Susceptibility of Staphylococcus epidermidis planktonic cells and biofilms to the lytic action of staphylococcus bacteriophage K

AIMS: To evaluate differences in biofilm or planktonic bacteria susceptibility to be killed by the polyvalent antistaphylococcus bacteriophage K. METHODS AND RESULTS: In this study, the ability of phage K to infect and kill several clinical isolates of Staphylococcus epidermidis was tested. Strains were grown in suspension or as biofilms to compare the susceptibility of both phenotypes to the phage lytic action. Most strains (10/11) were susceptible to phage K, and phage K was also effective in reducing biofilm biomass after 24 h of challenging. Biofilm cells were killed at a lower rate than the log-phase planktonic bacteria but at similar rate as stationary phase planktonic bacteria. CONCLUSIONS: Staphylococcus epidermidis biofilms and stationary growth phase planktonic bacteria are more resistant to phage K lysis than the exponential phase planktonic bacteria. SIGNIFICANCE OF STUDY: This study shows the differences in Staph. epidermidis susceptibility to be killed by bacteriophage K, when grown in biofilm or planktonic phenotypes.     

甲苯胺蓝介导的超声疗法对牙菌斑中细菌灭活效果的研究

目的：验证甲苯胺蓝介导超声对龈上牙菌斑中细菌的抑制作用。方法：从门诊20例牙周炎病人下前牙区取龈上菌斑一份,每个患者的菌斑于实验室培养后,在同一甲苯胺蓝浓度下与不同超声强度联合作用,利用涂板计数法分析其对菌斑中细菌的抑制作用。实验分组：空白组、单独甲苯胺蓝组、单独超声组、甲苯胺蓝＋超声组。结果：单独超声作用时没有灭菌效果,细菌数目反而有所增长（P〈0.05）,超声联合甲苯胺蓝作用时取得明显的灭菌效果,超声强度2W/CM2灭菌率可达到79%。结论：低强度超声能够促进细菌生长,甲苯胺蓝介导一定强度的超声疗法对龈上牙菌斑中的细菌有良好的抑制作用。     

Mechanism of low intensity ultrasound effect on mitochondria

Ultrasound of 0,2 Wt/cm2 intensity affects the ionic transport across the mitochondrial membrane in vitro. In the presence of 1 mM EGTA in the incubation medium ultrasound slows down K+ exit into the external medium after the addition of an uncoupling agent (2,4-dinitrophenol). With the addition of 100 divided by 400 mM Ca2+ to the starting medium ultrasound makes the amount of Ca2+ absorbed by mitochondrial decrease and the rate of Ca2+/H+ electroneutral exchange increase. Without Ca2+ ultrasound does not influence the rate of coupled and 2,4-dinitrophenol uncoupled respiration and oxidative phosphorylation, i.e. does not produce strong functional changes.     

Destruction of planktonic, adherent and biofilm cells of Staphylococcus epidermidis using a gliding discharge in humid air

AIMS: To determine the efficiency of an electric discharge of the gliding arc type for the destruction of Staphylococcus epidermidis planktonic, adherent and biofilm cells. METHODS AND RESULTS: Bacterial cells were treated in humid air and at atmospheric pressure by a nonthermal quenched plasma of the glidarc type. The kinetics of destruction (followed by plating) were modelled by an Add-inn for Microsoft Excel, GInaFiT. For planktonic cells, log-linear destruction was obtained, whereas biphasic kinetics were observed for sessile cells. An increased resistance of biofilm cells was observed: the reduction of 6 logarithm units of the population was obtained in 15, 30 and 70 min for planktonic, adherent and biofilm cells, respectively. The experiments also show that the cells destruction did not depend on the adhesion surface but was governed by the gap between the target and the plasma source. CONCLUSION: The complete destruction of planktonic, adherent and more resistant biofilm cells of Staph. epidermidis is achieved by a glidarc air plasma at atmospheric pressure. SIGNIFICANCE AND IMPACT OF THE STUDY: The glidarc plasma technology is a promising candidate among the emerging nonthermal techniques for decontamination, as it can destroy even biofilms that are known as particularly resistant to various antimicrobials.     

