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Osteomyelitis is a progressive infection of bone, that results in inflammatory destruction of the bone, bone necrosis, and new bone formation and may progress to a chronic and persistent state. The major categories of osteomyelitis are based on the source of infection (hematogenous or secondary to a contiguous focus of infection) and whether vascular insufficiency (either local or systemic) exists. While large-organism inoculation and/or host compromise can predispose patients to the development of osteomyelitis, the virulence of the infecting pathogen also has a significant role. One species in particular, Staphylococcus aureus , is able to cause an acute bone infection even with a low inoculum in a healthy host. In addition, through the timed expression of its arsenal of virulence factors and aided by its ability to develop antibiotic resistance rapidly, S. aureus progresses to a chronic, biofilm-mediated infection. Once a chronic infection develops, bacterial clearance cannot be attained by the host immune system or antimicrobial therapy. At this point, surgical removal of the nidus of infection is usually necessary for complete infection resolution.

L-forms of Enterococcus faecium, Bacillus subtilis and Pseudomonas syringae pv. phaseolicola were differentiated from their parent, cell-walled forms by a modified Gram staining technique. The addition of glutaraldehyde to the culture medium fixed the cells to prevent lysis of the L-forms. The cell-walled forms exhibited typical Gram staining reactions whereas the L-forms remained red due to the counterstain. L-forms were easily differentiated from cell-walled forms by their size and morphology which was made more obvious by the staining procedure. This is a very rapid and easy technique which distinguishes L-form bacteria from cell-walled organisms.

The severe problems and economic effects of corrosion and impeded fluid flow which biofilms can cause are well-known to industry, though relatively little is known of the physiology of the microorganisms involved or the mechanics of biofilm formation. Improved their detrimental effects to be more closely controlled, and even lead to applications exploiting their unique characteristics.

Infection-induced urinary stones form as a result of a urinary tract infection by urease-producing bacteria. These stones are not totally crystalline in nature but rather consist of an agglomeration of bacteria, organic matrix, and crystal of struvite (MgNH4PO4· 6H2O). Crystal formation is related to the ability of the bacteria to effect an increase in the urine pH. Another equally important bacterial role lies in their formation of a ‘biofilm’ which later becomes the organic matrix constituent of the stone. Results of the present in vitro study indicate that crystals are formed more readily if produced within the bacterial biofilm than in the surrounding urine. It is proposed that supersaturation, due in part to a bacterial-induced pH increase and in part to the metal binding tendency of the biofilm, leads to crystal formation via a gel growth mechanism within the biofilm itself. In time further bacterial cell division, microcolony.

The idea that bacteria grow preferentially on surfaces has come to the fore at regular intervals, for more than 150 years, whenever microbiologists have used direct methods to examine natural populations of these organisms growing in real ecosystems. In 1947, Antonie van Leuwenhoek used his primitive but effective microscope to describe aggregates of ‘animalcules’ that he had scraped from human tooth surfaces. Almost 100 years later, in 1934, Claude Zobell examined natural marine populations by direct microscopy, and concluded that these bacteria are attracted to the surfaces to which they sometimes adhere, to form sessile populations. Steadily, throughout the history of microbiology, a very small proportion of microbiologists have chosen to examine their subjects directly, by microscopy, and have found that bacteria grow differently after they adhere to a surface and initiate biofilm formation. Between 1935 and 1978, microbiologists at the Forsyth Dental Center, notably Ron Gibbons and van Houte, examined the microbial biofilms that constitute dental plaque and form macroscopic accretions on tooth surfaces. In 1964, Ralph Mitchell and Kevin Marshall examined the first stages of biofilm formation by bacteria in pure cultures, and distinguished between the reversible adsorption of bacteria to surfaces and the subsequent irreversible attachment that constitutes the first stage of biofilm formation. All of these bellwether studies of the association of bacterial cells with surfaces meet the criteria of the recent report of the taskforce of the American Academy of Microbiology (AAM). This AAM taskforce has taken the position that the discipline of microbiology has been seriously misled by its tendency to extrapolate from studies of planktonic cells in monospecies cultures, and these early studies that discovered biofilms constitute one of its most persuasive examples. The idea that some bacteria, in some natural ecosystems, lived preferentially on surfaces has been present consistently since the beginning of this relatively ‘young’ scientific discipline. However, partly because the subjects of this arcane science are rarely visible to the unaided eye, most practitioners have removed bacteria from their native ecosystems and have studied them in pure cultures, where they grow predominantly as floating ‘planktonic’ cells that are not, by definition, associated with surfaces.

