Mechanisms of microbial movement in subsurface materials

The biological factors important in the penetration of Escherichia coli through anaerobic, nutrient-saturated, Ottawa sand-packed cores were studied under static conditions. In cores saturated with galactose-peptone medium, motile strains of E. coli penetrated four times faster than mutants defective only in flagellar synthesis. Motile, nonchemotactic mutants penetrated the cores faster than did the chemotactic parental strain. This, plus the fact that a chemotactic galactose mutant penetrated cores saturated with peptone medium at the same rate with or without a galactose gradient, indicates that chemotaxis may not be required for bacterial penetration through unconsolidated porous media. The effect of gas production on bacterial penetration was studied by using motile and nonmotile E. coli strains together with their respective isogenic non-gas-producing mutants. No differences were observed between the penetration rates of the two motile strains through cores saturated with peptone medium with or without galactose. However, penetration of both nonmotile strains was detected only with galactose. The nonmotile, gas-producing strain penetrated cores saturated with galactose-peptone medium five to six times faster than did the nonmotile, non-gas-producing mutant, which indicates that gas production is an important mechanism for the movement of nonmotile bacteria through unconsolidated porous media. For motile strains, the penetration rate decreased with increasing galactose concentrations in the core and with decreasing inoculum sizes. Also, motile strains with the faster growth rates had faster penetration rates. These results imply that, for motile bacteria, the penetration rate is regulated by the in situ bacterial growth rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms of microbial movement in subsurface materials.

The biological factors important in the penetration of Escherichia coli through anaerobic, nutrient-saturated, Ottawa sand-packed cores were studied under static conditions. In cores saturated with galactose-peptone medium, motile strains of E. coli penetrated four times faster than mutants defective only in flagellar synthesis. Motile, nonchemotactic mutants penetrated the cores faster than did the chemotactic parental strain. This, plus the fact that a chemotactic galactose mutant penetrated cores saturated with peptone medium at the same rate with or without a galactose gradient, indicates that chemotaxis may not be required for bacterial penetration through unconsolidated porous media. The effect of gas production on bacterial penetration was studied by using motile and nonmotile E. coli strains together with their respective isogenic non-gas-producing mutants. No differences were observed between the penetration rates of the two motile strains through cores saturated with peptone medium with or without galactose. However, penetration of both nonmotile strains was detected only with galactose. The nonmotile, gas-producing strain penetrated cores saturated with galactose-peptone medium five to six times faster than did the nonmotile, non-gas-producing mutant, which indicates that gas production is an important mechanism for the movement of nonmotile bacteria through unconsolidated porous media. For motile strains, the penetration rate decreased with increasing galactose concentrations in the core and with decreasing inoculum sizes. Also, motile strains with the faster growth rates had faster penetration rates. These results imply that, for motile bacteria, the penetration rate is regulated by the in situ bacterial growth rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Grain Size on Bacterial Penetration, Reproduction, and Metabolic Activity in Porous Glass Bead Chambers

We determined the effects of grain size and nutritional conditions on the penetration rate and metabolic activity of Escherichia coli strains in anaerobic, nutrient-saturated chambers packed with different sizes of glass beads (diameters, 116 to 767 μm) under static conditions. The chambers had nearly equal porosities (38%) but different calculated pore sizes (range, 10 to 65 μm). Motile strains always penetrated faster than nonmotile strains, and nutrient conditions that resulted in faster growth rates (fermentative conditions versus nitrate-respiring conditions) resulted in faster penetration rates for both motile and nonmotile strains for all of the bead sizes tested. The penetration rate of nonmotile strains increased linearly when bead size was increased, while the penetration rate of motile strains became independent of the bead size when beads having diameters of 398 μm or greater were used. The rate of H₂ production and the final amount of H₂ produced decreased when bead size was decreased. However, the final protein concentrations were similar in chambers packed with 116-, 192-, and 281-μm beads and were only slightly higher in chambers packed with 398- and 767-μm beads. Our data indicated that conditions that favored faster growth rates also resulted in faster penetration times and that the lower penetration rates observed in chambers packed with small beads were due to restriction of bacterial activity in the small pores. The large increases in the final amount of hydrogen produced without corresponding increases in the final amount of protein made indicated that metabolism became uncoupled from cell mass biosynthesis as bead size increased, suggesting that pore size influenced the efficiency of substrate utilization.     

