Barotolerant Bacteria Research Articles

High hydrostatic pressure for disinfection of bone grafts and biomaterials: an experimental study.

Background:Autoclaving, heat, irradiation or chemical detergents are used to disinfect autografts, allografts and biomaterials for tissue reconstruction. These methods are often associated with deterioration of mechanical, physical, and biological properties of the bone grafts and synthetic implants. High hydrostatic pressure has been proposed as a novel method preserving biomechanical and biological properties of bone, tendon and cartilage. This is the first study to assess the inactivation of clinically relevant bacteria on biomaterials and human bone by high hydrostatic pressure.Methods:Bacterial suspensions of Staphylococcus aureus, Pseudomonas aeruginosa and Enterococcus faecium, implants covered with infected blood, human bone infected in vitro, and biopsies of patients with chronic osteomyelitis were subjected to different protocols of high hydrostatic pressure up to 600 MPa. Bacterial survival after high hydrostatic pressure treatment was determined and compared with bacterial growth in untreated controls.Results:S. aureus and P. aeruginosa in suspension were completely inactivated by high hydrostatic pressure (> 5log levels), whereas E. faecium showed barotolerance up to 600 MPa. Blood and adherence to metal implants did not significantly alter inactivation of bacteria, and complete disinfection was achieved with barotolerant bacteria (S. aureus and P. aeruginosa). However, osteoarthritic bone demonstrated a non-homogeneous baroprotective effect, with single bone samples resistant to treatment resulting in unaltered bacterial growth, and complete disinfection of artificially infected bone specimens was achieved in 66% for S. aureus, 60% for P. aeruginosa and 0% for E. faecium. Human bone samples of patients with chronic osteomyelitis could be completely disinfected in 2 of 37 cases.Conclusion:High hydrostatic pressure offers new perspectives for disinfection of sensitive biomaterials and bone grafts, and contamination by blood did not significantly affect bacterial inactivation rates. However, a significant baroprotective effect was demonstrated in bone. Effectiveness is currently limited to colonization and / or infection with barosensitive micro-organisms.

New Method of Screening for Pressure-sensitive Mutants at High Hydrostatic Pressure.

We have developed an efficient method of screening to detect pressure-sensitive mutants of barophilic or barotolerant bacteria using conventional agar medium plates. By this new method, 75 colonies can be screened per plate under high hydrostatic pressure.

Molecular analyses of the sediment of the 11,000-m deep Mariana Trench.

We have obtained sediment samples from the world's deepest sea-bottom, the Mariana Trench challenger point at a depth of 10,898 m, using the new unmanned submersible Kaiko. DNA was extracted from the sediment, and DNA fragments encoding several prokaryotic ribosomal RNA small-subunit sequences and pressure-regulated gene clusters, typically identified in deep-sea adapted bacteria, were amplified by the polymerase chain reaction. From the sequencing results, at least two kinds of bacterial 16S rRNAs closely related to those of the genus Pseudomonas and deep-sea adapted marine bacteria, and archaeal 16S rRNAs related to that of a planktonic marine archaeon were identified. The sequences of the amplified pressure-regulated clusters were more similar to those of deep-sea barophilic bacteria than those of barotolerant bacteria. These results suggest that deep-sea adapted barophilic bacteria, planktonic marine archaea, and some of the world's most widespread bacteria (the genus Pseudomonas) coexist on the world's deepest sea-bottom.

Isolation and properties of barophilic and barotolerant bacteria from deep-sea mud samples

Several barophilic and barotolerant bacteria were isolated from deep-sea mud samples of Suruga Bay (2485 m depth), the Ryukyu Trench (5110 m depth), and the Japan Trench (land-side 6356 m, and sea-side 6269 m depth, respectivelys. The barophilic bacteria, strains DB5501, DB6101, DB6705 and DB6906, were albe to grow better under high hydrostatic pressures than under atmospheric pressure (0.1 megapascals; MPa). The optimal growth pressures for the barophilic bacteria were approximately 50 MPa at 10°C. The barotolerant strains DSK1 and DSS12 were determined to be psychrophilic, and had optimal growth temperatures of 10°C and 8°C, respectively. The degree of barophily and barotolerance was shown to be very dependent on temperature. For example, at 4°C the barophilic strains were indistinguishable from barotolerant bacteria, whereas at 15°C the barotolerant strains behaved more like the barophilic strains. Based on sequence analysis of 16S ribosomal DNA, all of the strains included in this study belong to the gamma subgroup of the Proteobacteria. Phylogenetic relations between the isolated strains and the known gamma subgroup bacteria suggested that the isolated strains belong to a new sub-branch of this group.

