| 1. |
- Cao, Rong, et al.
(författare)
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Elevated Enterotoxin A Expression and Formation in Staphylococcus aureus and its Association with Prophage Induction.
- 2012
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Ingår i: Applied and Environmental Microbiology. - 0099-2240. ; 78:14, s. 4942-4948
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Tidskriftsartikel (refereegranskat)abstract
- Staphylococcus aureus strains producing the bacteriophage-encoded staphylococcal enterotoxin A (SEA) were divided into two groups, high- and low-SEA-producing strains, based on the amount of SEA produced. After growth under favorable conditions in batch cultures, ten of the 21 strains tested produced more than 1,000 ng/ml SEA and nine strains produced less than 10 ng/ml SEA; two enterotoxigenic strains, MRSA252 and Newman, produced intermediate levels of SEA (around 450 ng/ml). The differences in the production of SEA were found to be associated with the expression level of sea and whether they hosted the versions sea(1) or sea(2). Furthermore, differences in the nucleotide sequence in the Siphoviridae phage region showed two clonal lineages of the high-SEA-producing strains. One of these lines was correlated with the capacity for a massive increase in SEA levels by prophage induction as demonstrated using mitomycin C (MC). This was also confirmed by the occurrence of additional sea expression presumed to be initiated by a latent phage promoter located upstream of the endogenous sea promoter. Remarkably, the SEA level was increased by up to ten fold in some strains due to prophage induction. The low-SEA-producing group and the high-SEA-producing subgroup lacking phage activated sea transcription showed no increase in SEA formation after the addition of MC. This study demonstrates that sea expression in enterotoxigenic strains is correlated with the clonal lineage of sea-encoded phages. The high-SEA-producing group, and in particular the prophage inducible sea(1) group, may be more relevant in staphylococcal food poisoning than the low-SEA-producing group, mainly harboring sea(2).
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| 2. |
- Makariti, I P, et al.
(författare)
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Investigating boundaries of survival, growth and expression of genes associated with stress and virulence of Listeria monocytogenes in response to acid and osmotic stress.
- 2015
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Ingår i: Food Microbiology. - : Elsevier BV. - 1095-9998 .- 0740-0020. ; 45:Pt B, s. 231-244
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Tidskriftsartikel (refereegranskat)abstract
- The objective of this study was to correlate the relative transcription of Listeria monocytogenes (strains C5, 6179) stress- (gad2, sigB) and virulence- (prfA) associated genes following habituation at twenty-five pH (4.8, 5.0, 5.2, 5.5, 6.4) and NaCl (2, 4, 6, 8, 10% w/v) combinations at 7 °C, with the survival against subsequent exposure to severe acid stress (pH 2.0 at 37 °C). Our findings pointed out the inter-strain variation governing growth inhibiting conditions (pH ≤5.0 and NaCl ≥6%), where C5 was less affected (a reduction of 2.0-3.0 log CFU/mL) than 6179 which was reduced by 4.0-6.0 log CFU/mL at the end of storage. Nevertheless, the higher the habituation at the growth permitting (pH ≥5.5; NaCl ≤4% w/v) or growth inhibiting conditions, the higher the acquired acid resistance or sensitization, respectively. At day 2, gad2 increased relative transcriptional levels are more related to elevated acid resistance, while at day 6 both gad2 transcriptional levels and upregulation of sigB were correlated to low log reductions and high DpH:2.0-values against severe acid stress. Regarding virulence, the increased transcriptional levels of prfA at day 2 were correlated to adverse pH and NaCl combinations, while prolonged stay in suboptimal conditions as well as exposure to severe acid stress resulted in general activation of the virulence regulator. Such data could definitely contribute in designing safe intervention strategies and additionally integrate -omics aspects in quantitative microbial risk assessment.
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| 3. |
- Zeaki, Nikoleta, et al.
(författare)
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Assessment of high and low enterotoxin A producing Staphylococcus aureus strains on pork sausage.