Determination of in situ bacterial growth rates in aquifers and aquifer sediments

Laboratory and field-scale studies with stained cells were performed to monitor cell growth in groundwater systems. During cell division, the fluorescence intensity of the protein stain 5-(and 6-)-carboxyfluorescein diacetate succinimidyl ester (CFDA/SE) for each cell is halved, and the intensity can be tracked with a flow cytometer. Two strains of bacteria, Comamonas sp. strain DA001 and Acidovorax sp. strain OY-107, both isolated from a shallow aquifer, were utilized in this study. The change in the average generation or the average fluorescence intensity of the CFDA/SE-stained cells could be used to obtain estimates of doubling times. In microcosm experiments, the CFDA/SE-based doubling times were similar to the values calculated by total cell counting and were independent of cell concentration. Intact and repacked sediment core experiments with the same bacteria indicated that changes in groundwater chemistry were just as important as growth rates in determining planktonic cell concentrations. The growth rates within the sediment cores were similar to those calculated in microcosm experiments, and preferential transport of the daughter cells was not observed. The experiments indicated that the growth rates could be determined in systems with cell losses due to other phenomena, such as attachment to sediment or predation. Application of this growth rate estimation method to data from a field-scale bacterial transport experiment indicated that the doubling time was approximately 15 days, which is the first known direct determination of an in situ growth rate for bacteria in an aquifer.     

Action of low-frequency ultrasound on the peritoneum, peritoneal exudate cells and the course of experimental peritonitis

The authors studied the action of low-frequency ultrasound on rat and guinea-pig peritoneal exudate cells during aseptic peritonitis, on the intact peritoneum of these animals, and on experimental peritonitis in guinea-pigs. It was shown that ultrasound "hammers in" India ink solutions and antibacterial drugs into the peritoneum and in combination with antibiotics, it increases the guinea-pig survival rate in peritonitis. Ultrasound was not found to produce a direct bactericidal effect in vivo. Exposure of peritoneal exudate to ultrasound (1 s/cm2) demonstrated an increase in chemotaxis of neutrophil leukocytes to autologic serum and appreciable phagocytic activity. A longer exposure (up to 3-5 s/cm2 or 6-8 s/cm2) resulted in the partial damage to the peritoneum. Leukocytes, mesotheliocytes and subperitoneal striated muscles were found to be especially sensitive to ultrasound.     

Reducing bacterial resistance to antibiotics with ultrasound

The effect of erythromycin on planktonic cultures of Psedomonas aeruginosa, with and without application of 70 kHz ultrasound, was studied. Ultrasound was applied at levels that had no inhibitory effect on cultures of Ps. aeruginosa. Ultrasound in combination with erythromycin reduced the viability of Ps. aeruginosa by 1-2 orders of magnitude compared with antibiotic alone, even at concentrations below the minimum inhibitory concentration (MIC). Electron-spin resonance studies suggest that ultrasound induces uptake of antibiotic by perturbing or stressing the membrane. This application of ultrasound may be useful for expanding the number of drugs available for treating localized infections by rendering bacteria susceptible to normally ineffective antibiotics.     

Effect of chlorhexidine and benzalkonium chloride on bacterial biofilm formation

AIM: To study the effect of antiseptics on bacterial biofilm formation. METHODS AND RESULTS: Biofilm formation and planktonic growth were tested in microtiter plates in the presence of antiseptics. For Escherichia coli G1473 in the presence of chlorhexidine or benzalkonium chloride, for Klebsiella pneumoniae CF504 in the presence of chlorhexidine and for Pseudomonas aeruginosa PAO1 in the presence of benzalkonium chloride, biofilm development and planktonic growth were affected at the same concentrations of antiseptics. For PAO1 in the presence of chlorhexidine and CF504 in the presence of benzalkonium chloride, planktonic growth was significantly inhibited by a fourfold lower antiseptic concentration than biofilm development. For Staphylococcus epidermidis CIP53124 in the presence of antiseptics at the minimal inhibitory concentration (MIC), a total inhibition of biofilm formation was observed. For Staph. epidermidis exposed to chlorhexidine at 1/2, 1/4 and 1/8 MIC, or to benzalkonium chloride at 1/8, 1/16 or 1/32 MIC, biofilm formation was increased from 11.4% to 22.5% without any significant effect onto planktonic growth. CONCLUSIONS: Chlorhexidine and benzalkonium chloride inhibited biofilm formation of different bacterial species but were able to induce biofilm development for the Staph. epidermidis CIP53124 strain at sub-MICs. SIGNIFICANCE AND IMPACT OF THE STUDY: Sublethal exposure to cationic antiseptics may contribute to the persistence of staphylococci through biofilm induction.     