Effect of Sterilization by Dry Heat or Autoclaving on Bacterial Penetration through Berea Sandstone

A study was undertaken to determine why bacteria could penetrate lengths of consolidated sandstone (Berea) faster when the sandstone was sterilized by autoclaving than when dry heat (150°C, 3 h) was used. Changes in permeability, porosity, and pore entrance size of the rock as a result of autoclaving were not sufficient to explain the differences in penetration times observed, but electron dispersion spectroscopy and electron microscopy of the rock revealed changes in mineral composition and clay morphology. Autoclaved cores contained more chloride than dry-heated cores, and the clays of autoclaved cores were aggregated and irregularly shaped. Therefore, the decreases in bacterial penetration rates caused by autoclave sterilization were probably the result of a change in surface charge of the pores of the rock and of a reduction in surface area of clays available for adhesion. The results implied that dry-heat sterilization was preferable to autoclaving when examining biotic and abiotic interactions in a native-state rock model.     

Characterizing the adhesion of motile and nonmotile Escherichia coli to a glass surface using a parallel-plate flow chamber

A parallel-plate flow chamber was used to measure the attachment and detachment rates of Escherichia coli to a glass surface at various fluid velocities. The effect of flagella on adhesion was investigated by performing experiments with several E. coli strains: AW405 (motile); HCB136 (nonmotile mutant with paralyzed flagella); and HCB137 (nonmotile mutant without flagella). We compared the total attachment rates and the fraction of bacteria retained on the surface to determine how the presence and movement of the flagella influence transport to the surface and adhesion strength in this dynamic system. At the lower fluid velocities, there was no significant difference in the total attachment rates for the three bacterial strains; nonmotile strains settled at a rate that was of the same order of magnitude as the diffusion rate of the motile strain. At the highest fluid velocity, the effect of settling was minimized to better illustrate the importance of motility, and the attachment rates of both nonmotile strains were approximately five times slower than that of the motile bacteria. Thus, different processes controlled the attachment rate depending on the parameter regime in which the experiment was performed. The fractions of motile bacteria retained on the glass surface increased with increasing velocity, whereas the opposite trend was found for the nonmotile strains. This suggests that the rotation of the flagella enables cells to detach from the surface (at the lower fluid velocities) and strengthens adhesion (at higher fluid velocities), whereas nonmotile cells detach as a result of shear. There was no significant difference in the initial attachment rates of the two nonmotile species, which suggests that merely the presence of flagella was not important in this stage of biofilm development.     

In situ growth and activity and modes of penetration of Escherichia coli in unconsolidated porous materials.

Statistically reliable data on the in situ rates of growth, substrate consumption, and product formation are required to test the validity of the mathematical models developed for microbially enhanced oil recovery and in situ bioremediation processes. A simple, replicable porous-core system that could be aseptically divided into sections at various times was developed to follow the kinetics of microbial growth and metabolism in situ. This core system was used to study the kinetics of growth and the mode of penetration of strains of Escherichia coli through anaerobic, nutrient-saturated, fine Ottawa sand (permeability of 7.0 microns2 and porosity of 37%) under static conditions. The in situ rate of growth of a wild-type, motile, chemotactic strain, RW262, was two times slower inside cores than it was in liquid cultures. The mode of metabolism of galactose by strain RW262 was not altered inside cores, as acetate was the only product detected either inside the cores or in liquid cultures. Without applied advective force, strain RW262 grew exponentially and moved through cores at a rate of about 0.1 m/day. The cell population moved through cores in a band-like fashion, as the front of the moving cells consisted of high cell concentrations (greater than 10(5) cells per ml). Until the breakthrough of the cells occurred, galactose consumption and acetate production were observed only in the proximal sections of the core, showing that the cell propagation preceded the complete depletion of the substrate or the accumulation of large amounts of products.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microbial selective plugging and enhanced oil recovery

Summary The ability of indigenous populations of microorganisms in Berea sandstone to improve the volumetric sweep efficiency and increase oil recovery by in situ growth and metabolism following the injection of nutrients was studied. Cores of differing permeabilities connected in parallel without crossflow and slabs of sandstone with differing permeabilities in capillary contact to allow crossflow were used. The addition of a sucrosenitrate mineral salts medium stimulated the growth and metabolism of microorganisms in the sandstone systems. This resulted in a preferential decrease in permeability in the core or slab with the higher initial permeability, diverted flow into the lower-permeability core or slab and improved the volumetric sweep efficiency. Injectivity into the slab with the lower initial permeability in the crossflow system increased during subsequent nutrient injections. Thus, microbial selective plugging does occur in laboratory systems that have the complex flow patterns observed in petroleum reservoirs without losing the ability to inject fluids into the formation. In situ microbial growth and metabolism increased oil recovery 10 to 38% of the original oil in place. Biogenic gas production accompanied oil production, and much of the gas was entrained within the produced oil suggesting that gas production was an important factor leading to increased oil recovery. Quantitation of the amount of phospholipid in the core confirmed that microbial growth preferentially occurred throughout the core with the higher initial permeability. These data showed that in situ microbial growth in the high-permeability regions improved not only the volumetric sweep efficiency but also the microscopic oil displacement efficiency.     

