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 Pa?N (Phe-Arg ?-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.
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.
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.
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.
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.
A set of C. jejuni isolates of different origins and flaA-genotypes obtained throughout the broiler meat production chain was tested in this study for a possible correlation of their origin, phylogenetic relationship, and phenotypic properties. Interestingly, the results showed a correlation of the origin and the phylogenetic relationship between the C. jejuni isolates and their ability to form biofilm, but not in their ability to survive at -18, 5, 20, and 48 °C. Two strains, a broiler cloacae isolate and a broiler fillet isolate, were unable to develop biofilm, while most of the C. jejuni isolates originating from meat and surfaces of the slaughterhouse readily formed biofilms after both 24, 48, and 72 h. Interestingly, these biofilm-forming strains were closely related. Furthermore, two strains that were isolated after disinfection developed significantly more biofilms after 24 h of incubation than the remaining strains. A comparative genomic analysis using DNA microarrays showed that the gene contents of strains that efficiently formed biofilms were different from those that did not. The study suggests that biofilm formation might be a lineage specific property, allowing C. jejuni to both survive environmental stress at the slaughterhouse and to attach to the surface of meat.
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.
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