Fleroxacin, ampicillin, trimethoprim-sulfamethoxazole, and gentamicin were comparatively evaluated for effectiveness in treating experimentally induced catheter-associated urinary tract infection and bacteriuria in a rabbit model with a closed drainage system. Fleroxacin, ampicillin and gentamicin effectively eliminated a lactose-negative, streptomycin-resistant uropathogenic strain of Escherichia coli (WE6933) from bag urine and catheter port urine, while trimethoprim-sulfamethoxazole only marginally reduced urine bacterial counts when compared to rabbits that received no antibiotic therapy. Fleroxacin eliminated E. coli from the catheter surfaces and from tissues adjacent to the catheter. Ampicillin or gentamicin therapy also eliminated biofilm bacteria from the catheter surfaces, but did not eliminate th residual bacteria from tissue adjacent to the septic catheters despite achieving urine levels of antibiotics substantially higher than minimum bactericidal concentrations for this pathogen. Trimethoprim-sulfamethoxazole was ineffective in eliminating E. coli from the catheter surfaces and the adjacent tissues. The ability of fleroxacin to effectively eliminate biofilm bacteria from catheter surfaces and tissues adjacent to such medical devices in the urinary tract may prove useful in the treatment of catheter-associated urinary tract infection and bacteriuria in mammals and humans.

Cusack, F.M., U. of Calgary, Singh, S., Alberta Research Council, Novosad, J., Petroleum Recovery Inst., Petroleum Recovery Inst., Chmilar, M., Alberta Oil Sands Technology and Research Authority, and Blenkinsopp, S.A., and Costerton, J.W., U. of Calgary Abstract While the use of bacteria to plug the high permeability zones that cause water breakthrough in permeability zones that cause water breakthrough in waterflood operations has been suggested repeatedly for several decades, field operations have met with only intermittent success. We have used starvation to reduce normal-sized (1.0 m), naturally adherent, vegetative bacterial cells to very small (0.3 m), non-adherent ultramicro bacteria (UMB) that penetrate rock and sand matrices very readily. When UMB are injected into sand or rock greater than 180 milli Darcies inpermeability, these very small, non-adherent cells are readily transported deep into the pore spaces. As they penetrate, some UMB are retained in the pore structures and, when the matrix is flooded with a suitable nutrient, these small, starved cells return to their full vegetative size and their normal high level of exopolysaccharide slime production. Within days of nutrient flooding, the permeability of the bacteria-treated high permeability zones is reduced by at least 99%. This novel biotechnology is presently being licensed for field testing in the prevention of coning and in the selective plugging of high permeability zones in waterflood operations. Introduction Conventional oil production technologies are able to recover about one-third of the original oil-in-place in a reservoir, with the remaining two-thirds being the target for enhanced oil recovery (EOR) research and development. A major problem in EOR technologies first investigator to suggest that injected bacteria preferentially plug the high permeability zones. preferentially plug the high permeability zones. Bacteria grow exponentially and generate extracellular polymer (slime) when provided with a suitable polymer (slime) when provided with a suitable nutrient. Slime production in the high permeability zones will plug the pores in those areas and divert fluid flow to the unswept regions of the reservoir resulting in increased oil production. Numerous studies have shown that normal-sized vegetative bacterial cells reduce permeability in sandstone cores. Since vegetative cells generally produce a sticky exopolysaccharide, they adhere to the first surface they contact when injected into a porous matrix and form a skin plug near the injection face. This shallow plug prevents injection water from reaching the unswept zones, as the water quickly channels around the plug. Therefore, to effectively enhance oil recovery, a deep plug is required. To improve the depth of bacterial penetration, we applied a microbial ecology phenomenon first reported by Amy and Morita. p. 341

This study was designed to assess the risk of colonization and biofilm formation of central venous catheters left in situ for seven days vs those changed over a guidewire at three days and removed at seven days. Colonization was determined using scanning and transmission electron microscopy and compared to a special scraping/sonication culture method. Thirty-one catheters were examined, and no difference was found between catheters left in situ (9 of 16 colonized) and those changed over a guidewire (11 of 15 colonized). Colonization rates rose significantly from 4 of 15 catheters at the time of guidewire change to 11 of 15 at 7 days (p less than 0.001). Of the catheters defined as colonized by SEM, the special culture technique showed bacterial growth in only 35 percent, making a negative culture result of dubious value in ruling out catheter colonization. No beneficial effect of guidewire changes in reducing colonization could be demonstrated.

