Jenny Schelin

The formation of Staphylococcus aureus enterotoxin in food environments and advances in risk assessment

The recent finding that the formation of staphylococcal enterotoxins in food is very different from that in cultures of pure Staphylococcus aureus sheds new light on, and brings into question, traditional microbial risk assessment methods based on planktonic liquid cultures. In fact, most bacteria in food appear to be associated with surfaces or tissues in various ways, and interaction with other bacteria through molecular signaling is prevalent. Nowadays it is well established that there are significant differences in the behavior of bacteria in the planktonic state and immobilized bacteria found in multicellular communities. Thus, in order to improve the production of high-quality, microbiologically safe food for human consumption, in situ data on enterotoxin formation in food environments are required to complement existing knowledge on the growth and survivability of S. aureus. This review focuses on enterotoxigenic S. aureus and describes recent findings related to enterotoxin formation in food environments, and ways in which risk assessment can take into account virulence behavior. An improved understanding of how environmental factors affect the expression of enterotoxins in foods will enable us to formulate new strategies for improved food safety.

The ATP‐dependent Clp protease is essential for acclimation to UV‐B and low temperature in the cyanobacterium Synechococcus

ClpP is the proteolytic subunit of the ATP‐dependent Clp protease in eubacteria, mammals and plant chloroplasts. Cyanobacterial ClpP protein is encoded by a multigene family, producing up to four distinct isozymes. We have examined the importance of the first ClpP protein (ClpP1) isolated from the cyanobacterium Synechococcus sp. PCC 7942 for acclimation to ecologically relevant UV‐B and low‐temperature regimens. When the growth light of 50 μmol photons m−2 s−1 was supplemented with 0.5 W m−2 UV‐B for 8 h, the constitutive level of ClpP1 rose eightfold after an initial lag of 1 h. Wild‐type cells readily acclimated to this UV‐B level, recovering after the initial stress to almost the same growth rate as that before UV‐B exposure. Growth of a clpP1 null mutant (ΔclpP1), however, was severely inhibited by UV‐B, being eight times slower than the wild type after 8 h. In comparison, ClpP1 content increased 15‐fold in wild‐type cultures shifted from 37°C to 25°C for 24 h. Wild‐type cultures readily acclimated to 25°C after 24 h, whereas the ΔclpP1 strain did not and eventually lost viability with prolonged cold treatment. During acclimation to either UV‐B or cold, photosynthesis in the wild type was initially inhibited upon the shift but then recovered. Photosynthesis in ΔclpP1 cultures, however, was more severely inhibited by the stress treatment and failed to recover. Acclimation was also monitored by examining the exchange of photosystem II reaction centre D1 proteins that occurs in wild‐type Synechococcus during conditions of excitation stress. During both cold and UV‐B shifts, wild‐type cultures replaced the acclimative form of D1 (D1:1) with the alternative D1 form 2 (D1:2) within the first hours. Once acclimated to either 25°C or 0.5 W m−2 UV‐B, D1:2 was exchanged back for D1:1. In ΔclpP1 cultures, this second exchange between D1 forms did not occur, with D1:2 remaining the predominant D1 form. Our results demonstrate that the ATP‐dependent Clp protease is an essential component of the cold and UV‐B acclimation processes of Synechococcus.

Structure and Function of a Novel Type of ATP-dependent Clp Protease

The Clp protease is conserved among eubacteria and most eukaryotes, and uses ATP to drive protein substrate unfolding and translocation into a chamber of sequestered proteolytic active sites. The main constitutive Clp protease in photosynthetic organisms has evolved into a functionally essential and structurally intricate enzyme. The model Clp protease from the cyanobacterium Synechococcus consists of the HSP100 molecular chaperone ClpC and a mixed proteolytic core comprised of two distinct subunits, ClpP3 and ClpR. We have purified the ClpP3/R complex, the first for a Clp proteolytic core comprised of heterologous subunits. The ClpP3/R complex has unique functional and structural features, consisting of twin heptameric rings each with an identical ClpP33ClpR4 configuration. As predicted by its lack of an obvious catalytic triad, the ClpR subunit is shown to be proteolytically inactive. Interestingly, extensive modification to ClpR to restore proteolytic activity to this subunit showed that its presence in the core complex is not rate-limiting for the overall proteolytic activity of the ClpCP3/R protease. Altogether, the ClpP3/R complex shows remarkable similarities to the 20 S core of the proteasome, revealing a far greater degree of convergent evolution than previously thought between the development of the Clp protease in photosynthetic organisms and that of the eukaryotic 26 S proteasome.