深海耐圧性細菌の圧力応答

Although the deep - sea environment with high pressure and low temperature is rigorous for microorganisms in costal water, many barotolerant and barophilic bacteria are found to be inhabiting there. It is very important for the barophilic or barotolerant bacteria to maintain the physiological functions of cell membrane, because many vital functions are involved in the membrane. The fluidity of membrane composed by phospholipids and proteins is essential for the physiological functions. As high hydrostatic pressure raises the melting point of lipids and causes the phase transition of lipid within 100 MPa, barotolerant bacteria under high hydrostatic pressure appear to regulate the fluidity of membrane phospholipids. Therefore, the characterization of membrane at high pressure is indispensable to clarify the biochemical adaptation of bacteria to the environment. This report dealt with effects of pressure and temperature on the lipid composition of barotolerant deep - sea bacteria. The bacteria maintained the membrane fluidity by increasing unique fatty acids under high hydrostatic pressure.

Variability of pressure adaptation in deep sea bacteria

Isolations of pressure-adapted deep sea bacteria from depths of 1,400 to 5,100 m resulted in a variety of psychrophilic barotolerant and barophilic strains. Growth rates determined at different pressures indicated a gradual transition between the two types of pressure-adapted isolates. The presence of barotolerant bacteria in deep water, sustained by sinking particulate matter, causes the nonbarophilic response of natural populations, i.e., increased growth after decompression. With increasing pressure-adaptation in barophilic isolates the maximum growth rates at optimum pressures decrease. Thus, the observed general slow-down of microbial activity in the deep sea takes effect regardless of the common occurrence of psychrophilic and barophilic bacteria. The highest degree of barophilism was observed in isolates from nutrient-rich habitats such as intestinal tracts of deep sea animals or decaying carcasses. Detailed studies with an isolate, growing barophilically on a complex as well as a single-carbon-source medium, showed that (1) culturing at pressures lower than optimal for growth resulted in the formation of cell filaments, (2) growth was unaffected by repeated compression/decompression cycles and (3) no perceptible differences in the distribution of radiolabeled carbon from an amino acid mixture occurred in cells grown at, below and above the pressure optimal for growth.

Enzymatic adaptation by bacteria under pressure.

A study of enzymic adaptation under hydrostatic pressure by moderately barotolerant bacteria that can grow at pressure up to about 500 atm revealed that some adaptive processes are relatively insensitive to pressure, whereas others are sufficiently barosensitive to compromise survival capacity in situations requiring adaptation to new substrates under pressure. Examples of the former include adaptation of Escherichia coli to arabinose catabolism for growth and adaptation of Streptococcus faecalis to catabolism of lactose, ribose, or maltose. Examples of the latter include derepression of the lac operon in Escherichia coli and induction of penicillinase synthesis by Bacillus licheniformis. For both these barosensitive systems, pressure had little effect on enzyme levels in constitutive strains or in bacteria that had previously been induced at 1 atm. Moreover, it had no detectable effect on penicillinase secretion. However, pressures of 300 to 400 atm were found to reduce markedly rates and extents of enzyme synthesis by bacteria undergoing derepression or adaptation. This inhibitory effect of pressure was reflected in greater barosensitivity with extended lag and slower growth of initially unadapted Escherichia coli cells inoculated into minimal medium with lactose as sole source of carbon and fuel, and by major reductions in the minimal inhibitory concentrations of penicillin G for unadapted B. licheniformis cells inoculated into complex, antibiotic-containing media. Cyclic adenosine 5'-monophosphate did not reverse pressure inhibition of derepression of the lac operon, and catabolite repression was complete under pressure. However, derepression of the lac operon was more sensitive to pressure at low concentrations of inducer than at high concentrations. Apparent volume changes for derepression were 94 and 60 ml/mol at inducer concentrations of about 0.5 and 5 mM, respectively. Pressure was found not to be inhibitory for uptake of beta-galactosides; in fact, it was somewhat stimulatory. Therefore, results were interpreted in terms of inducer binding and subsequent conversion of an operator-inducer-repressor complex to inactive repressor and operator. Both reactions appeared to result in an increase in volume, the former more so than the latter. We found also that 200 atm was actually stimulatory for growth of Escherichia coli in minimal media, and the bacterium was in a sense barophilic.

Growth Response of Mycoplasma to Carotenoid Pigments and Carotenoid Intermediates

The effect of elevated hydrostatic pressure on biosynthesis of keto acids by barotolerant bacteria growing on glucose-mineral media is discussed in this paper. Strains which are more tolerant to the action of pressure can liberate exocellular keto acids. Keto acids are synthesized at a low content of glucose and a relatively high concentration of nitrogen in the presence of dissolved oxygen. Pressure may either induce or inhibit the synthesis. A shift towards oxalacetate in the composition of keto acids caused by the action of pressure shows that anabolism and catabolism are decoupled. Free exocellular keto acids are not found in bacteria, which do not grow at 500 atm. either at atmospheric or elevated pressures; therefore, their metabolism is more balanced. Differences in the growth of barotolerant bacteria, and in biosynthesis of keto acids at elevated pressure, may be caused by different response of various strains and species of barotolerant bacteria to the action of this factor.