Antibacterial synergy of glycerol monolaurate and aminoglycosides in Staphylococcus aureus biofilms.

Glycerol monolaurate (GML) is a natural surfactant with antimicrobial properties. At ∼0.3 mM, both GML and its component lauric acid were bactericidal for antibiotic-resistant Staphylococcus aureus biofilms. With the use of MICs of antibiotics obtained from planktonic cells, GML and lauric acid acted synergistically with gentamicin and streptomycin, but not ampicillin or vancomycin, to eliminate detectable viable biofilm bacteria. Images of GML-treated biofilms suggested that GML may facilitate antibiotic interaction with matrix-embedded bacteria.

Anoxia inhibits biofilm development and modulates antibiotic activity.

BACKGROUND: Many infections involve bacterial biofilms that are notoriously antibiotic resistant. Unfortunately, the mechanism for this resistance is unclear. We tested the effect of oxygen concentration on development of Staphylococcus aureus biofilms, and on the ability of gentamicin and vancomycin to inhibit biofilm development. MATERIALS AND METHODS: To mimic catheter-associated biofilms, silastic coupons were inoculated with 10(7)S aureus and incubated either aerobically (∼21% O2) or anaerobically (10% CO2, 5% H2, 85% N2) for 16 h at 37°C with varying concentrations of gentamicin and vancomycin. Viable colony-forming units were quantified from sonicated biofilms, and the crystal violet assay quantified biofilm biomass. Metabolomic profiles probed biochemical differences between aerobic and anaerobic biofilms. RESULTS: Control biofilms (no antibiotic) cultivated aerobically contained 8.1-8.6 log10S aureus. Anaerobiasis inhibited biofilm development, quantified by viable bacterial numbers and biomass (P < 0.05). Bactericidal concentrations of gentamicin inhibited biofilm development in normoxia but not anoxia, likely because bacterial uptake of gentamicin is oxygen dependent. The inhibitory effect of vancomycin was more uniform aerobically and anaerobically, although at high bactericidal concentrations, vancomycin effectiveness was decreased under anoxia. There were notable differences in the metabolomic profiles of biofilms cultivated under normoxia versus anoxia. CONCLUSIONS: Compared with aerobic incubation, anaerobiasis resulted in decreased biofilm development, and metabolomics is a promising tool to identify key compounds involved in biofilm formation. The effectiveness of a specific antibiotic depended on its mode of action, as well as on the oxygen concentration in the environment.

Interplay of antibiotics and bacterial inoculum on suture-associated biofilms.

BACKGROUND: Biofilms are often antibiotic resistant, and it is unclear if prophylactic antibiotics can effectively prevent biofilm formation. Experiments were designed to test the ability of high (bactericidal) concentrations of ampicillin (AMP), vancomycin (VAN), and oxacillin (OXA) to prevent formation of suture-associated biofilms initiated with low (10(4)) and high (10(7)) numbers of Staphylococcus aureus. MATERIALS AND METHODS: S. aureus biofilms were cultivated overnight on silk suture incubated in biofilm growth medium supplemented with bactericidal concentrations of AMP, VAN, or OXA. Standard microbiological methods were used to quantify total numbers of viable suture-associated S. aureus. Crystal violet staining followed by spectroscopy was used to quantify biofilm biomass, which includes bacterial cells plus matrix components. To observe the effects of antibiotics on the microscopic appearance of biofilm formation, biofilms were cultivated on glass slides, then stained with fluorescent dyes, and observed by confocal microscopy. RESULTS: In the presence of a relatively low inoculum (10(4)) of S. aureus cells, bactericidal concentrations of AMP, VAN, or OXA were effective in preventing development of suture-associated biofilms. However, similar concentrations of these antibiotics were typically ineffective in preventing biofilm development on sutures inoculated with 10(7)S. aureus, a concentration relevant to contaminated skin. Confocal microscopy confirmed that bactericidal concentrations of AMP, VAN, or OXA inhibited, but did not prevent, development of S. aureus biofilms. CONCLUSION: Bactericidal concentrations of AMP, VAN, or OXA inhibited formation of suture-associated biofilms initiated with low numbers (10(4)), but not high numbers (10(7)), of S. aureus cells.

Gentamicin promotes Staphylococcus aureus biofilms on silk suture.

