Abstract
BackgroundMalonate utilization, an important differential trait, well recognized as being possessed by six of the seven Cronobacter species is thought to be largely absent in Cronobacter sakazakii (Csak). The current study provides experimental evidence that confirms the presence of a malonate utilization operon in 24 strains of sequence type (ST) 64, obtained from Europe, Middle East, China, and USA; it offers explanations regarding the genomic diversity and phylogenetic relatedness among these strains, and that of other C. sakazakii strains.ResultsIn this study, the presence of a malonate utilization operon in these strains was initially identified by DNA microarray analysis (MA) out of a pool of 347 strains obtained from various surveillance studies involving clinical, spices, milk powder sources and powdered infant formula production facilities in Ireland and Germany, and dried dairy powder manufacturing facilities in the USA. All ST64 C. sakazakii strains tested could utilize malonate. Zebrafish embryo infection studies showed that C. sakazakii ST64 strains are as virulent as other Cronobacter species. Parallel whole genome sequencing (WGS) and MA showed that the strains phylogenetically grouped as a separate clade among the Csak species cluster. Additionally, these strains possessed the Csak O:2 serotype. The nine-gene, ~ 7.7 kbp malonate utilization operon was located in these strains between two conserved flanking genes, gyrB and katG. Plasmidotyping results showed that these strains possessed the virulence plasmid pESA3, but in contrast to the USA ST64 Csak strains, ST64 Csak strains isolated from sources in Europe and the Middle East, did not possess the type six secretion system effector vgrG gene.ConclusionsUntil this investigation, the presence of malonate-positive Csak strains, which are associated with foods and clinical cases, was under appreciated. If this trait was used solely to identify Cronobacter strains, many strains would likely be misidentified. Parallel WGS and MA were useful in characterizing the total genome content of these Csak O:2, ST64, malonate-positive strains and further provides an understanding of their phylogenetic relatedness among other virulent C. sakazakii strains.
Highlights
Malonate utilization, an important differential trait, well recognized as being possessed by six of the seven Cronobacter species is thought to be largely absent in Cronobacter sakazakii (Csak)
Identities and general features of malonate‐positive, ST64 C. sakazakii strains Twenty-three C. sakazakii strains were obtained from surveillance studies of dried milk powder, clinical, spice, and environmental samples taken from retail facilities, cheese and milk protein and powdered infant formula manufacturing facilities located in the United States of America (USA), Middle East, and Europe by various investigators [13, 15, 19,20,21,22, 35]
All strains were identified as C. sakazakii using the species-specific rpoβ polymerase chain reaction (PCR) assays as described by Stoop et al [23] and Lehner et al [24], and the diguanylate cyclase-encoding gene (cgcA) species-specific PCR assay as described by Carter et al [20]
Summary
An important differential trait, well recognized as being possessed by six of the seven Cronobacter species is thought to be largely absent in Cronobacter sakazakii (Csak). Cronobacter species are Gram-negative bacteria which can cause severe infantile septicemia, meningitis, and necrotizing enterocolitis and pose a serious threat to neonates and underweight infants [1, 2]. Because PIF is not manufactured as a sterile product, it poses a significant consumer risk should contaminated lots be prepared and handled inappropriately. This led to the publishing of guidelines for proper PIF preparation (http://www.who.int/foodsafety/publications/micro/ PIF_Bottle_en.pdf ) by the World Health Organization. Jason [10] reported that 8% (7/82) of infected infants studied during 2004–2010 presented with invasive disease (defined as a culture-positive, confirmed case of septicemia or meningitis) and consumed breast milk without any PIF or human milk fortifier supplementation prior to onset of illness. It is important that the food manufacturing and public health communities continue surveillance efforts to find the presence of these organisms in food products and within food processing environments
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