Inactivation of the clpP1 gene for the proteolytic subunit of the ATP-dependent Clp protease in the cyanobacterium Synechococcus limits growth and light acclimation

ClpP functions as the proteolytic subunit of the ATP-dependent Clp protease in eubacteria, mammals and plant chloroplasts. We have cloned a clpP gene, designated clpP1, from the cyanobacterium Synechococcus sp. PCC 7942. The monocistronic 591 bp gene codes for a protein 80% similar to one of four putative ClpP proteins in another cyanobacterium, Synechocystis sp. PCC 6803. The constitutive ClpP1 content in Synechococcus cultures was not inducible by high temperatures, but it did rise fivefold with increasing growth light from 50 to 175 µmol photons m-2 s-1. A clpP1 inactivation strain (ΔclpP1) exhibited slower growth rates, especially at the higher irradiances, and changes in the proportion of the photosynthetic pigments, chlorophyll a and phycocyanin. Many mutant cells (ca. 35%) were also severely elongated, up to 20 times longer than the wild type. The stress phenotype of ΔclpP1 when grown at high light was confirmed by the induction of known stress proteins, such as the heat shock protein GroEL and the alternate form of PSII reaction center D1 protein, D1 form 2. ClpP1 content also rose significantly during short-term photoinhibition, but its loss in ΔclpP1 did not exacerbate the extent of inactivation of photosynthesis, nor affect the inducible D1 exchange mechanism, indicating ClpP1 is not directly involved in D1 protein turnover.

Acetic acid increases the phage-encoded enterotoxin A expression in Staphylococcus aureus.

BackgroundThe effects of acetic acid, a common food preservative, on the bacteriophage-encoded enterotoxin A (SEA) expression and production in Staphylococcus aureus was investigated in pH-controlled batch cultures carried out at pH 7.0, 6.5, 6.0, 5.5, 5.0, and 4.5. Also, genomic analysis of S. aureus strains carrying sea was performed to map differences within the gene and in the temperate phage carrying sea.ResultsThe sea expression profile was similar from pH 7.0 to 5.5, with the relative expression peaking in the transition between exponential and stationary growth phase and falling during stationary phase. The levels of sea mRNA were below the detection limit at pH 5.0 and 4.5, confirmed by very low SEA levels at these pH values. The level of relative sea expression at pH 6.0 and 5.5 were nine and four times higher, respectively, in the transitional phase than in the exponential growth phase, compared to pH 7.0 and pH 6.5, where only a slight increase in relative expression in the transitional phase was observed. Furthermore, the increase in sea expression levels at pH 6.0 and 5.5 were observed to be linked to increased intracellular sea gene copy numbers and extracellular sea- containing phage copy numbers. The extracellular SEA levels increased over time, with highest levels produced at pH 6.0 in the four growth phases investigated. Using mitomycin C, it was verified that SEA was at least partially produced as a consequence of prophage induction of the sea-phage in the three S. aureus strains tested. Finally, genetic analysis of six S. aureus strains carrying the sea gene showed specific sea phage-groups and two versions of the sea gene that may explain the different sea expression and production levels observed in this study.ConclusionsOur findings suggest that the increased sea expression in S. aureus caused by acetic acid induced the sea-encoding prophage, linking SEA production to the lifecycle of the phage.

Culture-independent quantification of Salmonella enterica in carcass gauze swabs by flotation prior to real-time PCR