BACKGROUND: Communities of bacteria, termed biofilms, develop on biotic and abiotic surfaces, including medical devices and surgical suture. Biofilm-associated bacteria are typically recalcitrant to antibiotic therapy, and the effects of antibiotics on microbial biofilms are not clearly understood. There is emerging evidence that under specific conditions, aminoglycosides may actually promote biofilm development. Experiments were designed to study the effects of gentamicin on suture-associated Staphylococcus aureus biofilms. MATERIALS AND METHODS: S. aureus biofilms were formed after 24 h incubation of bacteria with silk suture. Susceptibility of planktonic S. aureus (from broth culture) to gentamicin was compared with the susceptibility of cells from mechanically dispersed S. aureus biofilms. Subinhibitory and inhibitory concentrations of gentamicin were subsequently incubated with intact suture-associated biofilms. S. aureus viability and metabolic capacity were assessed, and biofilm biomass was quantified with crystal violet (binds negatively charged surface molecules) and with the nucleic acid stain Syto 9. Scanning electron microscopy was used to assess the effect of gentamicin on the ultrastructure of suture-associated S. aureus biofilms. RESULTS: Planktonic cells and S. aureus cells from mechanically dispersed biofilms had similar susceptibility to gentamicin. However, after incubation of high concentrations of gentamicin with intact biofilms, high numbers of S. aureus remained both viable and metabolically active; biofilm biomass was increased and biofilm ultrastructure showed staphylococcal cells within copious amounts of extracellular material. CONCLUSION: Gentamicin does not effectively kill S. aureus within intact suture-associated biofilms, and gentamicin also promotes the biomass of S. aureus biofilms.

Role of Staphylococcus aureus protein A in adherence to silastic catheters.

BACKGROUND: Communities of bacteria, termed biofilms, frequently develop on central venous catheters, and bacterial contamination of central venous catheters is the most common cause of nosocomial bloodstream infections. Little is known about the initial events in bacterial adherence to the catheter surface, and experiments were designed to clarify the role of staphylococcal protein A, serum, and immunoglobulin in adherence of Staphylococcus aureus to silastic catheters. We hypothesized that S. aureus protein A in the presence of serum and immunoglobulin would alter S. aureus adherence to silastic catheters. MATERIALS AND METHODS: Three strains of S. aureus with varying expression of staphylococcal protein A were incubated 15 min at room temperature with silastic catheters, and bacterial adherence to the catheter surface was quantified. In addition, the effects of serum, albumin, and purified IgG on bacterial adherence were assessed. RESULTS: Both serum and albumin had an inhibitory effect on S. aureus adherence to the catheter surface, and protein A expression did not appreciably modulate these effects. Purified serum IgG also inhibited S. aureus adherence, with IgG having a greater inhibitory effect on the adherence of an S. aureus strain deficient in protein A compared with an S. aureus strain expressing high levels of protein A. CONCLUSION: S. aureus adherence to silastic catheters was inhibited by whole serum, albumin, and purified IgG. Expression of staphylococcal protein A interfered with IgG mediated inhibition of bacterial binding to the catheter surface. Protein A altered S. aureus adherence to silastic catheters in the presence of immunoglobulin, but not in the presence of serum or albumin.

Selected factors affecting Staphylococcus aureus within silastic catheters.

BACKGROUND: Catheter-related infections are frequent complications in hospitalized patients, and Staphylococcus aureus is a frequent etiologic agent. Little is known about factors that contribute to the growth and viability of S. aureus within contaminated catheters. MATERIALS AND METHODS: In vitro experiments assessed the ability of S. aureus to adhere to silastic catheter tubing. The effects of heparin, serum, and calcium on initial bacterial adherence were also assessed. Additional experiments quantified the effect of ethanol locking on S. aureus viability within catheter-associated biofilms produced after 48 to 72 h incubation of S. aureus with catheters under conditions of nutrient flow. Scanning electron microscopy visualized the effect of ethanol locking on the morphology of bacterial vegetations adherent to the catheter wall. RESULTS: S. aureus readily adhered (in a dose dependent manner) to silastic catheters incubated with bacteria for 15 min, and adherence was not affected by calcium or heparin (even though heparin adhered to the silastic tubing and S. aureus is known to express heparin-binding proteins). S. aureus adherence was inhibited by serum but not albumin. Ethanol locking (5 min to 24 h) of catheters containing mature 48 to 72 h S. aureus biofilms resulted in no detectable bacterial viability, although scanning electron microscopy revealed similar bacterial vegetations adherent to control and ethanol-treated catheters. CONCLUSION: S. aureus adherence to silastic catheters was inhibited by serum, but the active inhibitory component was not albumin. Ethanol locking efficiently sterilized S. aureus contaminated catheters, although nonviable bacterial vegetations remained on the ethanol-treated catheters.

