Marianne Thorup Cohn

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.

Planktonic aggregates of Staphylococcus aureus protect against common antibiotics.

Bacterial cells are mostly studied during planktonic growth although in their natural habitats they are often found in communities such as biofilms with dramatically different physiological properties. We have examined another type of community namely cellular aggregates observed in strains of the human pathogen Staphylococcus aureus. By laser-diffraction particle–size analysis (LDA) we show, for strains forming visible aggregates, that the aggregation starts already in the early exponential growth phase and proceeds until post-exponential phase where more than 90% of the population is part of the aggregate community. Similar to some types of biofilm, the structural component of S. aureus aggregates is the polysaccharide intercellular adhesin (PIA). Importantly, PIA production correlates with the level of aggregation whether altered through mutations or exposure to sub-inhibitory concentrations of selected antibiotics. While some properties of aggregates resemble those of biofilms including increased mutation frequency and survival during antibiotic treatment, aggregated cells displayed higher metabolic activity than planktonic cells or cells in biofilm. Thus, our data indicate that the properties of cells in aggregates differ in some aspects from those in biofilms. It is generally accepted that the biofilm life style protects pathogens against antibiotics and the hostile environment of the host. We speculate that in aggregate communities S. aureus increases its tolerance to hazardous environments and that the combination of a biofilm-like environment with mobility has substantial practical and clinical importance.

Contribution of Conserved ATP-Dependent Proteases of Campylobacter jejuni to Stress Tolerance and Virulence

ABSTRACT In prokaryotic cells the ATP-dependent proteases Lon and ClpP (Clp proteolytic subunit) are involved in the turnover of misfolded proteins and the degradation of regulatory proteins, and depending on the organism, these proteases contribute variably to stress tolerance. We constructed mutants in the lon and clpP genes of the food-borne human pathogen Campylobacter jejuni and found that the growth of both mutants was impaired at high temperature, a condition known to increase the level of misfolded protein. Moreover, the amounts of misfolded protein aggregates were increased when both proteases were absent, and we propose that both ClpP and Lon are involved in eliminating misfolded proteins in C. jejuni. In order to bind misfolded protein, ClpP has to associate with one of several Clp ATPases. Following inactivation of the ATPase genes clpA and clpX, only the clpX mutant displayed the same heat sensitivity as the clpP mutant, indicating that the ClpXP proteolytic complex is responsible for the degradation of heat-damaged proteins in C. jejuni. Notably, ClpP and ClpX are required for growth at 42°C, which is the temperature of the intestinal tract of poultry, one of the primary carriers of C. jejuni. Thus, ClpP and ClpX may be suitable targets of new intervention strategies aimed at reducing C. jejuni in poultry production. Further characterization of the clpP and lon mutants revealed other altered phenotypes, such as reduced motility, less autoagglutination, and lower levels of invasion of INT407 epithelial cells, suggesting that the proteases may contribute to the virulence of C. jejuni.

The Chaperone ClpX Stimulates Expression of Staphylococcus aureus Protein A by Rot Dependent and Independent Pathways

The Clp ATPases (Hsp100) constitute a family of closely related proteins that have protein reactivating and remodelling activities typical of molecular chaperones. In Staphylococcus aureus the ClpX chaperone is essential for virulence and for transcription of spa encoding Protein A. The present study was undertaken to elucidate the mechanism by which ClpX stimulates expression of Protein A. For this purpose, we prepared antibodies directed against Rot, an activator of spa transcription, and demonstrated that cells devoid of ClpX contain three-fold less Rot than wild-type cells. By varying Rot expression from an inducible promoter we showed that expression of Protein A requires a threshold level of Rot. In the absence of ClpX the Rot content is reduced below this threshold level, hence, explaining the substantially reduced Protein A expression in the clpX mutant. Experiments addressed at pinpointing the role of ClpX in Rot synthesis revealed that ClpX is required for translation of Rot. Interestingly, translation of the spa mRNA was, like the rot mRNA, enhanced by ClpX. These data demonstrate that ClpX performs dual roles in regulating Protein A expression, as ClpX stimulates transcription of spa by enhancing translation of Rot, and that ClpX additionally is required for full translation of the spa mRNA. The current findings emphasize that ClpX has a central role in fine-tuning virulence regulation in S. aureus.