- 2014
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Ingår i: International Journal of Food Microbiology. - : Elsevier BV. - 0168-1605. ; 182, s. 44-50
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Tidskriftsartikel (refereegranskat)abstract
- Three Staphylococcus aureus strains representing different alleles of the Siphoviridae prophage-encoded enterotoxin A (SEA) gene, including two high-SEA-producing strains and one low-SEA-producing strain were studied to investigate sea expression and SEA formation on a frankfurter type of sausage. The effect of lactic acid, an antimicrobial compound used as a preservative in food, was also investigated on the same product. All three strains were grown on pork sausages at 15°C for 14days in the presence or absence of lactic acid (1 or 2% v/v). Growth, sea mRNA expression and SEA formation were regularly monitored and compared between non-treated and treated sausages. For all experiments performed, the extracellular SEA formation significantly differed between the high- and low-SEA-producing strains, although growth and viability were overall the same. For the low producer (Sa51), the accumulated amount of extracellular SEA formed after 14days was close to the detection limit (less than 1ng/g) in all conditions; while Sa21 and Sa17, the two high-producing strains, formed 250±25.37ng/g and 750±82.65ng/g in non-treated sausage and 150±75.75ng/g and 300±83.89ng/g when treated with 1% lactic acid, respectively, after 14days. Sausages treated with 2% lactic acid followed the same pattern as above, but with an extended lag phase to 4days and reduced levels of enterotoxin formed for all strains. The difference in the level of SEA between the two high-producing strains is most likely due to the different clonal lineages of the sea-encoded Siphoviridae phages where induction of the prophage potentially could be the reason for higher production of SEA in one of the lines. Furthermore, a prolonged expression of sea gene in the two high-producing strains was observed during the entire incubation period, while the sea expression was under the detection limit in the low-producing strain. This study indicates that the high-SEA-producing strains, especially the strains with the putative capacity of prophage induction, could be more relevant in food safety aspects than low-producing type of strains on pork sausage.
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| 4. |
- Zeaki, Nikoleta, et al.
(författare)
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Evaluation of Potential Effects of NaCl and Sorbic Acid on Staphylococcal Enterotoxin A Formation
- 2015
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Ingår i: Microorganisms. - : MDPI AG. - 2076-2607. ; 3:3, s. 551-566
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Tidskriftsartikel (refereegranskat)abstract
- The prophage-encoded staphylococcal enterotoxin A (SEA) is recognized as the main cause of staphylococcal food poisoning (SFP), a common foodborne intoxication disease, caused by Staphylococcus aureus. Studies on the production of SEA suggest that activation of the SOS response and subsequent prophage induction affect the regulation of the sea gene and the SEA produced, increasing the risk for SFP. The present study aims to evaluate the effect of NaCl and sorbic acid, in concentrations relevant to food production, on SOS response activation, prophage induction and SEA production. The impact of stress was initially evaluated on steady state cells for a homogenous cell response. NaCl 2% was found to activate the SOS response, i.e., recA expression, and trigger prophage induction, in a similar way as the phage-inducer mitomycin C. In contrast, sorbic acid decreased the pH of the culture to a level where prophage induction was probably suppressed, even when combined with NaCl stress. The impact of previous physiological state of the bacteria was also addressed on cells pre-exposed to NaCl, and was found to potentially affect cell response upon exposure to further stress. The results obtained highlight the possible SFP-related risks arising from the use of preservatives during food processing.
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| 5. |
- Zeaki, Nikoleta, et al.
(författare)
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Prophage-Encoded Staphylococcal Enterotoxin A: Regulation of Production in Staphylococcus aureus Strains Representing Different Sea Regions.
- 2015
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Ingår i: Toxins. - : MDPI AG. - 2072-6651. ; 7:12, s. 5359-5376
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Tidskriftsartikel (refereegranskat)abstract
- The present study investigates the nature of the link between the staphylococcal enterotoxin A (SEA) gene and the lifecycle of Siphoviridae bacteriophages, including the origin of strain variation regarding SEA production after prophage induction. Five strains representing three different genetic lines of the sea region were studied under optimal and prophage-induced growth conditions and the Siphoviridae lifecycle was followed through the phage replicative form copies and transcripts of the lysogenic repressor, cro. The role of SOS response on prophage induction was addressed through recA transcription in a recA-disruption mutant. Prophage induction was found to increase the abundance of the phage replicative form, the sea gene copies and transcripts and enhance SEA production. Sequence analysis of the sea regions revealed that observed strain variances were related to strain capacity for prophage induction, rather than sequence differences in the sea region. The impact of SOS response activation on the phage lifecycle was demonstrated by the absence of phage replicative form copies in the recA-disruption mutant after prophage induction. From this study it emerges that all aspects of SEA-producing strain, the Siphoviridae phage and the food environment must be considered when evaluating SEA-related hazards.
