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Essential oils: their antibacterial properties and potential applications in foods--a review.

Antibiotics have always been considered one of the wonder discoveries of the 20th century. This is true, but the real wonder is the rise of antibiotic resistance in hospitals, communities, and the environment concomitant with their use. The extraordinary genetic capacities of microbes have benefitted from man's overuse of antibiotics to exploit every source of resistance genes and every means of horizontal gene transmission to develop multiple mechanisms of resistance for each and every antibiotic introduced into practice clinically, agriculturally, or otherwise. This review presents the salient aspects of antibiotic resistance development over the past half-century, with the oft-restated conclusion that it is time to act. To achieve complete restitution of therapeutic applications of antibiotics, there is a need for more information on the role of environmental microbiomes in the rise of antibiotic resistance. In particular, creative approaches to the discovery of novel antibiotics and their expedited and controlled introduction to therapy are obligatory.

Origins and Evolution of Antibiotic Resistance

Bacteriocins of gram-positive bacteria.

There is an urgent need in aquaculture to develop microbial control strategies, since disease outbreaks are recognized as important constraints to aquaculture production and trade and since the development of antibiotic resistance has become a matter of growing concern. One of the alternatives to antimicrobials in disease control could be the use of probiotic bacteria as microbial control agents. This review describes the state of the art of probiotic research in the culture of fish, crustaceans, mollusks, and live food, with an evaluation of the results obtained so far. A new definition of probiotics, also applicable to aquatic environments, is proposed, and a detailed description is given of their possible modes of action, i.e., production of compounds that are inhibitory toward pathogens, competition with harmful microorganisms for nutrients and energy, competition with deleterious species for adhesion sites, enhancement of the immune response of the animal, improvement of water quality, and interaction with phytoplankton. A rationale is proposed for the multistep and multidisciplinary process required for the development of effective and safe probiotics for commercial application in aquaculture. Finally, directions for further research are discussed.

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Probiotic Bacteria as Biological Control Agents in Aquaculture

Bacteriocins are bacterially produced antimicrobial peptides with narrow or broad host ranges. Many bacteriocins are produced by food-grade lactic acid bacteria, a phenomenon which offers food scientists the possibility of directing or preventing the development of specific bacterial species in food. This can be particularly useful in preservation or food safety applications, but also has implications for the development of desirable flora in fermented food. In this sense, bacteriocins can be used to confer a rudimentary form of innate immunity to foodstuffs, helping processors extend their control over the food flora long after manufacture.

Bacteriocins: developing innate immunity for food

Clostridium difficile is an important, emerging nosocomial pathogen. The transition from harmless colonization to disease is typically preceded by antimicrobial therapy, which alters the balance of the intestinal flora, enabling C. difficile to proliferate in the colon. One of the most perplexing aspects of the C. difficile infectious cycle is its ability to survive antimicrobial therapy and transition from inert colonization to active infection. Toxin-antitoxin (TA) systems have been implicated in facilitating persistence after antibiotic treatment. We identified only one TA system in C. difficile strain 630 (epidemic type X), designated MazE-cd and MazF-cd, a counterpart of the well-characterized Escherichia coli MazEF TA system. This E. coli MazF toxin cleaves mRNA at ACA sequences, leading to global mRNA degradation, growth arrest, and death. Likewise, MazF-cd expression in E. coli or Clostridium perfringens resulted in growth arrest. Primer extension analysis revealed that MazF-cd cleaved RNA at the five-base consensus sequence UACAU, suggesting that the mRNAs susceptible to cleavage comprise a subset of total mRNAs. In agreement, we observed differential cleavage of several mRNAs by MazF-cd in vivo, revealing a direct correlation between the number of cleavage recognition sites within a given transcript and its susceptibility to degradation by MazF-cd. Interestingly, upon detailed statistical analyses of the C. difficile transcriptome, the major C. difficile virulence factor toxin B (TcdB) and CwpV, a cell wall protein involved in aggregation, were predicted to be significantly resistant to MazF-cd cleavage.