Determination of In Situ Bacterial Growth Rates in Aquifers and Aquifer Sediments

Laboratory and field-scale studies with stained cells were performed to monitor cell growth in groundwater systems. During cell division, the fluorescence intensity of the protein stain 5-(and 6-)-carboxyfluorescein diacetate succinimidyl ester (CFDA/SE) for each cell is halved, and the intensity can be tracked with a flow cytometer. Two strains of bacteria, Comamonas sp. strain DA001 and Acidovorax sp. strain OY-107, both isolated from a shallow aquifer, were utilized in this study. The change in the average generation or the average fluorescence intensity of the CFDA/SE-stained cells could be used to obtain estimates of doubling times. In microcosm experiments, the CFDA/SE-based doubling times were similar to the values calculated by total cell counting and were independent of cell concentration. Intact and repacked sediment core experiments with the same bacteria indicated that changes in groundwater chemistry were just as important as growth rates in determining planktonic cell concentrations. The growth rates within the sediment cores were similar to those calculated in microcosm experiments, and preferential transport of the daughter cells was not observed. The experiments indicated that the growth rates could be determined in systems with cell losses due to other phenomena, such as attachment to sediment or predation. Application of this growth rate estimation method to data from a field-scale bacterial transport experiment indicated that the doubling time was approximately 15 days, which is the first known direct determination of an in situ growth rate for bacteria in an aquifer.     

Ultrasonic treatment stimulates multiple shoot regeneration and explant enlargement in recalcitrant squash cotyledon explants in vitro

Ultrasonic treatment (0.5–2 min) stimulated multiple shoot regeneration to high levels in vitro from recalcitrant cotyledon explants of commercial squash (Cucurbita pepo L.) cultivars Ma’yan and Bareqet, on Murashige and Skoog [Physiol Plant 15:473–497, 1962] (regeneration) medium augmented with 4.4 μM benzyladenine. At this stage, unsonicated control explants regenerated only a few very small shoots or bud-like structures. Ultrasound also stimulated massive explant growth. Ultrasound treatment resulted in further multiple shoot production (five times greater than control) after explant transfer to elongation medium (Murashige and Skoog [Physiol Plant 15:473–497, 1962] medium with 0.44 μM benzyladenine and 2.9 μM gibberellic acid). Longer ultrasonic treatments (5 or 10 min) promoted multiple shoot regeneration and explant growth accompanied by hyperhydration. Scanning electron microscope observations showed that 2 min ultrasound changed the joint area between epidermal cells and removed some of the surface from the cotyledon epidermal cells, without gross surface injury to the explants. Longer periods of ultrasound (5–10 min) caused further surface erosion. Rubbing the explant contact surface with chloroform or sandpaper emulated the effect of sonication on shoot regeneration and explant growth, demonstrating that ultrasound exerts its morphogenic influence by surface removal. Sonication of explants from other batches of squash seeds (of cultivars Ma'yan and True French), that regenerated without such treatment, reduced regeneration and caused hyperhydration. This is the first report of stimulation of in vitro regeneration by ultrasound treatment. Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Surface hydrophobicity and dispersal of Pseudomonas aeruginosa from biofilms

A novel method of cell culture was employed to control the growth-rate of bacterial biofilms [1]. Cell-surface hydrophobicity increased progressively with growth rate for planktonic, chemostatgrown Pseudomonas aeruginosa and also for cells, resuspended from the biofilms. Dependence of surface hydrophobicity upon growth rate was greater for the planktonic cells. Newly-formed daughter cells, shed from the biofilms, were in all cases more hydrophilic than their adherent counterparts and demonstrated only slight growth rate dependency for this property.     