Multiple functional domains of Enterococcus faecalis aggregation substance Asc10 contribute to endocarditis virulence.

Aggregation substance proteins encoded by sex pheromone plasmids increase the virulence of Enterococcus faecalis in experimental pathogenesis models, including infectious endocarditis models. These large surface proteins may contain multiple functional domains involved in various interactions with other bacterial cells and with the mammalian host. Aggregation substance Asc10, encoded by plasmid pCF10, is induced during growth in the mammalian bloodstream, and pCF10 carriage gives E. faecalis a significant selective advantage in this environment. We employed a rabbit model to investigate the role of various functional domains of Asc10 in endocarditis. The data suggested that the bacterial load of the infected tissue was the best indicator of virulence. Isogenic strains carrying either no plasmid, wild-type pCF10, a pCF10 derivative with an in-frame deletion of the prgB gene encoding Asc10, or pCF10 derivatives expressing other alleles of prgB were examined in this model. Previously identified aggregation domains contributed to the virulence associated with the wild-type protein, and a strain expressing an Asc10 derivative in which glycine residues in two RGD motifs were changed to alanine residues showed the greatest reduction in virulence. Remarkably, this strain and the strain carrying the pCF10 derivative with the in-frame deletion of prgB were both significantly less virulent than an isogenic plasmid-free strain. The data demonstrate that multiple functional domains are important in Asc10-mediated interactions with the host during the course of experimental endocarditis and that in the absence of a functional prgB gene, pCF10 carriage is actually disadvantageous in vivo.

Escherichia coli and TNF-alpha modulate macrophage phagocytosis of Candida glabrata.

BACKGROUND: The incidence of systemic nonalbicans Candida (especially C. glabrata) infections is increasing dramatically in intensive care units, but relatively little is known about the pathogenesis or host defenses associated with these life threatening infections. MATERIALS AND METHODS: The course of systemic C. glabrata infection was assessed as the fungal burden in the kidneys and livers of mice sacrificed 1, 8, and 15 d after intravenous C. glabrata. Sixteen hours before each sacrifice, half of the mice were injected intraperitoneally with intact viable or nonviable E. coli cells, or with E. coli lipopolysaccharide (LPS), or with tumor necrosis factor (TNF)-alpha. To clarify the effect of LPS and TNF-alpha on phagocytosis, resident (unstimulated) mouse peritoneal macrophages were harvested, cultivated ex vivo, and some cultures were treated with LPS or TNF-alpha prior to 30 min incubation with C. glabrata. RESULTS: Compared with mice injected with vehicle, each agent (intact E. coli cells or E. coli LPS or TNF-alpha) was consistently associated with decreased numbers of tissue C. glabrata, and some of these decreases were significant (P < 0.05). Compared with untreated macrophages, phagocytosis of C. glabrata was increased with LPS-treated macrophages (P < 0.01), and phagocytosis was also increased in the presence of TNF-alpha (P < 0.01). CONCLUSION: E. coli LPS and TNF-alpha may participate in host defense against C. glabrata by a mechanism involving increased macrophage phagocytosis, suggesting that stimulation of inflammatory cytokines may facilitate host clearance of C. glabrata.

An internal polarity landmark is important for externally induced hyphal behaviors in Candida albicans.

