Scanning Electron Microscopy

Studies of Earthly ice-bound microbes are relevant to the evolution and persistence of life on extraterrestrial bodies. Great diversity of icy environments make up Earth's cold biosphere. This chapter describes research conducted in laboratories on the newly discovered life associated with permanent Antarctic lake ice, glaciers and ice sheets (polar and temperate), and sub-glacial Antarctic lakes. Molecular-based approaches to microbial ecology yield data that measure the natural evolutionary relationships between microorganisms. The chapter illustrates the phylogenetic relatedness, based on 16S rDNA identity, between bacteria recovered in the laboratories and by others from Antarctica and permanently cold nonpolar locales. As indicated, these psychrophilic and psychrotrophic isolates originate from locations ranging from aquatic and marine ecosystems to terrestrial soils and glacial ice, with little in common between these environments except that all are permanently cold or frozen. Such information, coupled with a dedicated effort to further investigate microbial diversity within the planet's frozen realms, will provide the perspective necessary to understand the evolution and ecological impacts of microbial ecosystems residing within Earth's icy biosphere.

The medical importance of the enterococci is closely related to the propensity of these organisms to participate in the horizontal transfer of determinants for antibiotic resistance and virulence. Conjugative plasmids in enterococci tend to fall into two main groups. Members of one group encode recognition of recipient-produced peptide pheromones that initiate the mating process; the others do not make use of such signals. The pheromone-responding plasmids usually transfer efficiently in broth (liquid) matings, whereas the others transfer relatively poorly under these conditions. An exception is pMGl, a resistance plasmid in Enterococcus faecium that transfers well in broth despite the apparent absence of a pheromone system. Those plasmids that make use of sex pheromones (e.g., pADl and pCFlO) thus far appear to exhibit a narrow host range—primarily Enterococcus faecalis and closely related species-although information on this point is very limited. A partial list of pheromone-responding plasmids is presented in this chapter. The formation of mating aggregates relates to the induction of a protein "aggregation substance" (AS), which appears extensively over the donor surface and binds to "enterococcal binding substance" (EBS) on the recipient surface. Conjugative transposons are particularly common in enterococci and streptococci and play an important role in the dissemination of antibiotic resistance in these organisms. It is likely that the enterococci play a significant role as a hardy facultative reservoir of genetic information available to a variety of other genera.

In 1981, intravenous catheters infected with staphylococci by perfusion were investigated by scanning electron microscopy (SEM) to demonstrate the mode of adhesion. Bacterial cells, primarily those of staphylococci, followed by Acinetobacter calcoaceticus and Pseudomonas aeruginosa, were shown to be attached to the inner surface of the catheter. The thickest bacterial layers were found in catheters infected by coagulase-negative staphylococci (CoNS). Right heart flow-directed catheters removed from 18 critically ill patients after an average of 2.6 days after insertion were covered by a bacterial biofilm. In a neonatal intensive care unit, CoNS caused the majority of the nosocomial bacteremias. A study of arterial and central venous catheters removed from patients after 1 to 14 days revealed an extensive biofilm on all 42 arterial and 26 central venous catheters. By using special biofilm culture recovery methods, it was shown that 81% of the catheters were colonized by bacteria growing in slime-enclosed biofilms. It was speculated that the colonization represents a nidus for infection and bacteremia in these patients. Staphylococci also produced biofilm on polyvinyl chloride (PVC) endotracheal tubes used in neonates. Adherence of staphylococci to various intravascular catheter materials was investigated; these materials were composed of silicone elastomer, thermoplastic polyurethane, and polyurethane coated with Hydromer, a coating that absorbs water and provides a hydrophilic sheath around the catheter. Production of slime is necessary for Staphylococcus epidermidis colonization and is also observed with many other pathogens, including S. aureus.

For over 50 years microbiologists have been aware of a class of compounds, produced by microorganisms, called siderophores. This chapter examines the type of Iron(Fe) to which siderophores bind and the location where they do so. The assumption in the literature has been that the purpose of siderophores is to supply Fe to the cell-so often stated that this quote is rarely referenced. Even though the dissolution of Fe oxides has been reviewed extensively in the literature, it is worthwhile to briefly review dissolution mechanisms, since they apply to the potential involvement of siderophores. In a study, Fe release and siderophore production by Azotobacter vinelandii (a gram-negative, asymbiotic nitrogen-fixing soil bacterium with an absolute requirement for Fe) was investigated by using several Fe oxide minerals as sources of Fe. Undoubtedly, siderophores are used by microorganisms to acquire Fe and are produced by microorganisms in response to a limited availability of Fe. These two observations are supported by hundreds of publications in the open literature. However, the results discussed in the chapter suggest that siderophores may not be entirely responsible for Fe oxide dissolution, that the role that siderophores play in the dissolution of Fe oxides remains unclear, and thus that the microbial dissolution of Fe oxides merits further investigation.

The study of bacterial biofilms is more advanced in the engineering field than in the medical field, but the simple realization that biofilms are involved in chronic infections opens the way for a massive transfer of valuable information from the engineering realm to the medical realm and for its application to the treatment of infectious diseases. Pseudomonas aeruginosa first came to the attention of biofilm microbiologists because it predominates in cold alpine streams and grows predominantly (99.99%) in biofilms in this natural habitat. The Center for Biofilm Engineering (CBE) has established the fact that most biofilms assume this microcolony and water channel structure, including all biofilms formed by the few grampositive species examined to date, and the most significant consequence of this new observation is that we must now explain how these elaborate structures are established and maintained. If we try to imagine the bacterial survival strategies that would have been effective in the earliest stages of the development of life on this planet, growth in stationary biofilms that were protected from unfavorable conditions would prevent bacteria from being swept into acid or boiling downstream pools and from surges of threatening water from upstream sources. The role of host defenses in controlling biofilm infections is discussed in the chapter. There is a growing conviction that antibiotics are losing their ability to control bacterial infections because the bacteria have mobilized all of their survival strategies in the face of this frontal attack.

