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Surveillance, survival and adaptation of Cronobacter species in low-moisture environments

2014, Yan, QIongqiong, Fanning, Séamus

Cronobacter species (formerly known as Enterobacter sakazakii) are opportunistic pathogens that consist of seven species including C. sakazakii, C. malonaticus, C. muytjensii, C. turicensis, C. dublinensis, C. universalis, and C. condimenti. This bacterium can cause meningitis, necrotizing enterocolitis, bacteraemia and sepsis, predominantly in neonates with an under developed immune system, following the consumption of contaminated powdered infant formula (PIF). Therefore, Cronobacter represents an important challenge for the PIF industry.The reference method for the detection of Cronobacter takes up to seven days for the confirmation by using conventional bacteriological culture. Thus a detection platform, which can provide a reliable result within a shorter time frame, would be desirable for the PIF industry. Such a platform, known as Vitek Immuno Diagnostic Assay System (VIDAS®), was investigated for its specificity, sensitivity and accuracy, when Cronobacter was present in TSB culture or reconstituted PIF, or in the presence of other non-related competing species. Overall, VIDAS® Cronobacter kits can effectively detect most Cronobacter species within a 20-h period. This approach may provide a useful basis upon which to improve positive release protocols for the PIF industry generally.Serotyping often plays an essential role in the identification of bacteria of importance to human health. This strategy is based on the detection of cell surface antigens, thereby facilitating the epidemiologic classification of bacteria to the sub-species level. Previously our laboratory described the first two major serotypes for Cronobacter sakazakii, denoted as O:1 and O:2. Efforts in developing new serotyping protocols continue to be reported. In this study, five unique serotypes were identified, including C. turicensis O:3, C. muytjensii O:2, C. dublinensis O:1, C. dublinensis O:2, and C. universalis O:1.Monitoring the microbial ecology of PIF production sites is an important step in an attempt to limit the risk of contamination in the finished food products. Cronobacter species, like other microorganisms can adapt to the production environment. To date sequence type 4 (ST-4) has been linked with recorded cases of meningitis, which have been isolated from PIF and its production environment. In this thesis, we reported on a 26-month surveillance study performed in an effort to identify and characterise persistent ST type(s), cultured from four PIF production facilities, using molecular strategies including target-specific polymerase chain reaction (PCR), pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST) and multi-genome microarray. Phenotypes traits including bacterial motility, biofilm formation, as well as morphotypes, were further invesigated on all these isolates. These phenotypes were considered to be among the most relevant to support bacteria in such harsh environments. Results showed that C. sakazakii serotype O:1, ST-1 was the most commonly recognised sequence type in PIF and its production environment. Significant differences were noted based on the phenotypes expressed by ST-1 and -4 isolates. ST-1 isolates cultured from PIF, formed a stronger biofilm at both 28 and 37°C when compared to ST-4 of clinical origin; while the latter exhibited a higher swim activity and an increased Congo red dye binding. This may represent a form of patho-adaptation. Understanding how this pathogen adapts to the PIF production environment will support targeted improvements in food safety measures.Cronobacter species is known for its desiccation tolerance, a phenotype that can aid bacterial survival in the production site and in PIF itself. In this study the complete genome sequence of one such isolate found to persist in a PIF production facility, denoted as C. sakazakii SP291, along with its phenotypic characteristics were reported. The genome of C. sakazakii SP291 consists of a 4.3 Mb chromosome (56.9% GC) along with three plasmids, denoted as pSP291-1 (118.1 kb, 57.2% GC), pSP291-2 (52.1 kb, 49.2% GC) and pSP291-3 (4.4 kb, 54.0% GC). When compared with C. sakazakii ATCC® BAA-894, which is also of PIF origin, the annotated genome sequence identified two interesting functional categories, comprising of genes related to the bacterial stress response along with resistance to antimicrobial and toxic compounds. Using phenotypic microarray (PM), a full metabolic profile was provided comparing C. sakazakii SP291 with C. sakazakii ATCC® BAA-894. These data extend our understanding of the genome of this important neonatal pathogen and provide further insights into the genotypes associated with features that can contribute to its persistence in the PIF environments.Little is known about the mechanisms that Cronobacter species deploy to survive and persist in low-moisture environments, including the PIF production environment. The aim of this study was to explore the gene signalling contributing to the survival and persistence phenotype in low-moisture environments by using the well-characterised persistent PIF environmental isolate, C. sakazakii SP291. The gene expression profiles related to desiccation were investigated using RNA sequencing (RNA-seq). Overall the most up- regulated genes were identified and found to be involved in the osmotic stress response. These included the ProU system (composed of proV, proX, and proW) and the bet-encoding operon (betIAB), all of which have been reported previously in other microorganisms including E. coli and Salmonella species. Interestingly four stress response genes were involved with down-regulation, in particular yehW, a gene known to play a role in the osmoprotectant uptake system of E. coli. Our observations from RNA-seq were validated using reverse transcription PCR (RT-PCR) with a selected sub-set of these gene targets.Meanwhile a transposon-mutant library was constructed in C. sakazakii SP291 separately. Pools of random insertion mutants were similarly desiccated (as for the RNA-seq experiment) following series of passages in bacterial culture. The mutant library was screened by transposon-directed insertion site sequencing (TraDIS) and compared against the original, to identify those genes that are required to support survival in low-moisture conditions. TraDIS identified 258 genes required for fundamental biological processes, 133 advantageous genes for growth under standard laboratory conditions as well as 43 advantageous genes required for growth at the defined environmental conditions. Some 32 genes have significant fold-changes after desiccation as assayed by TraDIS. Comparing the data outputs from TraDIS with RNA-seq, 11 genes were mapping by RNA-seq and defined as being up-regulated, and 2 genes being greatly down-regulated. In conclusion, this study used two high-throughput technologies to investigate the gene signalling and requirements in Cronobacter sakazakii when exposed to low-moisture conditions. Together these findings highlight some of the important biomarkers that need to be further assessed for their roles in this process.In general data from these studies, will contribute to the future development of improved detection and identification strategies for this important neonatal pathogen. Furthermore the RNA-seq and TraDIS experiments will provide early insights that can be a solid foundation for later studies, to extend our understanding of how this bacterium remains viable in a low-moisture food matrix and the environment in which it is often found. In the future these data will contribute to reduce the risk of Cronobacter contamination in PIF and its production environments, thereby improving food safety and protecting public health.