Publisher Summary In the rumen, the nutrient substrates provided for microbial growth have a profound effect on the development of complex integrated communities of microorganisms. As scientists are beginning to understand these complicated and multifunctional systems, they are beginning to manipulate the rates of digestion of various feeds. The direct manipulation of the microbial component of the rumen ecosystem is difficult because the nutrient substrates dictate most ecological changes. Natural microbial populations are well adapted to their environment and are inherently very stable. Rumen populations react quickly to changes in nutrient substrates and the best immediate improvements in ruminant feed efficiency may emerge from concerted efforts to manipulate forages and cereal grains by plant breeding and/or chemical processing. Manipulation of feed materials is likely to control the rate of digestion in ruminants. The long-term control of this process will proceed from genetic engineering based on the emerging understanding of the molecular mechanisms of bacterial adhesion and specific enzyme activities.

Publisher Summary Numerous techniques are available for studying bacterial biofilms. Light and electron microscopy provide excellent means of examining the distribution of the bacteria on the surface. Scanning electron microscopy (SEM) can reveal some details about the mechanism(s) of attachment. Used in conjunction with intracellular formazan deposition techniques and a nalidixic acid sensitivity procedure, light microscopy can provide information about the percentage of actively metabolizing bacteria on a surface or concerning the sensitivity of the attached bacteria to biocidal agents. In this chapter, the methods are described that may be used to study the adherent biofilms on inert surfaces. Many of the procedures allow analysis of attached microbial consortia with minimum perturbation to the biofilm. Antibiotic (biocide) sensitivity testing is an especially useful application of procedures that measure the activity of biofilm bacteria.

During a program to improve injection into water injection wells in the East Beverly Hills and San Vicente fields, some empirical observations were made about plugging. Laboratory studies were performed that provided explanations about the nature and formation of microbial/fines plugging and its removal. It is demonstrated that the proper sequence of treatment is bleach first to degrade the organic coating, then the acid to dissolve the inorganic particulates.

Pre-treatment of straw with anhydrous ammonia increased its susceptibility to solubilization by the predominant cellulolytic bacteria from the rumen, Bacteroides succinogenes, Ruminococcus albus and R. flavefaciens. Ammonia treatment also increased the production of microbial protein and fermentation products by all three species. Scanning electron microscope observations of straw during digestion suggested that the attack of straw by these bacteria was accompanied by the formation of substantial numbers of adherent microcolonies.

Nueston microorganisms as part of a bituminous hydrocarbon degrading community of bacteria were observed using critical point drying and also thin sectioning in conjunction with transmission electron microscopy. Bacterionueston colonizing the undersurface of an oil film form a polysaccharide mat which is continuous over the surface in undisturbed cultures. The sequence of events during microbial degradation of hydrocarbons at the air-water interface may involve an initial hydrophobic interaction followed by a more permanent adhesion mediated by extracellular polymers.

A bacteriologically-stressed catheterized animal model was developed to evaluate the comparative importance of the intraluminal versus the extraluminal route of catheter-acquired urinary tract infections. This study indicated that in short-term catheterization (less than 7 days), contamination of the drainage spout or accidental disconnection of the drainage tube resulted in bacteriuria within a short time (32–48 hours). If a strict sterile closed drainage system was maintained, the extraluminal route assumed more importance in the development of bacteriuria, however this pathway was considerably slower (72–168 hours). It appeared that catheter-associated bacteriuria results from ascending bacterial colonization within glycocalyx-enclosed biofilm on the inside and/or outside surfaces of the catheter and drainage systems. Development of a biomaterial that inhibits bacterial adherence and does not allow upstream colonization of bacteria on the catheter drainage system would significantly influence the rate of catheter-acquired urinary tract infection.

Publisher Summary This chapter explores the microbial plugging problems associated with conventional water-flooding operations and evaluates the mechanisms responsible. The characteristics required of bacterial microbiologically enhanced oil recovery (MEOR) cultures for their successful injection are described and the microbial problems anticipated during other enhanced oil recovery (EOR) operations such as polymer flooding is discussed. There are two general mechanisms of microbial plugging likely to occur in the wellbore region. Two mechanisms of plugging can be distinguished: (1) particulate plugging by the microbial cells themselves and (2) viable bacterial plugging through biofilm formation. The mechanisms of microbial plugging is independently demonstrated in a model core system in which the average pore throat size is sufficiently large (33 pm) to ensure that particulate type plugging will be relatively insignificant for suspensions of singly dispersed bacteria. The importance of extracellular polymer production in microbial plugging has also been demonstrated using a culture not noted for biofilm formation.

