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Stress tolerant virulent strains of Cronobacter sakazakii from food.

ABSTRACT: Cronobacter sakazakii is considered as an emerging foodborne pathogen. The aim of this study was to isolate and characterize virulent strains of Cronobacter sakazakii from food samples of Bangladesh.Six (6) Cronobacter sakazakii was isolated and identified from 54 food samples on the basis of biochemical characteristics, sugar fermentation, SDS-PAGE of whole cell protein, plasmid profile and PCR of Cronobacter spp. specific genes (esak, gluA, zpx, ompA, ERIC, BOX-AIR) and sequencing. These strains were found to have moderately high antibiotic resistance against common antibiotics and some are ESBL producer. Most of the C. sakazakii isolates were capable of producing biofilm (strong biofilm producer), extracellular protease and siderophores, curli expression, haemolysin, haemagglutinin, mannose resistant haemagglutinin, had high cell surface hydrophobicity, significant resistance to human serum, can tolerate high concentration of salt, bile and DNase production. Most of them produced enterotoxins of different molecular weight. The isolates pose significant serological cross-reactivity with other gram negative pathogens such as serotypes of Salmonella spp., Shigella boydii, Shigella sonnei, Shigella flexneri and Vibrio cholerae. They had significant tolerance to high temperature, low pH, dryness and osmotic stress.Special attention should be given in ensuring hygiene in production and post-processing to prevent contamination of food with such stress-tolerant virulent Cronobacter sakazakii.

SUBMITTER: Fakruddin M

PROVIDER: S-EPMC4335510 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Stress tolerant virulent strains of Cronobacter sakazakii from food.

Fakruddin Md M Rahaman Mizanur M Ahmed Monzur Morshed MM Hoque Md Mahfuzul MM

Biological research 20141125

<h4>Background</h4>Cronobacter sakazakii is considered as an emerging foodborne pathogen. The aim of this study was to isolate and characterize virulent strains of Cronobacter sakazakii from food samples of Bangladesh.<h4>Result</h4>Six (6) Cronobacter sakazakii was isolated and identified from 54 food samples on the basis of biochemical characteristics, sugar fermentation, SDS-PAGE of whole cell protein, plasmid profile and PCR of Cronobacter spp. specific genes (esak, gluA, zpx, ompA, ERIC, BO ...[more]

PMID: 25723712

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Project description:Cronobacter (C.) sakazakii is an opportunistic pathogen and has been associated with serious infections with high mortality rates predominantly in pre-term, low-birth weight and/or immune compromised neonates and infants. Infections have been epidemiologically linked to consumption of intrinsically and extrinsically contaminated lots of reconstituted powdered infant formula (PIF), thus contamination of such products is a challenging task for the PIF producing industry. We present the draft genome of C. sakazakii H322, a highly persistent sequence type (ST) 83, clonal complex (CC) 65, serotype O:7 strain obtained from a batch of non-released contaminated PIF product. The presence of this strain in the production environment was traced back more than 4 years. Whole genome sequencing (WGS) of this strain together with four more ST83 strains (PIF production environment-associated) confirmed a high degree of sequence homology among four of the five strains. Phylogenetic analysis using microarray (MA) and WGS data showed that the ST83 strains were highly phylogenetically related and MA showed that between 5 and 38 genes differed from one another in these strains. All strains possessed the pESA3-like virulence plasmid and one strain possessed a pESA2-like plasmid. In addition, a pCS1-like plasmid was also found. In order to assess the potential in vivo pathogenicity of the ST83 strains, each strain was subjected to infection studies using the recently developed zebrafish embryo model. Our results showed a high (90-100%) zebrafish mortality rate for all of these strains, suggesting a high risk for infections and illness in neonates potentially exposed to PIF contaminated with ST83 C. sakazakii strains. In summary, virulent ST83, CC65, serotype CsakO:7 strains, though rarely found intrinsically in PIF, can persist within a PIF manufacturing facility for years and potentially pose significant quality assurance challenges to the PIF manufacturing industry.

Project description:Cronobacter sakazakii is an emerging foodborne pathogen, which is linked to life-threatening infections causing septicemia, meningitis, and necrotizing enterocolitis. These infections have been epidemiologically connected to ingestion of contaminated reconstituted powder infant formula. Even at low water activity C. sakazakii can survive for a long time; it is capable of protective biofilm formation and occasionally shows high virulence and pathogenicity even following stressful environmental conditions. Hence it is a challenging task for the food industry to control contamination of food ingredients and products through the entire production chain, since an increasing number of severe food-related outbreaks of C. sakazakii infections has been observed. The seemingly great capability of C. sakazakii to survive even strict countermeasures combined with its prevalence in many food ingredients requires a greater in depth understanding of its virulence factors to master the food safety issues related to this organism. In this context, we present the whole genome sequence (WGS) of two different C. sakazakii isolated from skimmed milk powder (C7) and ready-to-eat salad mix (C8), respectively. These are compared to other, already sequenced, C. sakazakii genomes. Sequencing of the fusA allele revealed that both isolates were C. sakazakii. We investigated the molecular characteristics of both isolates relevant for genes associated with pathogenesis and virulence factors, resistance to stressful environmental conditions (e.g., osmotic and heat), survival in desiccation as well as conducted a comparative genomic analysis. By using multi-locus sequence typing (MLST), the genetic type of both isolates is assessed and the number of unique genes is determined. DNA of C. sakazakii C8 is shown to hold a novel and unique sequence type; the number of unique genes identified in the genomic sequence of C. sakazakii C7 and C8 were 109 and 188, respectively. Some of the determined unique genes such as the rhs and VgrG genes are linked to the Type VI Secretion System cluster, which is associated with pathogenicity and virulence factors. Moreover, seven genes encoding for multi-drug resistance were found in both isolates. The finding of a number of genes linked to producing capsules and biofilm are likely related to the observed resistance to desiccation.

Project description:Background: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). 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. Results:In 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. Conclusions:Until 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.

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Stress tolerant virulent strains of Cronobacter sakazakii from food.

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Stress tolerant virulent strains of Cronobacter sakazakii from food.

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