Directional growth is a function of polarized cells such as neurites, pollen tubes, and fungal hyphae. Correct orientation of the extending cell tip depends on signaling pathways and effectors that mediate asymmetric responses to specific environmental cues. In the hyphal form of the eukaryotic fungal pathogen Candida albicans, these responses include thigmotropism and galvanotropism (hyphal turning in response to changes in substrate topography and imposed electrical fields, respectively) and penetration into semisolid substrates. During vegetative growth in C. albicans, as in the model yeast Saccharomyces cerevisiae, the Ras-like GTPase Rsr1 mediates internal cellular cues to position new buds in a prespecified pattern on the mother cell cortex. Here, we demonstrate that Rsr1 is also important for hyphal tip orientation in response to the external environmental cues that induce thigmotropic and galvanotropic growth. In addition, Rsr1 is involved in hyphal interactions with epithelial cells in vitro and its deletion diminishes the hyphal invasion of kidney tissue during systemic infection. Thus, Rsr1, an internal polarity landmark in yeast, is also involved in polarized growth responses to asymmetric environmental signals, a paradigm that is different from that described for the homologous protein in S. cerevisiae. Rsr1 may thereby contribute to the pathogenesis of C. albicans infections by influencing hyphal tip responses triggered by interaction with host tissues.

Candida glabrata colonizes but does not often disseminate from the mouse caecum.

Candida glabrata is the second or third most frequent cause of candidaemia. The gastrointestinal tract is considered to be a major portal of entry for systemic candidiasis, but relatively few studies have investigated the pathogenesis of C. glabrata. Experiments were designed to clarify the ability of C. glabrata to disseminate from the mouse intestinal tract. Following oral inoculation, C. glabrata readily colonized the caeca [approx. 10(7) cells (g caecum)(-1)] of antibiotic-treated mice, but extraintestinal dissemination was not detected. Superimposing several mouse models of trauma and/or immunosuppression known to induce dissemination of Candida albicans and other intestinal microbes did not cause C. glabrata to disseminate often, although one exception was mice given high doses of dexamethasone for 4 days. These data support the hypothesis that the antibiotic-treated mouse intestine may be an epidemiological reservoir for C. glabrata and that this yeast tends to disseminate under specific clinical conditions.

Neoadjuvant, surgery and adjuvant chemotherapy without radiation for esophageal cancer.

BACKGROUND: A phase II trial to evaluate neoadjuvant (NAD), surgery and adjuvant (AD) combination chemotherapy without radiation therapy (RT) for patients with esophageal adenocarcinoma staged with endoscopic ultrasound and CT as T3N1 was carried out. METHODS: Thirty-three eligible patients were enrolled. NAD therapy was administered in two 49-day cycles and included cisplatin, floxuridine, paclitaxel and leucovorin. Esophageal resection was performed followed by AD therapy. RESULTS: Thirty-three patients initiated NAD therapy; 10 experienced grade 3 and 4 toxicities, which included leucopenia, fatigue, nausea, diarrhea and stomatitis. Additionally, 16 patients experienced grade 1 and 2 hematologic and non-hematologic toxicities. Fifteen patients were down-staged, of whom five were T2, seven were T1, and three had nodal disease with no evidence of residual cancer in the esophageal bed. Fifteen patients remained T3, and two showed progressive disease. Thirty-two patients proceeded to surgery and 30 were resected. Although all resected patients were eligible for AD therapy, 15 did not receive it either because of patient refusal or surgeon recommendation. Fifteen patients received AD therapy: nine who had remained T3 and six who had down-staged. Three patients experienced grade 3 and 4 toxicities similar to those in NAD therapy. Six patients had grade 1 and 2 toxicities. Kaplan-Meier estimates of overall survival at 1, 3 and 5 years were 73% (95% CI: 58-88%), 52% (95% CI: 34-69%) and 29% (95% CI: 13-45%), respectively. Median survival was 42 months. CONCLUSION: Deletion of RT may safely allow for more aggressive chemotherapy and increase chances of survival. The results need to be confirmed in a randomized phase II or larger phase III trial.

Enterococcus faecalis readily colonizes the entire gastrointestinal tract and forms biofilms in a germ-free mouse model.