Clostridium difficile MazF Toxin Exhibits Selective, Not Global, mRNA Cleavage

Bavaricin MN, a bacteriocin produced by Lactobacillus bavaricus MN, reached titres of 2000 AU ml-1 in APT broth maintained at pH 6.0, 30°C in a batch fermenter. Levels of bavaricin MN at pH 5.5 and 6.5 were lower despite comparable levels of producer cells. The addition of 3.0 g l-1 beef extract to APT broth resulted in increases in both the growth rate of the culture and the production of bavaracin MN. The titre of bavaricin MN in batch fermenters controlled at pH 6.0 in APT broth plus 3 g l-1 beef extract reached 3200 AU ml-1 at 30°C. This level was reduced to 800 AU ml-1 by 76 h. Glucose-limited continuous culture of Lact. bavaricus MN under the same conditions resulted in an increase in the titre of bavaricin MN to 6400 AU ml-1. This level was maintained, independent of growth rate, for 345 h. Growth rates of 0.205, 0.118, 0.169 and 0.058 h-1 were examined.

The influence of pH and growth rate on production of the bacteriocin, bavaricin MN, in batch and continuous fermentations

Nisin is a bacteriocin produced by many strains of Lactococcus lactis. This study examined the effect of nisin on Mycobacterium smegmatis, a non-pathogenic species of Mycobacterium. Nisin had a minimum inhibitory concentration of 8.0 micrograms ml-1 and a minimum inhibitory dose of 7.5 micrograms ml-1 against Myco. smegmatis. Treatment with 25.0 micrograms ml-1 nisin caused partial inhibition of Myco smegmatis; the survivors were nisin-sensitive when tested in a separate experiment. Mycobacterium smegmatis cells exposed to 50.0 micrograms ml-1 of nisin, lost their viability. the effect of nisin on the growth of Myco. smegmatis was both time- and concentration-dependent. Nisin (10.0 micrograms ml-1) caused 97.7 +/- 2.0% reduction in internal ATP and leakage of intracellular ATP out of Myco. smegmatis cells after several hours of treatment. These data suggest that nisin inhibits Myco. smegmatis by the same mechanism by which it inhibits other bacteria and warrants further investigation as a possible antitubercular agent.

Nisin A depletes intracellular ATP and acts in bactericidal manner against Mycobacterium smegmatis.

Influence of pH, Salt, and Temperature on Nisin Resistance in Listeria monocytogenes †.

Listeria monocytogenes is a food-borne pathogen that has been implicated in many outbreaks associated with ready-to-eat products. Listeria adjusts to various stresses by adjusting its membrane fluidity, increasing the uptake of osmoprotectants and cryoprotectants, and activating the sigma(B) stress factor. The present work examines the regulation of membrane fluidity through direct measurement based on fluorescent anisotropy. The membrane fluidities of L. monocytogenes Scott A, NR30, wt10403S, and cld1 cells cultured at 15 and 30 degrees C were measured at 15 and 30 degrees C. The membrane of the cold-sensitive mutant (cld1) was more rigid than the membranes of the other strains when grown at 30 degrees C, but when grown at 15 degrees C, it was able to adjust its membrane to approach the rigidity of the other strains. The difference in rigidities, as determined at 15 and 30 degrees C, was greater in liposomes than in whole cells. The rates of fluidity adjustment and times required for whole cells to adjust to a different temperature were similar among strains but different from those of liposomes. This suggests that the cells had a mechanism for homeoviscous adaptation that was absent in liposomes.

Thomas J. Montville

Papers

Clostridium difficile MazF Toxin Exhibits Selective, Not Global, mRNA Cleavage

The influence of pH and growth rate on production of the bacteriocin, bavaricin MN, in batch and continuous fermentations

Nisin A depletes intracellular ATP and acts in bactericidal manner against Mycobacterium smegmatis.

Influence of pH, Salt, and Temperature on Nisin Resistance in Listeria monocytogenes †.

Changes in Listeria monocytogenes Membrane Fluidity in Response to Temperature Stress