Molecular Characterization of Cronobacter sakazakii Strains Isolated from Powdered Milk

Abstract

Cronobacter spp. are opportunistic pathogens of the Enterobacteriaceae family. The organism causes infections in all age groups, but the most serious cases occur in outbreaks related to neonates with meningitis and necrotizing enterocolitis. The objective was to determine the in silico and in vitro putative virulence factors of six Cronobacter sakazakii strains isolated from powdered milk (PM) in the Czech Republic. Strains were identified by MALDI-TOF MS and whole-genome sequencing (WGS). Virulence and resistance genes were detected with the Ridom SeqSphere+ software task template and the Comprehensive Antibiotic Resistance Database (CARD) platform. Adherence and invasion ability were performed using the mouse neuroblastoma (N1E-115 ATCCCRL-2263) cell line. The CRISPR-Cas system was searched with CRISPRCasFinder. Core genome MLST identified four different sequence types (ST1, ST145, ST245, and ST297) in six isolates. Strains 13755-1B and 1847 were able to adhere in 2.2 and 3.2 × 106 CFU/mL, while 0.00073% invasion frequency was detected only in strain 1847. Both strains 13755-1B and 1847 were positive for three (50.0%) and four virulence genes, respectively. The cpa gene was not detected. Twenty-eight genes were detected by WGS and grouped as flagellar or outer membrane proteins, chemotaxis, hemolysins, and invasion, plasminogen activator, colonization, transcriptional regulator, and survival in macrophages. The colistin-resistance-encoding mcr-9.1 and cephalothin-resis-encoding blaCSA genes and IncFII(pECLA) and IncFIB(pCTU3) plasmids were detected. All strains exhibited CRISPR matrices and four of them two type I-E and I-F matrices. Combined molecular methodologies improve Cronobacter spp. decision-making for health authorities to protect the population.

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

“…These values are similar to those reported by Holý et al (2019) , where 100% and 66.7% of the C. sakazakii clinical strains evaluated adhered to and invaded the N1E-115 cell line, respectively. Recently, Holý et al (2021) , when evaluating clinical strains of C. sakazakii with the N1E-115 cell line, found a value of 2.2 × 10 6 CFU/mL for adherence, which is much lower than those found in our study. However, the rates of invasion of C. sakazakii in our study are lower than those reported by various authors, such as Mange et al (2006) ; Townsend et al (2007) , Parra-Flores et al (2018a) , and Holý et al (2019) .…”

“…The cgMLST scheme analysis revealed a cluster of six C. sakazakii ST1 strains with one to three allele differences and ST83 strain with 3,043 allele difference ( Figure 1 ). C. sakazakii ST1 has been most frequently isolated from commercial PIF in various countries, from processing equipment for PIF manufacturing, and from patients with fatal meningitis, septicemia, or urinary tract infections ( Joseph and Forsythe, 2012 ; Fei et al, 2015 ; Holý et al, 2021 ; Parra-Flores et al, 2021 ). Lepuschitz et al (2017) , in their multicenter study of infections caused by C. sakazakii in Europe, studied eight isolates identified as C. sakazakii ST1 that included two isolates from newborns’ feces with an epidemiological link to an outbreak in Austria in 2009.…”

“…Key aspects for the prognosis and development of the disease caused by C. sakazakii include the presence of virulence factors which may be plasmidborne ( Shi et al, 2018 ; Aly et al, 2019 ), its adherence and invasiveness in cell lines ( Cruz et al, 2011 ; Parra-Flores et al, 2018a ; Holý et al, 2021 ), the presence of genes such as ompA , cpa , fliC , hly , sip , aut , plas , and inv ( Cruz et al, 2011 ; Franco et al, 2011 ; Aldubyan et al, 2017 ; Holý et al, 2019 ), sialic acid utilization, as well as its capsule and endotoxin production ( Ogrodzki and Forsythe, 2015 ). Another key aspect is resistance to beta-lactam antibiotics such as cephalothin, cefotaxime, ceftazidime, and ampicillin ( Flores et al, 2011 ; Lee et al, 2012 ; Fei et al, 2017 ; Holý et al, 2021 ).…”

Study of Antimicrobial Susceptibility And Biofilm Formation of Cronobacter Sakazakii Isolates From Neonatal Sepsis In Southwest Iran

“…In the current study, 14% of C. sakazakii isolates were high-level colistin-resistant, which is in good agreement with some former surveys (27,28). Microbial cells within bio lms have been exhibited to be 10-1000 times more resistant to antibiotics than the planktonic cells (29).…”

Draft Genome Sequences of Seven Cronobacter sakazakii Strains Carrying the mcr 9.1 Gene Isolated in Chile

“…The species were characterized using multilocus sequence typing (MLST) ( 13 ) extracted from the PubMLST Cronobacter database ( https://pubmlst.org/organisms/cronobacter-spp ) and core genome MLST (cgMLST) using SeqSphere+ v6.0. with default settings as previously described ( 7 , 14 ). All isolates had >97% good cgMLST targets, sequence type 1 (ST1) and ST83, and clonal complex 1 (CC1) and CC83.…”