The mammalian gastrointestinal (GI) tract is a complex organ system with a twist-a significant portion of its composition is a community of microbial symbionts. The microbiota plays an increasingly appreciated role in many clinically-relevant conditions. It is important to understand the details of biofilm development in the GI tract since bacteria in this state not only use biofilms to improve colonization, biofilm bacteria often exhibit high levels of resistance to common, clinically relevant antibacterial drugs. Here we examine the initial colonization of the germ-free murine GI tract by Enterococcus faecalis-one of the first bacterial colonizers of the naïve mammalian gut. We demonstrate strong morphological similarities to our previous in vitro E. faecalis biofilm microcolony architecture using 3 complementary imaging techniques: conventional tissue Gram stain, immunofluorescent imaging (IFM) of constitutive fluorescent protein reporter expression, and low-voltage scanning electron microscopy (LV-SEM). E. faecalis biofilm microcolonies were readily identifiable throughout the entire lower GI tract, from the duodenum to the colon. Notably, biofilm development appeared to occur as discrete microcolonies directly attached to the epithelial surface rather than confluent sheets of cells throughout the GI tract even in the presence of high (>109) fecal bacterial loads. An in vivo competition experiment using a pool of 11 select E. faecalis mutant strains containing sequence-defined transposon insertions showed the potential of this model to identify genetic factors involved in E. faecalis colonization of the murine GI tract.

Ability of the heparan sulfate proteoglycan syndecan-1 to participate in bacterial translocation across the intestinal epithelial barrier.

Although hundreds of microbial species reside in the human intestinal tract, comparatively few (e.g., Escherichia coli and other enterobacteria, Enterococcus faecalis, etc.) are typically associated with systemic infection in postsurgical, shock, and trauma patients. Syndecan-1 is the predominant cell surface heparan sulfate proteoglycan expressed on epithelia, and there is substantial evidence that heparan sulfate participates in interactions of a variety of frankly pathogenic microbes with mammalian cells. To investigate the role of syndecan-1 in interactions of enteric flora with intestinal epithelium, bacteria that might use the enterocyte as a portal of entry for systemic infection (including E. faecalis, E. coli, and other enterobacteria, and several species of staphylococci and streptococci) were studied for their abilities to interact with syndecan-1. Streptococcus bovis, S. agalactiae, S. pyogenes, Staphylococcus aureus, and S. epidermidis showed increased adherence to ARH-77 cells transfected to express syndecan-1. Heparin, a heparan sulfate analog, inhibited internalization of S. bovis, S. agalactiae, S. pyogenes, and S. aureus by HT-29 enterocytes (prominent syndecan-1 expression), but not Caco-2 enterocytes (relatively low syndecan-1 expression). Data from experiments with Chinese hamster ovary cells with altered glycosaminoglycan expression indicated that heparan sulfate and chondroitin sulfate (glycosaminoglycans on the syndecan-1 ectodomain) participated in bacterial interactions with mammalian cells. Thus, although E. faecalis, E. coli, and other gram-negative enterobacteria did not appear to interact with syndecan-1, this heparan sulfate proteoglycan may mediate enterocyte interactions with some staphylococci and streptococci that are known to cause systemic infections in specific populations of high-risk, immunosuppressed, postsurgical, and trauma patients.

Escherichia coli modulates extraintestinal spread of Staphylococcus aureus.

Staphylococcus aureus remains one of the most frequent causes of life-threatening systemic infection in surgical and trauma patients. It is understood that S. aureus colonization predisposes to complicating infection, but extraintestinal dissemination of S. aureus from the intestinal lumen to the draining mesenteric lymph nodes has not been systematically studied. After oral inoculation with high numbers of S. aureus, otherwise normal mice had low levels of cecal S. aureus (6.7 log10/g) and the incidence of extraintestinal dissemination was 30%. As expected, parenteral Escherichia coli lipopolysaccharide (LPS) was associated with increased numbers of cecal S. aureus, but the incidence of translocation remained unchanged. Purified LPS had no effect on S. aureus internalization by cultured HT-29 enterocytes and no effect on S. aureus transmigration through confluent enterocytes. To begin to clarify the effect of alterations in cecal bacteria on S. aureus translocation, mice were orally inoculated with E. coli and S. aureus. Compared with mice inoculated with S. aureus alone, these mice had increased numbers of cecal E. coli and S. aureus, and the incidence of S. aureus translocation nearly doubled from 46% to 88%. Experiments with HT-29 enterocytes indicated that viable E. coli had no effect on S. aureus internalization, but viable E. coli was at least 40 times more potent in inducing S. aureus transmigration across confluent enterocytes compared with a corresponding amount of purified LPS. Thus, S. aureus disseminated from the intestinal tract of normal mice by a mechanism that could involve paracellular migration across the intestinal epithelial barrier.

The Natural Surfactant Glycerol Monolaurate Significantly Reduces Development of Staphylococcus aureus and Enterococcus faecalis Biofilms.