In any study of the effects of the introduction of an organic compound, such as oil, into a particular environment, such as the Arctic we should, at the outset separate two basic responses: the responses of those organisms (largely bacteria and fungi) to whom the oil is a nutrient to be attacked and eventually decomposed, from the responses of those organisms (largely plants and animals) to whom the oil is a physical and chemical agent of potential toxicity to be tolerated with varying degrees of success. ... both groups really function as mixed populations that exhibit dynamic responses to environmental changes, such as oil spills, but our perception of the effects of these changes is largely population-oriented in the decomposers and species-oriented among higher organisms. ... The actual removal of oil from the Arctic environment depends on a combination of physical weathering and microbial decomposition .... Thus a general principle of microbial ecology is sustained here in that the addition of an organic material to a system stimulates the development of a specific microbial population capable of using that material as a nutrient. The rate of this decomposition process is of maximum importance and it obviously depends on the robustness of the initial microbial population and on nutrient limitation. ... One of the special problems of the Arctic is the very slow rate at which these decomposer populations develop significant activities ... and accessory nutrient supplementations may be required to achieve acceptable rates of hydrocarbon decomposition. A very important facet of oil degradation is the relative rates at which the different components of oil are broken down by bacteria and fungi. ... There are many reasons why oil may be toxic to animals .... Oil appears to constitute a fairly general "contact herbicide" whose direct application is most often toxic to plants. ... plants vary in their sensitivity to this "contact herbicide" and sensitivity mapping ... and bioassays of the sensitivity of specific plants under field conditions are very valuable. ... oil exerts direct and immediate toxic effects on certain plants and animals, in both aquatic and terrestrial systems, and ... more subtle toxic effects are often detected only with the passage of time. Whole populations react in the expected manner in that oil-resistant forms proliferate and then lead the recolonization of the system as the toxic hydrocarbons are removed by weathering or by microbial decomposition. The extent of severe ecological damage from oil spills is, therefore, a function both of the oil-sensitivity of the plant and animal populations and of the rates at which oil is removed by human intervention, weathering or microbial decomposition. ... In the decomposition studies perhaps the most promising development is the advent of rate studies which should be extended to cover the major classes of oil constituents and a very wide variety of ecological systems. ... In many cases it is clear that microbial decomposition, aided by fertilizer application ... will reduce the level of hydrocarbons below the toxic level for the indigenous plants and animals at a satisfactory rate. ... This entire program, with its emphasis on rates of microbial decomposition and on differential sensitivity of both species and populations of higher organisms, is basically well designed and offers a scientific basis for the development ... [of] rational oil spill clean-up policies in the sensitive Alaskan ecosystem.

Immediately following the introduction of cells of a pyelonephritogenic strain of Escherichia coli (expressing both GS and MS adhesins) into the mouse bladder, these cells were found to be randomly distributed on the mucosal surfaces of both the bladder and the kidney. After 24 hr. these adherent bacterial cells had proliferated to form nonrandomly distributed adherent microcolonies on both mucosal surfaces and large masses within the renal ducts. Large amounts of amorphous material, which we presume to be of both host and bacterial origin, was associated with the bacterial microcolonies on the infected tissues. The removal of urinary slime and tissue coat material by acid pretreatment increased both the degree and the apparent avidity of bacterial adhesion to the bladder mucosa. The adherent bacteria on the bladder mucosa were radically elongated, while those on the kidney mucosa usually retained their short rod-like morphological characteristics. These data suggest that pyelonephritogenic bacteria adhere to mucosal surfaces in both the bladder and kidney, and proliferate to form adherent microcolonies within which bacteria are associated with large amounts of amorphous extracellular material.

• In this study we have shown extensive microbial colonization of the surface of urine droppers and urine collecting systems from bacteriuric patients. In all instances there was an extensive background matrix and fibrous strands interconnected the bacteria. A "slime" layer completely encased bacteria on occasion. These observations suggest that incorporation of blockers of bacterial adhesion into the surfaces of these devices should be explored as one method of delaying, or in some cases preventing, catheter-acquired bacteriuria. ( Arch Intern Med 1983;143:1135-1141)