Few microorganisms are as versatile as Escherichia coli. An important member of the normal intestinal microflora of humans and other mammals, E. coli has also been widely exploited as a cloning host in recombinant DNA technology. But E. coli is more than just a laboratory workhorse or harmless intestinal inhabitant; it can also be a highly versatile, and frequently deadly, pathogen. Several different E. coli strains cause diverse intestinal and extraintestinal diseases by means of virulence factors that affect a wide range of cellular processes.

Pathogenic Escherichia coli

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

In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.

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Biological properties of extracellular vesicles and their physiological functions

Gram-negative bacteria characteristically are surrounded by an additional membrane layer, the outer membrane. Although outer membrane components often play important roles in the interaction of symbiotic or pathogenic bacteria with their host organisms, the major role of this membrane must usually be to serve as a permeability barrier to prevent the entry of noxious compounds and at the same time to allow the influx of nutrient molecules. This review summarizes the development in the field since our previous review (H. Nikaido and M. Vaara, Microbiol. Rev. 49:1-32, 1985) was published. With the discovery of protein channels, structural knowledge enables us to understand in molecular detail how porins, specific channels, TonB-linked receptors, and other proteins function. We are now beginning to see how the export of large proteins occurs across the outer membrane. With our knowledge of the lipopolysaccharide-phospholipid asymmetric bilayer of the outer membrane, we are finally beginning to understand how this bilayer can retard the entry of lipophilic compounds, owing to our increasing knowledge about the chemistry of lipopolysaccharide from diverse organisms and the way in which lipopolysaccharide structure is modified by environmental conditions.

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

Polyphasic taxonomy, a consensus approach to bacterial systematics.

The transmission of Escherichia coli O157:H7 from manure-contaminated soil and irrigation water to lettuce plants was demonstrated using laser scanning confocal microscopy, epifluorescence microscopy, and recovery of viable cells from the inner tissues of plants. E. coli O157:H7 migrated to internal locations in plant tissue and was thus protected from the action of sanitizing agents by virtue of its inaccessibility. Experiments demonstrate that E. coli O157:H7 can enter the lettuce plant through the root system and migrate throughout the edible portion of the plant.

Transmission of Escherichia coli O157:H7 from contaminated manure and irrigation water to lettuce plant tissue and its subsequent internalization.

Membrane vesicles released by Escherichia coli O157:H7 into culture medium were purified and analyzed for protein and DNA content. Electron micrographs revealed vesicles that are spherical, range in size from 20 to 100 nm, and have a complete bilayer. Analysis of vesicle protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrates vesicles that contain many proteins with molecular sizes similar to outer membrane proteins and a number of cellular proteins. Immunoblot (Western) analysis of vesicles suggests the presence of cell antigens. Treatment of vesicles with exogenous DNase hydrolyzed surface-associated DNA; PCR demonstrated that vesicles contain DNA encoding the virulence genes eae, stx1 and stx2, and uidA, which encodes for β-galactosidase. Immunoblot analysis of intact and lysed, proteinase K-treated vesicles demonstrate that Shiga toxins 1 and 2 are contained within vesicles. These results suggest that vesicles contain toxic material and transfer experiments demonstrate that vesicles can deliver genetic material to other gram-negative organisms.

Export of Virulence Genes and Shiga Toxin by Membrane Vesicles of Escherichia coli O157:H7

Many gram-negative bacteria produce membrane vesicles, suggesting that vesicle production is not purposeless; indeed, studies during the last two decades have presented strong evidence supporting the importance of vesicles. Typical vesicles released from the surfaces of gram-negative bacteria are 50 to 250 nm, spherical, and made up of outer membrane and encapsulated periplasmic components (4, 26). Vesicle components include outer membrane proteins, lipopolysaccharide, periplasmic proteins, phospholipids, DNA, and RNA (9, 12, 15, 22, 34, 40). Vesicles from gram-negative bacteria were reported to fuse to both gram-positive and gram-negative bacteria and in some instances to promote lysis of the target cell (28). Moreover, vesicles may function as an alternative secretory pathway (3, 23) and promote adherence of the parent cell to host cells (17, 32). By virtue of their small size, bilayer protecting envelope, and ability to integrate into the membranes of foreign bacteria and to adhere to or be engulfed by eukaryotic cells, a potential role of vesicles in delivery of virulence factors, including enzymes and toxins, is not unlikely (23). In fact, virulence factors associated with the parent strain, including proteases, phospholipases, autolysin, hemolysins, and Shiga toxins, have been isolated from vesicles (3, 22, 26, 28).