BACKGROUND: Bacterial biofilms are involved in a large proportion of clinical infections, including device-related infections. Unfortunately, biofilm-associated bacteria are typically less susceptible to antibiotics, and infected devices must often be removed. On the basis of a recent observation that lipid-rich biofilm matrix material is present in early biofilm formation and may protect a population of bacteria from interacting with ordinarily diffusible small molecules, we hypothesized that surfactants may be useful in preventing biofilm development. METHODS: Experimental Staphylococcus aureus or Enterococcus faecalis biofilms were cultivated on surgical suture suspended in a growth medium supplemented with the natural surfactant glycerol monolaurate (GML) or with a component molecule, lauric acid. After 16 h incubation, the numbers of viable biofilm-associated bacteria were measured by standard microbiologic techniques and biofilm biomass was measured using the colorimetric crystal violet assay. RESULTS: Both GML and lauric acid were effective in inhibiting biofilm development as measured by decreased numbers of viable biofilm-associated bacteria as well as decreased biofilm biomass. Compared with lauric acid on a molar basis, GML represented a more effective inhibitor of biofilms formed by either S. aureus or E. faecalis. CONCLUSIONS: Because the natural surfactant GML inhibited biofilm development, resulting data were consistent with the hypothesis that lipids may play an important role in biofilm growth, implying that interfering with lipid formation may help control development of clinically relevant biofilms.

High-resolution visualization of the microbial glycocalyx with low-voltage scanning electron microscopy: dependence on cationic dyes.

The microbial glycocalyx is composed of a variety of polyanionic exopolysaccharides and plays important roles in microbial attachment to different substrata and to other cells. Here we report the successful use of low-voltage scanning electron microscopy (LVSEM) to visualize the glycocalyx in two microbial models (Klebsiella pneumoniae and Enterococcus faecalis biofilms) at high resolution, and also the dependence on fixation containing polycationic dyes for its visualization. Fixation in a paraformaldehyde-glutaraldehyde cocktail without cationic dyes was inadequate for visualizing the glycocalyx, whereas addition of various dyes (alcian blue, safranin, and ruthenium red) to the aldehyde cocktail appeared necessary for stabilization. The cationic dyes varied in size, shape, and charge density, and these factors appeared responsible for different phenotypic appearances of the glycocalyx with each dye. These results suggest that aldehyde fixation with cationic dyes for high-resolution LVSEM will be a useful tool for investigation of microbial biofilms as well as investigation of the extent and role of the glycocalyx in microbial attachment to surfaces.

Impact of the indigenous flora in animal models of shock and sepsis.

Septicemia is currently the 10th leading cause of death in the United States, and shock and trauma patients are the source of much of the morbidity and mortality associated with septicemia. There is substantial evidence that the composition of the indigenous flora plays an important role in modulating outcome variables in animal models of shock and sepsis. Germ-free animals that lack an indigenous flora are not as susceptible to shock as their conventionally reared counterparts. And, in conventionally reared animals, the composition of the intestinal flora can also modulate outcome in shock and sepsis. For example, certain bacterial species/strains disseminate from the intestinal tract more easily than others, antibiotic-induced alterations of the flora can modulate the incidence of systemic spread, and a certain threshold number of intestinal bacteria facilitates extraintestinal dissemination. The composition of the intestinal flora can also affect intestinal permeability, the production of inflammatory mediators, and the responses of immune cells in extraintestinal sites. And, there is evidence that prior exposure to endotoxin, via either the oral or systemic route, can influence outcome in animals challenged with parenteral endotoxin, a widely used model of endotoxin shock. The general composition of intestinal flora of experimental animals can be characterized with relative ease. This knowledge can aid data interpretation, either to help explain irreproducible or expected results or to verify that observed differences are likely related to the dependent variable studied rather than the composition of the indigenous flora.

Hypoxia and extraintestinal dissemination of Candida albicans yeast forms.

