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Identification of genes involved in serum tolerance in the clinical strain Cronobacter sakazakii ES5.

ABSTRACT: BACKGROUND: Cronobacter spp. are opportunistic pathogens that can cause septicemia and infections of the central nervous system primarily in premature, low-birth weight and/or immune-compromised neonates. Serum resistance is a crucial virulence factor for the development of systemic infections, including bacteremia. It was the aim of the current study to identify genes involved in serum tolerance in a selected Cronobacter sakazakii strain of clinical origin. RESULTS: Screening of 2749 random transposon knock out mutants of a C. sakazakii ES 5 library for modified serum tolerance (compared to wild type) revealed 10 mutants showing significantly increased/reduced resistance to serum killing. Identification of the affected sites in mutants displaying reduced serum resistance revealed genes encoding for surface and membrane proteins as well as regulatory elements or chaperones. By this approach, the involvement of the yet undescribed Wzy_C superfamily domain containing coding region in serum tolerance was observed and experimentally confirmed. Additionally, knock out mutants with enhanced serum tolerance were observed. Examination of respective transposon insertion loci revealed regulatory (repressor) elements, coding regions for chaperones and efflux systems as well as the coding region for the protein YbaJ. Real time expression analysis experiments revealed, that knock out of the gene for this protein negatively affects the expression of the fimA gene, which is a key structural component of the formation of fimbriae. Fimbriae are structures of high immunogenic potential and it is likely that absence/truncation of the ybaJ gene resulted in a non-fimbriated phenotype accounting for the enhanced survival of this mutant in human serum. CONCLUSION: By using a transposon knock out approach we were able to identify genes involved in both increased and reduced serum tolerance in Cronobacter sakazakii ES5. This study reveals first insights in the complex nature of serum tolerance of Cronobacter spp.

SUBMITTER: Schwizer S

PROVIDER: S-EPMC3621496 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Identification of genes involved in serum tolerance in the clinical strain Cronobacter sakazakii ES5.

Schwizer Sarah S Tasara Taurai T Zurfluh Katrin K Stephan Roger R Lehner Angelika A

BMC microbiology 20130215

<h4>Background</h4>Cronobacter spp. are opportunistic pathogens that can cause septicemia and infections of the central nervous system primarily in premature, low-birth weight and/or immune-compromised neonates. Serum resistance is a crucial virulence factor for the development of systemic infections, including bacteremia. It was the aim of the current study to identify genes involved in serum tolerance in a selected Cronobacter sakazakii strain of clinical origin.<h4>Results</h4>Screening of 27 ...[more]

PMID: 23414256

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Project description:Cronobacter sakazakii is a typical example of a xerotolerant bacterium. It is epidemiologically linked to low-moisture foods like powdered infant formula (PIF) and is associated with high fatality rates among neonates. We characterized the xerotolerance in a clinically isolated strain, Cronobacter sakazakii ATCC™29544T, and compared the desiccation tolerance with that of an environmental strain, C. sakazakii SP291, whose desiccation tolerance was previously characterized. We found that, although the clinical strain was desiccation-tolerant, the level of tolerance was compromised when compared with that of the environmental strain. Transcriptome sequencing (RNA-seq)-based deep transcriptomic characterization identified a unique transcriptional profile in the clinical strain compared with what was already known for the environmental strain. As RNA-seq was also carried out under different TSB growth conditions, genes that were expressed specifically under desiccated conditions were identified and denoted as desiccation responsive genes (DRGs). Interestingly, these DRGs included transcriptomic factors like fnr, ramA, and genes associated with inositol metabolism, a phenotype as yet unreported in C. sakazakii. Further, the clinical strain did not express the proP gene, which was previously reported to be very important for desiccation survival and persistence. Interestingly, analysis of the plasmid genes showed that the iron metabolism in desiccated C. sakazakii ATCC™29544T cells specifically involved the siderophore cronobactin, encoded by the iucABCD genes. Confirmatory studies using quantitative reverse transcription-PCR (qRT-PCR) determined that, though the secondary desiccation response genes were upregulated in C. sakazakii ATCC™29544T, the level of upregulation was lower than that in C. sakazakii SP291. All these factors may collectively contribute to the compromised desiccation tolerance in the clinical strain. IMPORTANCE Cronobacter sakazakii has led to outbreaks in the past, particularly associated with foods that are low in moisture content. This species has adapted to survive in low water conditions and can survive in such environments for long periods. These characteristics have enabled the pathogen to contaminate powder infant formula, a food matrix with which the pathogen has been epidemiologically associated. Even though clinically adapted strains can also be isolated, there is no information on how the clinical strains adapt to low moisture environments. Our research assessed the adaptation of a clinically isolated strain to low moisture survival on sterile stainless steel coupons and compared the survival with that of a highly desiccation-tolerant environmental strain. We found that, even though the clinical strain is desiccation-tolerant, the rate of tolerance was compromised compared with that of the environmental strain. A deeper investigation using RNA-seq identified that the clinical strain used pathways different from that of the environmental strain to adapt to low-moisture conditions. This shows that the adaptation to desiccation conditions, at least for C. sakazakii, is strain-specific and that different strains have used different evolutionary strategies for adaptation.