Effect of chicken meat environment on gene expression of Campylobacter jejuni and its relevance to survival in food

Poultry meat is the major food source responsible for gastrointestinal infections caused by the human pathogen Campylobacter jejuni. Even though C. jejuni does not grow below 30 °C, the bacterium survives on raw meat surfaces at refrigerated temperatures and thus poses a risk to the consumer. Previously, we have shown that chicken meat juice prolongs survival of C. jejuni at 5 °C compared to laboratory medium, suggesting that compounds present in meat juice influence adaptation to low temperatures. In the present study we have used chicken meat juice to identify C. jejuni genes that are differentially expressed in a typical chicken meat environment encountered by consumers. The analysis showed that chicken meat juice increased expression of luxS involved in quorum sensing, as well as a gene involved in O-linked flagellin glycosylation in C. jejuni, while expression of haemin uptake and the peroxide stress response genes were reduced. Furthermore, we propose that LuxS may play a key role in adaptation to the chicken meat juice environment, as lack of the luxS gene reduces the ability of C. jejuni to survive in chicken meat juice at low temperature. Finally, our data suggest that part of an ABC transport system is induced and we speculate that uptake of cryoprotectants may be important for C. jejuni to adapt to low temperature. In summary, we found that C. jejuni has a specific but limited transcriptional response to chicken meat juice and that luxS has an impact on the prolonged survival of C. jejuni in this important environment in the food chain.

Method Enabling Gene Expression Studies of Pathogens in a Complex Food Matrix

ABSTRACT We describe a simple method for stabilizing and extracting high-quality prokaryotic RNA from meat. Heat and salt stress of Escherichia coli and Salmonella spp. in minced meat reproducibly induced dnaK and otsB expression, respectively, as observed by quantitative reverse transcription-PCR (>5-fold relative changes). Thus, the method is applicable in studies of bacterial gene expression in a meat matrix.

Clp-dependent proteolysis of the LexA N-terminal domain in Staphylococcus aureus

The SOS response is governed by the transcriptional regulator LexA and is elicited in many bacterial species in response to DNA damaging conditions. Induction of the SOS response is mediated by autocleavage of the LexA repressor resulting in a C-terminal dimerization domain (CTD) and an N-terminal DNA-binding domain (NTD) known to retain some DNA-binding activity. The proteases responsible for degrading the LexA domains have been identified in Escherichia coli as ClpXP and Lon. Here, we show that in the human and animal pathogen Staphylococcus aureus, the ClpXP and ClpCP proteases contribute to degradation of the NTD and to a lesser degree the CTD. In the absence of the proteolytic subunit, ClpP, or one or both of the Clp ATPases, ClpX and ClpC, the LexA domains were stabilized after autocleavage. Production of a stabilized variant of the NTD interfered with mitomycin-mediated induction of sosA expression while leaving lexA unaffected, and also significantly reduced SOS-induced mutagenesis. Our results show that sequential proteolysis of LexA is conserved in S. aureus and that the NTD may differentially regulate a subset of genes in the SOS regulon.