Effect of Motility on Surface Colonization and Reproductive Success of Pseudomonas fluorescens in Dual-Dilution Continuous Culture and Batch Culture Systems

The colonization of glass surfaces by motile and nonmotile strains of Pseudomonas fluorescens was evaluated by using dual-dilution continuous culture (DDCC), competitive and noncompetitive attachment assays, and continuous-flow slide culture. Both strains possessed identical growth rates whether in the attached or planktonic state. Results of attachment assays using radiolabeled bacteria indicated that both strains obeyed first-order (monolayer) adsorption kinetics in pure culture. However, the motile strain attached about four times more rapidly and achieved higher final cell densities on surfaces than did the nonmotile strain (2.03 × 10⁸ versus 5.57 × 10⁷ cells vial^-1) whether evaluated alone or in cocultures containing motile and nonmotile P. fluorescens. These kinetics were attributed to the increased transport of motile cells from the bulk aqueous phase to the hydrodynamic boundary layer where bacterial attachment, growth, and recolonization could occur. First-order attachment kinetics were also observed for both strains by using continuous-flow slide culture assays analyzed by image analysis. The DDCC system contained both aqueous and particulate phases which could be diluted independently. DDCC results indicated that when cocultures containing motile and nonmotile P. fluorescens colonized solid particles, the motile strain replaced the nonmotile strain in the system over time. Increasing the aqueous-phase rates of dilution decreased the time required for extinction of the nonmotile strain while concurrently decreasing the overall carrying capacity of the DDCC system for both strains. These results confirmed that bacterial motility conveyed a selective advantage during surface colonization even in aqueous-phase systems not dominated by laminar flow.     

Flagellar Motility Confers Epiphytic Fitness Advantages upon Pseudomonas syringae

The role of flagellar motility in determining the epiphytic fitness of an ice-nucleation-active strain of Pseudomonas syringae was examined. The loss of flagellar motility reduced the epiphytic fitness of a normally motile P. syringae strain as measured by its growth, survival, and competitive ability on bean leaf surfaces. Equal population sizes of motile parental or nonmotile mutant P. syringae strains were maintained on bean plants for at least 5 days following the inoculation of fully expanded primary leaves. However, when bean seedlings were inoculated before the primary leaves had expanded and bacterial populations on these leaves were quantified at full expansion, the population size of the nonmotile derivative strain reached only 0.9% that of either the motile parental or revertant strain. When fully expanded bean primary leaves were coinoculated with equal numbers of motile and nonmotile cells, the population size of a nonmotile derivative strain was one-third of that of the motile parental or revertant strain after 8 days. Motile and nonmotile cells were exposed in vitro and on plants to UV radiation and desiccating conditions. The motile and nonmotile strains exhibited equal resistance to both stresses in vitro. However, the population size of a nonmotile strain on leaves was less than 20% that of a motile revertant strain when sampled immediately after UV irradiation. Epiphytic populations of both motile and nonmotile P. syringae declined under desiccating conditions on plants, and after 8 days, the population size of a nonmotile strain was less than one-third that of the motile parental or revertant strain.     

Analysis of DNA-DNA homologies in obligate methylotrophic bacteria

The genotypic affinity of 19 bacterial strains obligately dependent on methanol or methylamine as carbon and energy sources was studied by techniques of molecular DNA hybridization. The high homology level (35-88%) between motile strain Methylophilus methanolovorus V-1447D and nonmotile strain Methylobacillus sp. VSB-792 as well as other motile strains (Pseudomonas methanolica ATCC 21704, Methylomonas methanolica NRRL 5458, Pseudomonas sp. W6, strain A3) indicates that all of them belong to one genus. Rather high level of homology (62-63%) was found between Methylobacillus glycogenes ATCC 29475 and Pseudomonas insueta ATCC 21276 and strain G-10. The motile strain Methylophilus methylotrophus NCIB 10515 has a low homology (below 20%) to other of the studied obligate methylobacteria. Therefore, at least two genetically different genera of obligate methylobacteria can be distinguished, namely Methylophilus and Methylobacillus, the latter being represented by both motile and nonmotile forms.     

