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

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.

Genomic Characterization of Cronobacter spp. and Salmonella spp. Strains Isolated From Powdered Infant Formula in Chile

“…Only the C. sakazakii ST1 strains carried the COL(pHHAD28) plasmids, which are associated with antibiotic resistance genes; these have previously been detected in C. sakazakii strains isolated from dairy products in Chile ( Ramsamy et al, 2020 ; Khezri et al, 2021 ; Parra-Flores et al, 2021b ). The C. malonaticus strain harbored a plasmid homologous to pESA3 called IncFIB (pCTU1), which encodes iron acquisition virulence genes necessary for pathogen survival, but not an external protease known as plasminogen activator ( cpa ) that enhances pathogen propagation in the host ( Franco et al, 2011 ).…”

“…This may reflect either the widespread occurrence of this sequence type in PIF manufacturing environment or that a common ingredient was contaminated with the same strain ( Sonbol et al, 2013 ). Cronobacter sakazakii ST1 has frequently been found in PIF commercialized in different countries, in the PIF processing environment, and in invasive clinical cases such as fatal meningitis and septicemia ( Joseph and Forsythe, 2012 ; Sonbol et al, 2013 ; Fei et al, 2015 ; Csorba et al, 2021 ; Holý et al, 2021 ; Parra-Flores et al, 2021b ). A survey of Cronobacter in the Americas showed the majority of reports isolations were from North America (57.4%, n = 465) and Brazil (42.6%, n = 465).…”

“…The marA gene, whose transcription function regulates multidrug efflux, modulates membrane permeability and activates the transcription of the AcrAB-TolC efflux pump that plays an important role in antibiotic resistance ( Wang X. et al, 2021 ). Several authors have detected the msbA , emrR , H-NS , emrB , marA , CRP , PBP3 , H-NS , and msrB genes that are associated with antibiotic resistance by using efflux pumps, regulatory systems, and antibiotic target protection genes ( Aly et al, 2019 ; Lepuschitz et al, 2019 ; Parra-Flores et al, 2021b ). All strains exhibited the glpT gene that encodes fosfomycin resistance.…”

“…It is known that Salmonella is recognized as an intestinal pathogen; therefore, this is an ecosystem where bacteria can acquire external genetic material and integrate it into their genome by various horizontal transfer mechanisms and by other means such as CRISPR-Cas systems ( Louwen et al, 2014 ). The cas1 and cas2 genes are indispensable for integrating and processing the information acquired by the bacteria; in their absence, the system loses the ability to acquire information ( Parra-Flores et al, 2021b ). The search for prophages in the studied genomes showed that they may have few intact phages.…”

Infection of Cronobacter sakazakii ST1 Producing SHV-12 in a Premature Infant Born from Triplet Pregnancy

“…Our genomic analysis of global C. sakazakii ST1 population, revealed 36 (~30%) isolates from ten countries with single acquired gene, mainly mcr-9.1 (n = 35; Table S1). The majority of MCR-producers (>60%) were collected recently, between 2017 and 2020 [10]. The exception was an isolate from Switzerland with six acquired genes conferring resistance to different antimicrobial compounds.…”