Profiling the Virulence and Antibiotic Resistance Genes of Cronobacter sakazakii Strains Isolated From Powdered and Dairy Formulas by Whole-Genome Sequencing.

Julio Parra-Flores,Jetsi Mancilla-Rojano,Ondrej Holý,Eduard Maury-Sintjago,Juan Xicohtencatl-Cortes,Alejandra Rodríguez-Fernández,Francisca Riffo,Guillermo Figueroa,Stephen Forsythe,Ariadnna Cruz-Córdova,Miriam Troncoso,Werner Ruppitsch,Sarah Lepuschitz

doi:10.3389/fmicb.2021.694922

Abstract

Cronobacter sakazakii is an enteropathogen that causes neonatal meningitis, septicemia, and necrotizing enterocolitis in preterm infants and newborns with a mortality rate of 15 to 80%. Powdered and dairy formulas (P-DF) have been implicated as major transmission vehicles and subsequently the presence of this pathogen in P-DF led to product recalls in Chile in 2017. The objective of this study was to use whole genome sequencing (WGS) and laboratory studies to characterize Cronobacter strains from the contaminated products. Seven strains were identified as C. sakazakii, and the remaining strain was Franconibacter helveticus. All C. sakazakii strains adhered to a neuroblastoma cell line, and 31 virulence genes were predicted by WGS. The antibiograms varied between strains. and included mcr-9.1 and blaCSA genes, conferring resistance to colistin and cephalothin, respectively. The C. sakazakii strains encoded I-E and I-F CRISPR-Cas systems, and carried IncFII(pECLA), Col440I, and Col(pHHAD28) plasmids. In summary, WGS enabled the identification of C. sakazakii strains and revealed multiple antibiotic resistance and virulence genes. These findings support the decision to recall the contaminated powdered and dairy formulas from the Chilean market in 2017.

Highlights

On June 2, 2017, the Chilean Ministry of Health issued a national and international food safety alert as a result of the presence of Cronobacter sakazakii in one batch of powdered infant formula (PIF) and one batch of dairy products
Due to the later recognition of the Franconibacter genus, the fusA sequence is designated as ‘Franconibacter helveticus’ and this indicates that the original polymerase chain reaction (PCR) probes lacked genus specificity
From whole genome sequencing (WGS) data and using average nucleotide identity (ANI), ribosomal multilocus sequence typing (MLST), and core genome MLST, six strains were confirmed as C. sakazakii ST1, one as C. sakazakii ST83, and the remaining strain as Franconibacter helveticus ST345

Summary

Introduction

On June 2, 2017, the Chilean Ministry of Health issued a national and international food safety alert as a result of the presence of Cronobacter sakazakii in one batch of powdered infant formula (PIF) and one batch of dairy products (powder milk). This preventive measure was adopted due to the risk of disease associated with C. sakazakii in vulnerable populations (Parra-Flores et al, 2018b). The source of contamination is closely associated with powdered milk (PM) manufacturing plants and the ingredients used in its manufacture (Holý and Forsythe, 2014). In Chile, its incidence in PIF was 9.5% in 2015, 35% in 2017, and 4.7% in 2020 (Parra et al, 2015; Parra-Flores et al, 2018b, 2020)

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Methods

Results