Effect of bovine sperm separation by either swim-up or Percoll method on success of in vitro fertilization and early embryonic development

The objectives of these experiments were to characterize separation of frozen-thawed bovine spermatozoa on a Percoll gradient and then to compare sperm separation by either a swim-up or Percoll gradient procedure for the ability of spermatozoa to fertilize oocytes in vitro. The Percoll gradient was a 45 and 90% discontinuous gradient. Initial experiments found that centrifugation of semen on the Percoll gradient for 15 min at 700 g was sufficient to obtain optimal recovery of motile spermatozoa. Most of the nonmotile spermatozoa were recovered at the interface of the 45 and 90% Percoll layers, while the motile spermatozoa were primarily in the sperm pellet at the bottom of the gradient. When frozen-thawed semen from each of 7 bulls was separated by swimup, a mean +/- SEM of 9% +/- 1 of the motile spermatozoa were recovered after the procedure. In contrast, more spermatozoa were recovered after Percoll gradient separation (P < 0.05), with 40% +/- 4 of the motile spermatozoa recovered. The effect of separation procedure on in vitro fertilization found swim-up separated spermatozoa penetrated a mean +/- SEM of 74% +/- 5 of the oocytes, while fewer oocytes were penetrated by Percoll separated spermatozoa at 52% +/- 8 (P < 0.05). There was no effect of the separation procedure on the rates of polyspermy as measured by sperm/penetrated ova, with a mean +/- SEM of 1.25 +/-.09 for swim-up separated spermatozoa and 1.14 +/-.07 for Percoll separated spermatozoa (P>0.05). A carry over of Percoll into the fertilization medium with the Percoll separated spermatozoa was found not the cause for the decreased penetration of oocytes by these spermatozoa. In 2 of 3 bulls tested, the decreased penetration of oocytes by Percoll separated spermatozoa could be overcome by increasing the sperm concentration during fertilization from 1 x 10(6) to 5 x 10(6)/ml. When development of embryos fertilized by either swim-up or Percoll separated spermatozoa was compared for the semen from 2 bulls, a difference in cleavage rate was found in favor of swim-up separated spermatozoa (P < 0.05), but there was no effect of separation procedure on development (Day 7) to the morula + blastocyst or blastocyst stage (P>0.05). The disadvantages of the Percoll procedure could easily be overcome and the procedure was faster and yielded a six-fold greater recovery of motile spermatozoa than the swim-up method.     

Selectivity and depth of microbial plugging in Berea sandstone cores

Summary The depth of plugging by the in situ growth of either injected or indigenous microorganisms was investigated using Berea sandstone cores with pressure taps located along the length of the core. The continuous injection of aerobically prepared sucrose-mineral salts medium with 5% NaCl and 0.1% NaNO₃ resulted in large permeability reductions (70–98%). The plugging was localized at the inlet and outlet faces of the cores, and was attributed to microbial biomass production at the inlet face and biogas accumulation at the outlet face. Batch addition of aerobic medium resulted in more uniform permeability reduction along the core's length, but the magnitude of the permeability reduction was not as large (about 65%). The semi-continuous injection of oxygen-free medium resulted in a slower but a more uniform permeability reduction throughout the core compared to cores which received aerobically prepared medium. The selectivity of the process was investigated in a dual core system where two cores of 240 and 760 mdarcy permeability were connected parallel to each other without crossflow. Initially, about 85% of the total fluid flow passed through the high permeability core. After the addition ofBacillus species and medium, the flow pattern changed and about 85% of the total fluid passed through the low permeability core. These results show that the in situ growth of microorganisms can selectively plug high permeability zones and that control of the process may be achieved by alterations in the method of nutrient injection.     