Residual Antibiotics Disrupt Meat Fermentation and Increase Risk of Infection

ABSTRACT Fermented sausages, although presumed safe for consumption, sometimes cause serious bacterial infections in humans that may be deadly. Not much is known about why and when this is the case. We tested the hypothesis that residual veterinary antibiotics in meat can disrupt the fermentation process, giving pathogenic bacteria a chance to survive and multiply. We found that six commercially available starter cultures were susceptible to commonly used antibiotics, namely, oxytetracycline, penicillin, and erythromycin. In meat, statutorily tolerable levels of oxytetracycline and erythromycin inhibited fermentation performance of three and five of the six starter cultures, respectively. In model sausages, the disruption of meat fermentation enhanced survival of the pathogens Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium compared to successful fermentations. Our work reveals an overlooked risk associated with the presence of veterinary drugs in meat. IMPORTANCE Antibiotics have for a long time been used as growth promoters in farm animals, and while they are banned as such in Europe, their clinical use in farm animals still accounts for the majority of consumption. Here, we examined how acceptable levels of antibiotics in meat influence fermentation. Our results show that commonly used bacterial starter cultures are sensitive to residual antibiotics at or near statutorily tolerable levels, and as a result, processed sausages may indeed contain high levels of pathogens. Our findings provide a possible explanation for outbreaks and disease cases associated with consumption of fermented sausages and offer yet another argument for limiting the use of antimicrobials in farm animals. Antibiotics have for a long time been used as growth promoters in farm animals, and while they are banned as such in Europe, their clinical use in farm animals still accounts for the majority of consumption. Here, we examined how acceptable levels of antibiotics in meat influence fermentation. Our results show that commonly used bacterial starter cultures are sensitive to residual antibiotics at or near statutorily tolerable levels, and as a result, processed sausages may indeed contain high levels of pathogens. Our findings provide a possible explanation for outbreaks and disease cases associated with consumption of fermented sausages and offer yet another argument for limiting the use of antimicrobials in farm animals.

Cellular Response of Campylobacter jejuni to Trisodium Phosphate

ABSTRACT The highly alkaline compound trisodium phosphate (TSP) is used as an intervention to reduce the load of Campylobacter on poultry meat in U.S. poultry slaughter plants. The aim of the present study was to investigate the cellular responses of Campylobacter jejuni NCTC11168 when exposed to sublethal concentrations of TSP. Preexposure of C. jejuni to TSP resulted in a significant increase in heat sensitivity, suggesting that a combined heat and TSP treatment may increase reduction of C. jejuni. A microarray analysis identified a limited number of genes that were differently expressed after sublethal TSP exposure; however, the response was mainly associated with ion transport processes. C. jejuni NCTC11168 nhaA1 (Cj1655c) and nhaA2 (Cj1654c), which encode orthologues to the Escherichia coli NhaA cation/proton antiporter, were able to partially restore TSP, alkaline, and sodium resistance phenotypes to an E. coli cation/proton antiporter mutant. In addition, inhibition of resistance-nodulation-cell division (RND) multidrug efflux pumps by the inhibitor PaβN (Phe-Arg β-naphthylamide dihydrochloride) decreased tolerance to sublethal TSP. Therefore, we propose that NhaA1/NhaA2 cation/proton antiporters and RND multidrug efflux pumps function in tolerance to sublethal TSP exposure in C. jejuni.

Simple method for correct enumeration of Staphylococcus aureus

Optical density (OD) measurement is applied universally to estimate cell numbers of microorganisms growing in liquid cultures. It is a fast and reliable method but is based on the assumption that the bacteria grow as single cells of equal size and that the cells are dispersed evenly in the liquid culture. When grown in such liquid cultures, the human pathogen Staphylococcus aureus is characterized by its aggregation of single cells into clusters of variable size. Here, we show that aggregation during growth in the laboratory standard medium tryptic soy broth (TSB) is common among clinical and laboratory S. aureus isolates and that aggregation may introduce significant bias when applying standard enumeration methods on S. aureus growing in laboratory batch cultures. We provide a simple and efficient sonication procedure, which can be applied prior to optical density measurements to give an accurate estimate of cellular numbers in liquid cultures of S. aureus regardless of the aggregation level of the given strain. We further show that the sonication procedure is applicable for accurate determination of cell numbers using agar plate counting of aggregating strains.