Ultrasonic enhancement of antibiotic action on several species of bacteria

The effect of the antibiotics gentamicin, streptomycin, kanamycin, tetracycline, and ampicillin on planktonic cultures of Enterobacter aerogenes, Serratia marcescens, Salmonella derby, Streptococcus mitis, and Staphylococcus epidermidis with and without an application of 70 kHz ultrasound was studied. The ultrasound was applied at levels that had no inhibitory effect on planktonic cultures of bacteria. Measurements of viability at, above, and below the minimum inhibitory concentration of the above antibiotics on the planktonic cultures of these bacteria showed that a simultaneous application of 70 kHz ultrasound and antibiotic significantly increased the effectiveness of selected antibiotics. Bacterial viability was reduced several orders of magnitude when harmless levels of ultrasound were combined with some antibiotics, especially the aminoglycosides. Similar synergistic effects of combined ultrasound and antibiotic treatment were seen in both Gram-positive and Gram-negative bacteria with several classes of antibiotics. These results may have application in the treatment of bacterial infections normally resistant to some antibiotics.     

Stimulation of gramicidin incorporation into lipid bilayer membranes by ultrasound

Ultrasound effect on gramicidin incorporation into a bilayer lipid membrane has been investigated. The observed increase in the channel opening frequency points to the incorporation rate growth due to the thickness diminishing of near-membrane non-stirred layers. The dependence of ultrasound intensity on the layer thickness is presented.     

Polymer multilayers with pH-triggered release of antibacterial agents

We report on the layer-by-layer design principles of poly(methacrylic acid) (PMAA) ultrathin hydrogel coatings that release antimicrobial agents (AmAs) in response to pH variations. The studied AmAs include gentamicin and an antibacterial cationic peptide L5. Adipic acid dihydrazide (AADH) is a cross-linker which, relative to ethylenediamine (EDA), increases the hydrogel hydrophobicity and introduces centers for hydrogen bonding to AmAs. AmA retention in AADH-cross-linked hydrogels in high-salt solutions was enhanced while AmA release at low pH was suppressed. L5 retains its antibacterial activity toward planktonic Staphylococcus epidermidis after release from PMAA hydrogels in response to pH decreases in the surrounding medium due to bacterial growth. Staphylococcus epidermidis adhesion and colonization was almost completely inhibited by L5 loading of hydrogels. The AmA-releasing and AmA-retaining properties of these hydrogel coatings provide new opportunities to study the fundamental mechanisms of AmA-coating-bacteria interactions and develop a new class of clinically relevant antibacterial coatings for medical devices.     

The use of glass wool as an attachment surface for studying phenotypic changes in Pseudomonas aeruginosa biofilms by two-dimensional gel electrophoresis

Two-dimensional polyacrylamide gel electrophoresis was used to demonstrate phenotypic differences between Pseudomonas aeruginosa biofilm cells and the planktonic counterpart cells under defined culture conditions. Glass wool was used as a substratum for cell attachment as it affords a large surface-to-volume ratio (1 g with a mean diameter of 15 microns = 1300 cm2), supports the growth of biofilms, allows for free movement of cells between the inter-strand spaces, and it facilitates the exchange of nutrients and oxygen. It also allows for the separation of the biofilm biomass from the surrounding surface influenced planktonic (SIP) cells for further characterization. Comparative analysis of the respective proteomes indicated striking differences in the protein patterns of planktonic, biofilm and SIP cells. We selected 41 proteins, the levels of which varied in a significant and reproducible way in the respective protein profiles. In the biofilm cells, a general up-regulation of the spots was seen, but in SIP cells expression of these spots were generally down-regulated. Altogether six unique proteins were seen in the planktonic cells, while the biofilm and SIP cells contained five and two unique proteins, respectively. Glass wool, therefore, appears to be an ideal attachment surface for the study of biofilm development.     

Possible involvement of the division cycle in dispersal of Escherichia coli from biofilms.

