments）.EspYZU13@Fe3O4 exhibited peroxidaselike activity and catalyzed the color development reaction of 3,3’.5.5’-tetramethylbenzidine（TMB）in the presence of H2O2.Based on the specific recognition and capture ability of EspYZU13@Fe3O4 to the host,C.sakazakii could specifically and efficiently inhibit the peroxidase-like activity of EspYZU13@Fe3O4,and suppress the above colorimetric reaction.Based on this strategy,EspYZU13@Fe3O4 could be used for the colorimetric detection of C.sakazakii in the 3.2×102-3.2×107 CFU/mL with a detection limit of 2.6×10 CFU/mL.The sample recovery of EspYZU13@Fe3O4 for the colorimetric detection of C.sakazakii in PIF ranged from 95.8%to 106.8%with a relative standard deviation of less than 3.2%.The relative standard deviation was less than 3.2%,indicating that the method is highly reliable.The results agreed with the plate count method,indicating that the method has good accuracy.It was demonstrated that EspYZU13@Fe3O4 could be used to detect C.sakazakii in PIF.In addition,based on the magnetic and specific adsorption ability of EspYZU13@Fe3O4,the nanomicrospheres have specific enrichment and separation ability for Cronobacter sakazakii in complex fractions.In summary,four C.sakazakii phages with high lytic activity were isolated,and the whole genome sequencing of the four phages were determined by using bioinformatics technology,the phage isolates were found to be highly genetically distinct and did not contain resistance and virulence genes,providing a candidate biocontrol agent for the bioreduction of C.sakazakii during PIF production.Based on C.sakazakii phage EspYZU12,a PAA-qPCR assay was successfully established to rapidly and accurately detect live C.sakazakii in PIF.Two peroxidase-like enzymes based on phage EspYZU13 and EspYZU 15 as biorecognition elements were successfully prepared for the rapid and accurate colorimetric（visualization）detection of C.sakazakii.This study provides a new method and theoretical basis for the bioreduction and specific and inexpensive rapid detection of C.sakazakii.