Showing papers by "Thomas J. Montville published in 2006"

Bioenergetic Mechanism for Nisin Resistance, Induced by the Acid Tolerance Response of Listeria monocytogenes

Abstract: Listeria monocytogenes causes ∼2,500 cases/year of listeriosis out of an estimated 76 million cases/year of food-borne disease in the United States (23). The pathogen targets mainly newborn, pregnant, elderly, and immunocompromised individuals, and it is associated with mortality rates of up to 37%. Control of L. monocytogenes is difficult, due to its widespread presence in nature, intrinsic physiologic resistance, adaptation capacity, and ability to grow at low temperatures (37). The responses of L. monocytogenes to acid, osmotic, and thermal stresses increase its resistance and virulence (16, 28). The acid tolerance response (ATR) is the abnormal resistance to lethal acid after an exposure to mild acidic conditions (21). The regulation of stress response proteins changes during induction of the ATR (29, 31). These proteins include chaperones, transcriptional regulators (13), the glutamic acid decarboxylase system, and the FoF1 ATPase enzyme complex (10, 31). The ATR also increases virulence and cross-protects listeriae from other stressors, such as elevated temperatures (16) and antimicrobials (28). The specific acids affect the pH range at which the ATR is induced and the range within which the pH becomes lethal; lactic acid is a stronger inducer than HCl (2). The ATR also confers resistance to the bacteriocin nisin, an antimicrobial peptide that is approved for food use in >40 countries (6). ATR-induced L. monocytogenes cells (ATR+) but not the control cells (ATR−) survive for at least 30 days at 4°C in a model fermented system where Lactococcus lactis produced lactic acid (pH 5.7) and nisin (50 μg/ml) (2). The mechanism by which the ATR protects L. monocytogenes against nisin is uncertain. Analysis of membrane lipids of constitutively nisin-resistant listeriae shows that their membrane is more rigid, due to changes in the proportion of fatty acids (11, 24, 25). Similar temperature-induced changes in membrane composition cause measurable changes in membrane fluidity as demonstrated by fluorescence anisotropy (22). However, these changes in membrane lipid composition do not fully explain the increased nisin resistance of ATR+ listeriae (38). Cell membranes have low permeability to protons, which are subjected to specific transport mechanisms such as FoF1 ATPase, Na+/H+ antiporters, and electron transport systems (31). This enables living cells to build a potential across their membranes, which is essential for energy transduction (41). The peptide nisin targets energized cell membranes, and its insertion is activated by the difference in free available energy across the membrane (12). Nisin molecules insert cooperatively into the cell membrane, which is disrupted by transient pore formation (4). Destruction of the membrane integrity collapses the proton motive force (PMF), causing cell death. The PMF-dependent action of nisin suggested a bioenergetic contribution to nisin resistance in ATR+ listeriae. We hypothesized that decreased PMF contributes to the increased nisin resistance of ATR+ L. monocytogenes. In L. monocytogenes, the PMF is generated primarily by the membrane-associated FoF1 ATPase, which builds a PMF using energy derived from ATP hydrolysis (8). We present surprising evidence that ATR+ listeriae have significantly higher intracellular ATP (ATPi) concentrations than ATR− cells. This vital ATP sparing in ATR+ L. monocytogenes is correlated to the downregulation of the FoF1 ATPase c subunit.

Evidence for quorum sensing in Clostridium botulinum 56A

Abstract: Aims: Experiments were designed to detect quorum-sensing signals produced by Clostridium botulinum. Methods and Results: Clostridium botulinum 56A cell-free supernatants obtained at the end of lag phase, the mid-exponential phase and early stationary phase of growth were assayed for bioluminescence in the Vibrio harveyi quorum-sensing assay system. Twelve and 16-h culture supernatants induced bioluminescence in the auto-inducer 2 (AI-2) but not the auto-inducer 1 (AI-1) assay. Intra-species quorum sensing was also assayed as the ability of the supernatants to promote spore germination and outgrowth in a microtitre plate system. Spore populations exposed to C. botulinum supernatant from the end of lag phase became positive for growth sooner than controls. Conclusions: The influence of cell-free supernatant on ungerminated spores and detection of bioluminescence in the AI-2 assay are evidence for a signalling molecule(s) and provide a first step in characterizing C. botulinum quorum sensing. Significance and Impact of the Study: This study suggests that spores do not behave independently of each other and may explain the inocula size effects observed in challenge studies. Whether AI-2 production in C. botulinum serves as an inter-species signal or as a detoxification mechanism remains to be determined.

Research Note Growth Characteristics of Virulent Bacillus anthracis and Potential Surrogate Strains

Abstract: The objectives of this study were to compare generation and lag times of virulent Bacillus anthracis strains with those of other Bacillus strains, to identify possible surrogates for growth studies, and to determine if the B. cereus module of the U.S. Department of Agriculture Pathogen Modeling Program (PMP) had predictive value for B. anthracis. Growth characteristics of B. anthracis, B. cereus, B. mycoides, and B. subtilis strains in brain heart infusion broth at pH 6.5, 6.0, and 5.5 were determined by absorbance measurements. Growth curves of B. anthracis Sterne and B. cereus strains appeared similar, and the generation times for strain Sterne fell within the PMP’s 95% confidence interval for B. cereus. However, the virulent B. anthracis strains Vollum and Pasteur had shorter generation times than the avirulent Sterne strain and most other surrogates and were lower than the PMP’s 95% confidence interval for B. cereus. Growth curves of B. cereus ATCC 9818 and B. subtilis ATCC 6633 were more similar to those of virulent B. anthracis strains, but all potential surrogates had significantly different generation times and lag times under some conditions.