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| 6. |
- Zeaki, Nikoleta
(författare)
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Stress induced virulence of Staphylococcus aureus and Listeria monocytogenes
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Control of the dissemination of pathogens in the food production chain is an on-going process with new challenges emerging as pathogenic bacteria evolve and express enhanced virulence characteristics. Staphylococcus aureus and Listeria monocytogenes are two notorious pathogens responsible for staphylococcal food poisoning (SFP) and listeriosis: a foodborne intoxication and an infectious disease respectively. In this work, S. aureus was investigated regarding the regulatory mechanism of the prophage-encoded enterotoxin A (SEA), and the impact of the food environment on the SEA produced. Moreover, L. monocytogenes’ capacity to survive and grow was evaluated under conditions of osmotic and acid stress. The expression of stress and virulence-related genes under these conditions was evaluated, and related to acquired resistance towards further stress. In the S. aureus studies, a strain-dependent SEA production was demonstrated, and linked to the different sea gene alleles (sea1, sea2). The investigated strains were grouped as high and low SEA-producers, carrying the sea1 and sea2 allele respectively. Furthermore, it was shown how the life cycle of the sea-carrying phages, and specifically prophage induction, increases the levels of SEA produced in some of the high SEA-producers by increasing both sea gene expression and the number of copies of the gene in the induced cells. A phage-activated transcript, alongside the transcript from the endogenous P1 sea promoter, characterized these strains, which were further categorized as inducible high SEA-producers. Prophage induction was found to be linked to the SOS response, through the activation of the RecA protein. A study of S. aureus on pork sausages led to the observation that SEA production and growth rates should not be considered as coupled events, and that food components may lead to prophage induction and increased SEA formation in the food product. A study of the impact of NaCl and sorbic acid on prophage induction and SEA production showed that increased NaCl activates the phage and increases the potential risk for SFP, however, the presence of sorbic acid in pH conditions critical for S. aureus growth lowers this risk. L. monocytogenes was found to be more susceptible to sequential stress applications, where acid and osmotic shifts combined with low temperature delayed the initiation of growth. Strain dependence characterized the survival and growth potential of the pathogen under adverse conditions, and adaptive responses were triggered only under mild stress habituation that allowed growth. However, stress adaptation could potentially increase virulence, even in cases where the phenotypic responses indicate a low risk of pathogen survival. In summary, the genetic complexity of bacterial phenotypic responses when exposed to stress in the food processing environment was demonstrated in this work. The obtained knowledge could be incorporated into predictive modeling for more accurate hazard evaluation and effective control of pathogenic bacteria in food.
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| 7. |
- Zeaki, Nikoleta, et al.
(författare)
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The Role of Regulatory Mechanisms and Environmental Parameters in Staphylococcal Food Poisoning and Resulting Challenges to Risk Assessment
- 2019
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Ingår i: Frontiers in Microbiology. - : Frontiers Media SA. - 1664-302X. ; 10, s. 1-13
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Forskningsöversikt (refereegranskat)abstract
- Prevention, prediction, control, and handling of bacterial foodborne diseases – an ongoing, serious, and costly concern worldwide – are continually facing a wide array of difficulties. Not the least due to that food matrices, highly variable and complex, can impact virulence expression in diverse and unpredictable ways. This review aims to present a comprehensive overview of challenges related to the presence of enterotoxigenic Staphylococcus aureus in the food production chain. It focuses on characteristics, expression, and regulation of the highly stable staphylococcal enterotoxins and in particular staphylococcal enterotoxin A (SEA). Together with the robustness of the pathogen under diverse environmental conditions and the range of possible entry routes into the food chain, this poses some of the biggest challenges in the control of SFP. Furthermore, the emergence of new enterotoxins, found to be connected with SFP, brings new questions around their regulatory mechanisms and expression in different food environments. The appearance of increasing amounts of antibiotic resistant strains found in food is also highlighted. Finally, potentials and limitations of implementing existing risk assessment models are discussed. Various quantitative microbial risk assessment approaches have attempted to quantify the growth of the bacterium and production of disease causing levels of toxin under various food chain and domestic food handling scenarios. This requires employment of predictive modeling tools, quantifying the spatiotemporal population dynamics of S. aureus in response to intrinsic and extrinsic food properties. In this context, the armory of predictive modeling employs both kinetic and probabilistic models to estimate the levels that potentiate toxin production, the time needed to reach that levels, and overall, the likelihood of toxin production. Following risk assessment, the main challenge to mitigate the risk of S. aureus intoxication is first to prevent growth of the organism and then to hamper the production of enterotoxins, or at least prevent the accumulation of high levels (e.g., >10–20 ng) in food. The necessity for continued studies indeed becomes apparent based on the challenges to understand, control, and predict enterotoxin production in relation to the food environment. Different types of food, preservatives, processing, and packaging conditions; regulatory networks; and different staphylococcal enterotoxin-producing S. aureus strains need to be further explored to obtain more complete knowledge about the virulence of this intriguing pathogen.
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