Growth rate control of adherent, sessile populations was achieved by the controlled perfusion of membrane-associated bacterial biofilms by the method of Gilbert et al. (P. Gilbert, D. G. Allison, D. J. Evans, P. S. Handley, and M. R. W. Brown, Appl. Environ. Microbiol. 55:1308-1311, 1989). Changes in cell surface hydrophobicity were evaluated with respect to growth rate for such sessile Escherichia coli cells and compared with those of suspended (planktonic) populations grown in a chemostat. Newly formed daughter cells shed at the various growth rates from the biofilm during its growth and development were also included in the study. Surface hydrophobicity decreased with growth rate similarly for both planktonic and sessile E. coli; no significant differences were noted between the two. Daughter cells dislodged from the biofilm, however, were significantly more hydrophilic than those remaining, indicating that hydrophobicity changed during the division cycle. Our data support the hypothesis that dispersal of cells from adhesive biofilms and recolonization of new surfaces reflect cell-cycle-mediated events.     

Functional, mechanical, and biochemical assessment of ultrasound therapy on tendon healing in the chicken toe

We studied the therapeutic effect of ultrasound treatment on the functional recovery of surgically repaired profundus tendon in the right third toe of mature White Leghorn hens. Ultrasound treatment was given daily for 5 minutes for a total of 20 treatment days, starting immediately after 4 weeks immobilization. Ultrasound (frequency 3.0 mHz; intensity 0.75 W/cm2) was delivered to the right leg by a 5-cm2 probe through distilled water in a bath kept at 35 degrees C. The flexion of the toe was measured preoperatively for 4 consecutive days and daily for 6 weeks after the start of ultrasound treatment for the calculation of percent of functional recovery of the flexor tendon. The non-ultrasound-treated group of birds went through the same manipulations except that no ultrasound was given when the right leg was immersed in the water bath. Ultrasound treatment significantly (p less than 0.01) improved the functional recovery of the repaired tendons (less than 5-mm gap) starting on the third week of treatment (94 +/- 2 percent) compared with the non-ultrasound-treated group (79 +/- 4 percent). Ultrasound treatment had no effect on gap formation or breaking (tensile) strength of the repaired tendons. It is concluded that ultrasound enhanced functional return of repaired flexor tendons in the hen, and the clinical implication is discussed.     

Planktonic-cell yield of a pseudomonad biofilm

Biofilm cells differ phenotypically from their free-floating counterparts. Differential growth rates in biofilms are often referred to, particularly in response to limited diffusion of oxygen and nutrients. We observed growth rates of attached Pseudomonas sp. strain CT07 cells that were notably higher than the maximum specific growth rate measured in batch culture. Despite dilution rates in continuous flow cells that exceeded the maximum planktonic specific growth rate by 58 times, sampling of the effluent revealed >10(9) cells ml(-1), suggesting that biofilms function as a source of planktonic cells through high cell yield and detachment. Further investigation demonstrated considerable planktonic cell yield from biofilms as young as 6 h, indicating that detachment is not limited to established biofilms. These biofilm-detached cells were more sensitive to a commercial biocide than associated biofilm- and chemostat-cultivated populations, implying that detached biofilm cells exhibit a character that is distinct from that of attached and planktonic cell populations.     

Bacterial adhesion, intracellular survival and cytokine induction upon stimulation of mononuclear cells with planktonic or biofilm phase Staphylococcus epidermidis

Staphylococcus epidermidis is a leading cause of hospital-acquired and biofilm-associated infections. Interactions of peripheral blood mononuclear cells (PBMCs) and monocyte-derived macrophages with planktonic or biofilm phase S.?epidermidis cells were studied. Biofilm phase bacteria exhibited higher attachment, as well as, a 10-fold higher intracellular survival in monocyte-derived macrophages than their planktonic counterparts. Stimulation of PBMCs and monocyte-derived macrophages was performed with live or formalin-fixed bacterial cells. Supernatant concentration of selected cytokines was measured by Luminex(?) xMAP(?) technology at different time points. As compared to planktonic phase, biofilm phase bacteria elicited lower amounts of proinflammatory cytokines and Th1 response cytokines, such as TNFα, IL-12p40, IL-12p70 and IFN-γ, whereas they enhanced production of IL-8, GM-CSF and IL-13. This phenomenon was independent of formalin pretreatment. Taken together, these results may contribute to interpretation of observed silent course of biofilm-associated infections.