To facilitate quantitative risk assessment in the meat production chain, there is a need for culture-independent quantification methods. The aim of this study was to evaluate the use of flotation, a non-destructive sample preparation method based on traditional buoyant density centrifugation, for culture-independent quantification of intact Salmonella in pig carcass gauze swabs (100 cm(2)) prior to quantitative PCR (qPCR). A novel approach was investigated, excluding the homogenization step prior to flotation, to improve the detection limit and speed up the quantification procedure. The buoyant density of two Salmonella strains in different growth conditions was determined to be 1.065-1.092 g/ml. Based on these data, an optimal discontinuous flotation with three different density layers, ~1.200, 1.102 and 1.055 g/ml, was designed for extracting intact Salmonella cells from pig carcass swabs. The method allowed accurate quantification from 4.4 × 10(2) to at least 2.2 × 10(7)CFU Salmonella per swab sample using qPCR (without preceding DNA extraction) or selective plating on xylose lysine deoxycholate agar. Samples with 50CFU could be detected occasionally but fell outside the linear range of the standard curve. The swab samples showed a broad biological diversity; for seven samples not inoculated with Salmonella, the microbial background flora (BGF) was determined to 5.0 ± 2.2 log CFU/ml sample withdrawn after flotation. It was determined that the proceeding PCR step was inhibited by BGF concentrations of ≥ 6.1 × 10(8)CFU/swab sample, but not by concentrations ≤ 6.1 × 10(6)CFU/swab sample. By using the gauze swabs directly in the flotation procedure, the homogenization step normally used for preparation of food-related samples could be excluded, which simplified the culture-independent quantification method considerably.

Elevated Enterotoxin A Expression and Formation in Staphylococcus aureus and Its Association with Prophage Induction

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, 10 of the 21 strains tested produced more than 1,000 ng/ml SEA, and 9 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 the strains hosted the sea 1 or sea 2 version. Furthermore, differences in 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 up to 10-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-carrying phages. The high-SEA-producing group, in particular the prophage-inducible sea 1 group, may be more relevant to staphylococcal food poisoning than the low-SEA-producing group, harboring mainly sea 2.

Extended staphylococcal enterotoxin D expression in ham products.

Staphylococcal enterotoxin D (SED) is one of the most frequently recovered enterotoxins in staphylococcal food poisoning (SFP) outbreaks. The expression and production of SED were investigated in three ham products, i.e. boiled ham, smoked ham and dry-cured Serrano ham incubated at room temperature for seven days. Staphylococcus aureus was also, as a reference, grown in cultivation broth during optimal growth conditions for seven days. In boiled and smoked ham, continuous sed expression was observed throughout the incubation period with a second increase in sed expression found after five days of incubation. In smoked ham, nine times less SED per colony-forming unit of S. aureus was detected than in boiled ham. In boiled ham, the SED levels unpredictably decreased after three days of incubation. In the Serrano ham, SED was detected after five days of incubation although S. aureus growth was poor and sed expression was too low to determine. After five days of incubation, all three products contained enough SED to cause SFP. These results show that the specific production levels of SED vary in the different ham products, and that toxin production was in part uncoupled from bacterial growth.

Novel Form of ClpB/HSP100 Protein in the Cyanobacterium Synechococcus

Synechococcus sp. strain PCC 7942 has a second clpB gene that encodes a 97-kDa protein with novel features. ClpBII is the first ClpB not induced by heat shock or other stresses; it is instead an essential, constitutive protein. ClpBII is unable to complement ClpBI function for acquired thermotolerance. No truncated ClpBII version is normally produced, unlike other bacterial forms, while ectopic synthesis of a putative truncated ClpBII dramatically decreased cell viability.

Effects of Carbon Dioxide on Growth of Proteolytic Clostridium botulinum, Its Ability To Produce Neurotoxin, and Its Transcriptome

ABSTRACT The antimicrobial gas carbon dioxide is frequently used in modified atmosphere packaging. In the present study, the effects of CO2 (10 to 70%, vol/vol) on gene expression (measured using quantitative reverse transcription-PCR and a whole-genome DNA microarray) and neurotoxin formation (measured using an enzyme-linked immunosorbent assay [ELISA]) by proteolytic Clostridium botulinum type A1 strain ATCC 3502 were studied during the growth cycle. Interestingly, in marked contrast to the situation with nonproteolytic C. botulinum types B and E, CO2 had little effect on any of these parameters. At all CO2 concentrations, relative expression of neurotoxin cluster genes peaked in the transition between exponential and stationary phases, with evidence of a second rise in expression in late stationary phase. Microarray analysis enabled identification of coding sequences whose expression profiles matched those of the neurotoxin cluster. Further research is needed to determine whether these are connected to neurotoxin formation or are merely growth phase associated.