Project description:The genus Cronobacter (formerly called Enterobacter sakazakii) is composed of five species; C. sakazakii, C. malonaticus, C. turicensis, C. muytjensii, and C. dublinensis. The genus includes opportunistic human pathogens, and the first three species have been associated with neonatal infections. The most severe diseases are caused in neonates and include fatal necrotizing enterocolitis and meningitis. The genetic basis of the diversity within the genus is unknown, and few virulence traits have been identified. We report here the first sequence of a member of this genus, C. sakazakii strain BAA-894. The genome of Cronobacter sakazakii strain BAA-894 comprises a 4.4 Mb chromosome (57% GC content) and two plasmids; 31 Kb (51% GC) and 131 Kb (56% GC). The genome was used to construct a 385,000 probe oligonucleotide tiling DNA microarray covering the whole genome. Comparative genomic hybridization (CGH) was undertaken on five other C. sakazakii strains, and representatives of the four other Cronobacter species. Among 4,382 annotated genes inspected in this study, about 55% of genes were common to all C. sakazakii strains and 43% were common to all Cronobacter strains, with 10 - 17% absence of genes. CGH highlighted 15 clusters of genes in C. sakazakii BAA-894 that were divergent or absent in more than half of the tested strains; six of these are of probable prophage origin. Putative virulence factors were identified in these prophage and in other variable regions. A number of genes unique to Cronobacter species associated with neonatal infections (C. sakazakii, C. malonaticus and C. turicensis) were identified. These included a copper and silver resistance system known to be linked to invasion of the blood-brain barrier by neonatal meningitic strains of Escherichia coli. In addition, genes encoding for multidrug efflux pumps and adhesins were identified that were unique to C. sakazakii strains from outbreaks in neonatal intensive care units. Comparative genomic hybridization highlighted 15 clusters of genes in C. sakazakii BAA-894 that were divergent or absent in more than half of the tested strains; six of these are of probable prophage origin. Putative virulence factors were identified in these prophage and in other variable regions. A number of genes unique to Cronobacter species associated with neonatal infections (C. sakazakii, C. malonaticus and C. turicensis) were identified. These included a copper and silver resistance system known to be linked to invasion of the blood-brain barrier by neonatal meningitic strains of Escherichia coli. In addition, genes encoding for multidrug efflux pumps and adhesins were identified that were unique to C. sakazakii strains from outbreaks in neonatal intensive care units. Ten Cronobacter samples were analyzed, including total genomic DNA of six C. sakazakii strains, one C. malonaticus strain, one C. muytjensii strain, one C. dublinensis strain and one C. turicensis strain.

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Identification of genes involved in serum tolerance in the clinical strain Cronobacter sakazakii ES5.

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Identification of genes involved in serum tolerance in the clinical strain Cronobacter sakazakii ES5.

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