Plugging of a Model Rock System by Using Starved Bacteria

The effects of starvation on bacterial penetration through artificial rock cores were examined. Klebsiella pneumoniae was starved in a simple salts solution for a duration of up to 4 weeks. These cell suspensions were injected into sintered glass bead cores, and the resulting reductions in core permeabilities were recorded. Vegetative cell cultures of K. pneumoniae grown in a sodium citrate medium were injected into other, similar cores, and the reductions in core permeabilities were recorded. The starved cell suspensions did not completely block the core pores, whereas the vegetative cultures reduced core permeability to less than 1%. Scanning electron microscopy of core sections infiltrated with either vegetative or starved cells showed that the former produced shallow “skin” plugs and copious amounts of glycocalyx at the inlet face, whereas the latter produced very little glycocalyx and the cells were distributed evenly throughout the length of the core. The use of a DNA assay to produce a cell distribution profile showed that, compared with the vegetative cells, starved bacteria were able to penetrate deeper into the cores. This was due to the smaller size of the cells and the reduction in biofilm production. This ability of starved bacteria to penetrate further into cores than the normal-size vegetative cells can be usefully applied to selective plugging for enhanced oil recovery. To further test the suitability of starved cells for use in selective plugging, the activities of starved cells present within cores were monitored before and after nutrient stimulation. Our data indicate that with nutrient stimulation, the starved cells lose their metabolic dormancy and produce reductions in core permeability due to cell growth and polymer production.     

Nutrient Resuscitation and Growth of Starved Cells in Sandstone Cores: a Novel Approach to Enhanced Oil Recovery

Klebsiella pneumoniae, which was reduced in size (0.25 by 0.5 μm) by carbon deprivation, was injected into a series of sandstone cores and subjected to separate treatments. Scanning electron microscopy of 400-mD cores showed these small starved cells in nearly every core section. The cells were a mixture of small rods and cocci with little or no biofilm production. Continuous or dose stimulation with sodium citrate allowed the cells to grow throughout the sandstone and completely plug the length of the core. The resuscitated cells were larger than the starved cells (up to 1.7 μm) and were encased in glycocalyx. Scanning electron microscopic results of resuscitation in situ with half-strength brain heart infusion broth showed that a shallow “skin” plug of cells formed at the core inlet and that fewer cells were located in the lower sections. Starved cells also penetrated 200-mD cores and were successfully resuscitated in situ with sodium citrate, so that the entire core was plugged. Nutrient resuscitation of injected starved cells to produce full-size cells which grow and block the rock pores may be successfully applied to selective plugging and may effectively increase oil recovery.     

Penetration of mouse eggs at various intervals after insemination as influenced by concentration of sperm and strain of male

This study was conducted to determine the extent to which the percentage of mouse eggs that were penetrated by sperm at the end of the period of sperm penetration was due to the proportion of eggs penetrated per unit of time and to the span of time of sperm penetration. Female mice of ICR strain were inseminated 1.5 hr after ovulation with 5 X 10(6) sperm/50 microliter from males of DBA/2N, CF1 or C57BL/6N strains to determine the effect of the male. To determine the effect of concentration of sperm ICR females were inseminated with 2, 4, 6, or 8 X 10(6) sperm/50 microliter from CF1 males. Females were killed at various intervals after insemination and the eggs were recovered and examined for evidence of penetration by a sperm. The time intervals from both insemination to the onset of egg penetration and from insemination to cessation of penetration were similar for the three strains of males. Throughout the period of penetration of eggs a constant percentage of eggs was penetrated per hour for a particular strain of male. The relative percentage penetrated per hour very closely approximated the relative percentage of eggs finally penetrated for each strain of male. The percentage of eggs penetrated per hour was linearly positively related to the concentration of sperm inseminated. The final percentage of eggs penetrated depended primarily on the rate at which the eggs were penetrated during the period of sperm penetration and not on the length of the period of egg penetration which was constant.     

Separation of canine epididymal spermatozoa by Percoll gradient centrifugation

The objective was to characterize the separation of canine epididymal spermatozoa on a Percoll gradient. Epididymal spermatozoa were overlaid on a 45 and 90% discontinuous Percoll gradient and centrifuged at 700 x g for 20 min. The Percoll column was separated into six fractions (top to bottom, A-F) after centrifugation. Fractions A-C contained few spermatozoa. Spermatozoa with bent or folded tails and a large amount of granular debris were observed in Fraction B. Fraction D contained many nonmotile spermatozoa, erythrocytes and round epithelial cells. Spermatozoa in Fraction E had significantly lower motility than those in the initial layer. Spermatozoa in Fraction F had motility similar to those before separation. Fraction F contained 40.6% of the motile spermatozoa layered and 67.5% of all motile spermatozoa recovered. There was no significant difference between Fraction F and the initial layer in sperm membrane integrity. In the sperm-oocyte penetration assay, spermatozoa from Fraction F had a significantly higher penetration rate into the immature homologous oocytes than those from Fraction E. Although the recovery rate of the motile spermatozoa was low, the canine epididymal spermatozoa with motility, membrane integrity and penetrating capability could be separated by two-layer discontinuous Percoll gradient centrifugation.     