Aside from toxic compounds, DNA has also been isolated from vesicles. Vesicles produced by Pseudomonas aeruginosa were reported to contain DNA (22). Vesicles released by Neisseria gonorrhoeae harbor both linear and circular DNA, including 4.2- and 7.1-kb plasmids (12). Chromosomal and bacteriophage-associated virulence genes were detected in Escherichia coli O157:H7 vesicles (26). Moreover, this research demonstrated that DNA was protected from digestion by DNase, suggesting that DNA is packaged within vesicles (26).

Bacterial evolution often proceeds by horizontal gene transfer between different genera and species (1, 7). Antibiotic resistance genes and pathogenicity islands have been acquired by a variety of pathogens, including E. coli, Salmonella enterica serovar Typhimurium, Yersinia pestis, Dichelobacter nodosis, and Helicobacter pylori (19). Virulence factors contributing to the pathogenicity of E. coli O157:H7, including Shiga toxins (45, 46) and intimin (31, 44), are encoded on pathogenicity islands in the O157 chromosome and are thought to have been acquired by horizontal transfer. Results of previous studies suggest that vesicles may be involved in the transfer of genetic material among similar bacterial species (8, 12, 26). The hypothesis has been put forth that vesicles influence antibiotic resistance in other bacteria in two ways: by physical dissemination of preformed antibiotic-inactivating enzymes into the recipient periplasm and by delivery of antibiotic resistance plasmids (3, 12). Competent Haemophilus influenzae produces vesicles which are released into the medium when cells are returned to normal growth conditions or a noncompetent state (8). Specific DNA-binding peptides were reported to be present on the surfaces of H. influenzae vesicles (24, 25) and to be associated with vesicles from N. gonorrhoeae (11).

Previously, it was reported that vesicles released by E. coli O157:H7 into culture medium contain virulence genes and Shiga toxin (26). In the present study, we demonstrate that E. coli O157:H7 vesicles mediate the transfer of virulence genes, which are subsequently expressed by recipient enteric bacteria. Moreover, the origin of the DNA in E. coli O157:H7 vesicles is elucidated. Observations show that in addition to bacteriophage-associated genes, E. coli O157:H7 vesicles contain plasmids and fragments of chromosomal DNA.

Vesicle-Mediated Transfer of Virulence Genes from Escherichia coli O157:H7 to Other Enteric Bacteria

Staphylococcus aureus Invasion of Bovine Mammary Epithelial Cells

The multidisciplinary nature of food microbiology is one of the things that make it so fascinating as a career. Food microbiologists must understand basic microbiology, the roles of beneficial microbes, food safety regulations and policy, and the proper practices that ensure safe and healthy food for billions of people. They must also be nimble thinkers, willing to embrace new analytical methods, eager to solve problems, and ever vigilant about keeping the food supply safe. 
The fourth edition of Food Microbiology: An Introduction is designed for undergraduate courses in food science, nutrition, and microbiology. This edition has been substantially updated with new information on topics like the Food Safety Modernization Act and the use of bacteriophage as antimicrobial agents, while retaining the pedagogy that students and professors appreciate. Written in a clear and easy-to-understand style, the textbook is divided into four sections: 
Basics of food microbiology presents the growth processes of food microorganisms, the biology of spores and sporeformers, and the establishment of microbiological criteria in food safety programs, and it introduces students to some of the methods used to detect and enumerate microbes in food and food handling equipment. Foodborne pathogenic bacteria opens with a discussion about the regulatory agencies and surveillance systems responsible for keeping the United States food supply safe. The remainder of the section is a rogue's gallery of pathogenic bacteria found in food. 
Other microbes important in food examines the many beneficial and detrimental ways that microorganisms affect our food supply. The section opens with a look at numerous foods, like beer, bread, pickles, and cheeses, created by the fermentation reactions of lactic acid bacteria and yeast. The rest of the section looks at microbes that are less desirable: the spoilers of food, toxigenic molds, and foodborne parasites. This section closes with a look at viruses and prions. 
Control of microorganisms in food discusses the tactics used to inhibit microbial growth in food. The section ends with a chapter on the essentials of developing quality sanitation and HACCP programs in food processing facilities.

Karl R. Matthews

Papers

Transmission of Escherichia coli O157:H7 from contaminated manure and irrigation water to lettuce plant tissue and its subsequent internalization.

Export of Virulence Genes and Shiga Toxin by Membrane Vesicles of Escherichia coli O157:H7

Vesicle-Mediated Transfer of Virulence Genes from Escherichia coli O157:H7 to Other Enteric Bacteria

Staphylococcus aureus Invasion of Bovine Mammary Epithelial Cells

Food Microbiology : An Introduction