Candida albicans is a pleomorphic fungus with budding yeast and filamentous forms, and is a frequent cause of complicating infections in patients who are postsurgical, in shock, and have trauma. Many cases of systemic candidiasis are thought to orginate from the intestine, but it is unclear if the filament or the yeast is the more invasive form. Because C. albicans is relatively noninvasive and because mesenteric ischemia is thought to facilitate extraintestinal microbial dissemination, wild-type C. albicans CAF2 and mutant HLC54 (defective in filament formation) were orally inoculated into antibiotic-treated mice that were housed exclusively in room air, or were intermittently exposed to 10% oxygen for 1-h intervals. Both strains of C. albicans colonized the cecum in similar numbers (approximately 10(6.7)/g). C. albicans translocation to the draining mesenteric lymph nodes was not detected in mice inoculated with CAF2 (normoxic or hypoxic) or in normoxic mice inoculated with HLC54, but was detected in 33% (P < 0.01) of hypoxic mice inoculated with HLC54. Using Caco-2 and HT-29 enterocytes cultivated on plastic dishes and pretreated for 48 h in 10% oxygen, adherence of C. albicans HLC54 was decreased compared with wild-type CAF2, and hypoxia had no noticeable effect on adherence of either CAF2 or HLC54. Using enterocytes cultivated on permeable 8-microm filters, transepithelial migration of C. albicans CAF2 and HLC54 appeared similar. Thus, C. albicans HLC54 (defective in filament formation) was more invasive in hypoxic mice compared with wild-type CAF2, and host factors (e.g., mesenteric ischemia) rather than an innate ability to interact with enterocytes might play a more important role in extraintestinal dissemination of C. albicans yeast forms.

Adaptation of FUN-1 and Calcofluor white stains to assess the ability of viable and nonviable yeast to adhere to and be internalized by cultured mammalian cells.

The FUN-1 and Calcofluor white stains can be used in concert to assess the ability of viable and nonviable yeast to adhere to, and be internalized by, host mammalian cells in vitro. With this method, only extracellular yeast stain with Calcofluor, dead yeast cells have diffuse cytoplasmic yellow-green fluorescence, and live yeast have cytoplasmic orange-red or yellow-orange fluorescent intravacuolar structures.

Aminoglycoside inhibition of Staphylococcus aureus biofilm formation is nutrient dependent.

Biofilms represent microbial communities, encased in a self-produced matrix or extracellular polymeric substance. Microbial biofilms are likely responsible for a large proportion of clinically significant infections and the multicellular nature of biofilm existence has been repeatedly associated with antibiotic resistance. Classical in vitro antibiotic-susceptibility testing utilizes artificial growth media and planktonic microbes, but this method may not account for the variability inherent in environments subject to biofilm growth in vivo. Experiments were designed to test the hypothesis that nutrient concentration can modulate the antibiotic susceptibility of Staphylococcus aureus biofilms. Developing S. aureus biofilms initiated on surgical sutures, and in selected experiments planktonic cultures, were incubated for 16 h in 66 % tryptic soy broth, 0.2 % glucose (1× TSBg), supplemented with bactericidal concentrations of gentamicin, streptomycin, ampicillin or vancomycin. In parallel experiments, antibiotics were added to growth medium diluted one-third (1/3× TSBg) or concentrated threefold (3× TSBg). Following incubation, viable bacteria were enumerated from planktonic cultures or suture sonicates, and biofilm biomass was assayed using spectrophotometry. Interestingly, bactericidal concentrations of gentamicin (5 µg gentamicin ml(-1)) and streptomycin (32 µg streptomycin ml(-1)) inhibited biofilm formation in samples incubated in 1/3× or 1× TSBg, but not in samples incubated in 3× TSBg. The nutrient dependence of aminoglycoside susceptibility is not only associated with biofilm formation, as planktonic cultures incubated in 3× TSBg in the presence of gentamicin also showed antibiotic resistance. These findings appeared specific for aminoglycosides because biofilm formation was inhibited in all three growth media supplemented with bactericidal concentrations of the cell wall-active antibiotics, ampicillin and vancomycin. Additional experiments showed that the ability of 3× TSBg to overcome the antibacterial effects of gentamicin was associated with decreased uptake of gentamicin by S. aureus. Uptake is known to be decreased at low pH, and the kinetic change in pH of growth medium from biofilms incubated in 5 µg gentamicin ml(-1) in the presence of 3× TSBg was decreased when compared with pH determinations from biofilms formed in 1/3× or 1× TSBg. These studies underscore the importance of environmental factors, including nutrient concentration and pH, on the antibiotic susceptibility of S. aureus planktonic and biofilm bacteria.