Dilauroylphosphatidylcholine liposome effects on the acrosome reaction and in vitro penetration of zona-free hamster eggs by bull sperm: I. A fertility assay for fresh semen

Fresh sperm from five bulls having nonreturn rates ranging from 48% to 77% were treated with 15.7, 21.0, 26.2, 31.5, 36.7, and 42.0 μM dilauroylphosphatidylcholine (PC12) to induce the sperm acrosome reaction (AR). Treated sperm were incubated 3 hr with zona-free hamster eggs at 39°C prior to fixation. The eggs were then stained and examined for sperm penetration. Differences in the percentages of motile sperm and of sperm exhibiting an AR among bulls were small when compared on a within-liposome-concentration basis. Increasing the PC12 concentration from 15.7 μM to 42.0 μM increased the percentage of sperm exhibiting an AR for all bulls. At the lowest lipid concentration (15.7 μM), the percentage of eggs penetrated by sperm from the five bulls was 6% to 36%, with 0% in controls. When sperm were incubated with increasing lipid concentrations, the egg penetration rate increased to over 80%, and the total number of sperm increased to over 100 per 36 eggs in each treatment for every bull. These penetration rates decreased at the highest lipid concentration. A correlation between the PC12 concentration maximizing egg penetration and the nonreturn rate of ?.63 was found. The correlation between the PC12 concentration maximizing the total number of penetrated sperm per treatment and the bull nonreturn rate was ?.96. It was concluded that PC 12 liposomes induce the AR in bull spermatozoa, which enables them to penetrate zona-free hamster eggs. High fertility bulls required less lipid to induce the AR than did lower fertility bulls. Consequently, this assay of fresh semen could provide a laboratory method to estimate the fertility of a bull.     

Identification of EPEC and non-EPEC serotypes in the EPEC O serogroups by PCR-RFLP analysis of the fliC gene

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the flagellin gene (fliC) was performed in 233 strains of enteropathogenic Escherichia coli (EPEC) O serogroups for determining their flagellar antigen (H) status. The serological detection of flagellin is the basis for the H-codes typing system in E. coli. Thus, it is impossible to serotype nonmotile bacteria (i.e. to assign H-codes). Twenty-eight fliC restriction patterns were obtained for motile (H2, H4, H6, H7, H8, H9, H10, H11, H12, H18, H21, H27, H32, H34, H35, H40 and H51) and nonmotile serotypes (H(-)). Each motile serotype was characterized by one or two fliC specific restriction patterns. The only exception was serogroup O128ab, where a common restriction pattern was found for serotypes O128ab:H2 and O128ab:H35, even after digestion with RsaI, AluI and Sau3AI endonucleases. These two serotypes were, however, discriminated by single strand conformation polymorphism (SSCP) analysis of RsaI restriction fragments. Nonmotile strains showed fliC restriction patterns identical to some known H serotypes. The PCR-RFLP analysis of fliC gene proved to be a useful method for identifying the H variants in motile and nonmotile EPEC O serogroups.     

Penetration of β-lactamase inhibitors into the periplasm of Gram-negative bacteria

The effectiveness of a beta-lactamase inhibitor/beta-lactam combination against Gram-negative pathogens depends on many interplaying factors, one of which is the penetration of the inhibitor across the outer membrane. In this work we have measured the relative penetrations of clavulanic acid, sulbactam, tazobactam and BRL 42715 into two strains of Escherichia coli producing TEM-1 beta-lactamase, two strains of Klebsiella pneumoniae producing either TEM-1 or K-1, and two strains of Enterobacter cloacae each producing a Class C beta-lactamase. It was shown that clavulanic acid penetrated the outer membranes of all these strains more readily than the other beta-lactamase inhibitors. For the strains of E. coli and K. pneumoniae clavulanic acid penetrated approximately 6 to 19 times more effectively than tazobactam, 2 to 9 times more effectively than sulbactam and 4 to 25 times more effectively than BRL 42715. The superior penetration of clavulanic acid observed in this study is likely to contribute to the efficacy of clavulanic acid/beta-lactam combinations in combating beta-lactam resistant bacterial pathogens.