Presumptive exposure to toxins produced by Pfiesteria piscicida, a small dinoflagellate found in mid-Atlantic estuarine waters, emerged as the cause of a novel human health syndrome of impaired cognition associated with skin rash, headache, gastrointestinal symptoms, and respiratory complaints following fish-kill events in Maryland's Chesapeake Bay in 1997. Dinoflagellates meet their nutritional needs either by photosynthesis (autotrophy), phagocytosis of food sources (heterotrophy), or combinations of these strategies (mixotrophy). Blooms of populations of these and other organisms that affect environmental quality and, at times, human health are often considered collectively under the general topic of harmful algal bloom (HAB). Toxin production by marine and estuarine dinoflagellates is responsible for four human health syndromes. Many fish collected during Pfiesteria-related fish-kill events display aberrant swimming behavior and have diffuse epithelial injury, with hemorrhage and necrosis. No consistent or unexpected abnormalities were found on physical examination. As outlined in this chapter, much remains to be learned about the organism, its presumed toxins, the environmental conditions which promote toxin expression, and the mechanisms by which these toxins might cause disease in humans. At the same time, there is an increasing convergence of data which support the idea of a link between exposure to toxins produced by this and related dinoflagellates and the occurrence of illness in humans.

Despite the low incidence of clinical cases, transmissible murine colonic hyperplasia is of interest to microbiologists and gastroenterologists alike because of the unusual pathogenesis of the disease. Koch’s postulates have also been fulfilled with Citrobacter freundii biotype 4280, which produces transmissible murine colonic hyperplasia when pure cultures are inoculated into either conventional or germ free mice. The transient hyperplastic state which is induced by C. freundii biotype 4280 has a profound effect on the cytokinetics of the mucosal epithelial cells in the large bowel. These changes also serve to increase the susceptibility of the colonic epithelial cells to the carcinogenic effect of 1,2-dimethylhydrazine (DMH). The cytokinetics of the hyperplastic mucosa in transmissible murine colonic hyperplasia have been characterized by autoradiography of histological sections labeled with [3H]thymidine. The role of eaeA in the pathogenesis of transmissible murine colonic hyperplasia has been examined by the construction of an isogenic mutant of C. freundii biotype 4280. The molecular basis of transmissible murine colonic hyperplasia remains enigmatic. Characteristic histopathological lesions are produced by C. freundii biotype 4280 in the mucosa of the large bowel of laboratory mice prior to the onset of gross hyperplasia. At least one common, indigenous organism has the ability to cause histopathological changes in enterocytes at the site of bacterial adherence which are reminiscent of AE lesions. It seems likely that a better understanding of the molecular pathogenesis of transmissible murine colonic hyperplasia will also bring a better understanding of the role of bacteria in proliferative bowel disease.

During their interaction with host cells, the pathogenic Yersinia spp. export a set of proteins known as the Yersinia outer membrane proteins, or Yops. This chapter reviews the work that led to the discovery of the Yops and discusses recent progress made in understanding the expression and function of these proteins. Bacterial infection of the lungs results in pneumonic plague, a form of the disease that is transmissible by aerosolization and that is often fatal. Yersinia enterocolitica is responsible for a variety of human illnesses ranging in severity from mild gastroenteritis to acute terminal ileitis. Various rodents, farm animals, and birds are the normal reservoirs for Y. pseudotuberculosis. The product of yscC shares significant homology with PulD, a protein required for the export of pullulanase by Klebsiella pneumoniae. A number of additional polypeptides (later to be identified as the Yops) were detected in sucrose gradient-purified outer membranes from pYV-containing Y. enterocolitica and Yersinia pseudotuberculosis grown under LCR conditions. The ability of Yersinia strains to induce rounding and detachment of cultured mammalian cells has been referred to as "cytotoxicity". A partial cytotoxic activity is reconstituted if these protein preparations are introduced into HeLa cells by the use of glass carrier beads. This indicates that YopE must enter the cytoplasm of the host cell to be active. Signal transduction is critically involved in a number of cellular host responses to microbial infection.

Bacteriophages are very useful models for studying the protein-derived principles of form determination in virus assembly. Bacteriophage models cannot be ignored because of the assembly of the virus tail at the prohead portal vertex or connector, the entry point of double-stranded DNA. Bacteriophage ø29 of Bacillus subtilis, illustrated in the family portrait with bacteriophage T2 of Escherichia coli, features simplicity, efficient in vitro assembly, and advanced genetic and biochemical characterizations. This chapter emphasizes ø29 structure and the mechanisms of prohead assembly and DNA packaging, the major thrusts of current ø29 research in Minneapolis. The basic problem of ø29 morphogenesis is the mechanism by which a prolate shell of particular dimensions is assembled from subunits that are also capable of being assembled into incorrect structures. Mutants of asporogenous B. subtilis that cannot assemble ø29 (vam) were selected by the use of antibodies that reacted more strongly with the free connectors than with the portal vertex of proheads or phage. Bacteriophage DNA-packaging machines seem to have in common two packaging proteins and a connector with sixfold symmetry. Restriction enzyme digestion of DNA molecules extracted from DNase I-treated proheads shows that DNA-gp3 packaging is oriented with respect to the physical map. A gyrase action would be needed to generate supercoiled DNA for packaging.