A Pure Life: The Microbial Ecology of High Purity Industrial Waters.

The microbial ecology of various natural environments has been an active area of research since the earlier part of the twentieth century. Remote and sometimes extreme environments such as the deep ocean and the deep terrestrial subsurface have revealed a remarkable array of microorganisms. The majority of these environments are nutrient limited, and microorganisms-principally, bacteria-have developed a number of survival strategies that enable their survival and, in some cases, replication. While planktonic microorganisms exist in oligotrophic environments, the predominant mode of survival and growth is associated with biofilms. There are a number of similarities between the physicochemistry of industrial water systems and some natural aquatic ecosystems, and these similarities extend to the microbial populations and the survival mechanisms that are employed. The "starvation-survival" mechanisms, including biofilm formation, may be associated with deleterious effects on industrial water systems. These effects include heat transfer inhibition, microbially influenced corrosion, and contamination of various products manufactured in a wide array of industries. Biological fouling of industrial water systems has significant direct and indirect (through antimicrobial chemical applications) impacts on engineered materials and on the etiology of some waterborne diseases. This review provides an overview of the microbial ecology of purified waters and discusses the impacts of biological activity on industrial systems.

Biological characterization of a novel biodegradable antimicrobial polymer synthesized with fluoroquinolones.

Biomaterial-related infections continue to represent a significant challenge to the medical community. Several approaches have been utilized to incorporate antimicrobial agents at the surface of implant devices in attempts to delay or eliminate the formation of biofilms. To date, most of these strategies have focused on drug conjugation or diffusion-limited systems for the delivery of such pharmaceutical agents. More recently, work has been presented on the feasibility of incorporating drugs into the backbone of polymers as a main-chain monomer. When sequenced into the backbone of the polymer with other monomers that are hydrolytically sensitive to enzyme-catalyzed breakdown, it is thought that drugs may be able to be selectively released. Specifically, degradable polyurethanes have been synthesized with fluoroquinolone antibiotics and have shown an ability to kill bacteria when released following degradation of the polymer chains by the macrophage-derived enzyme cholesterol esterase. However, specificity of the cleavage sites in the polymer was difficult to control. Since cholesterol esterase has specificity for hydrophobic moieties, it is desirable to alter the formulation of the polyurethanes to incorporate long hydrophobic monomers immediately adjacent to the ciprofloxacin molecule. Hence, the current study focuses on evaluating the enzyme-catalyzed degradation of a degradable polyurethane synthesized with 1,12 diisocyanatododecane as a substitute for 1,6 diisocyanatohexane, which was used in previous work. Validation of specific ciprofloxacin release and the generation of antimicrobial are shown. A preliminary cell study to assess the cytotoxicity of this biodegradable antibiotic polymer shows that the material has no observable effects on cell proliferation or cell membrane structure.

Localized drug delivery using crosslinked gelatin gels containing liposomes: factors influencing liposome stability and drug release.

We describe a drug-delivery vehicle that combines the sustained release properties of liposomes with the structural advantages of crosslinked gelatin gels that can be implanted directly or coated onto medical devices. Liposome inclusion in gelatin gels does not compromise thermal stability nor does it interfere with the resiliency of gels to tensile force. However, electron spin resonance analysis of sequestered DPPC liposomes revealed a slight depression (ca. 1.0 degrees C) of the gel-to-fluid phase transition relative to liposomes in suspension. The level of liposome release from gels was determined by liposome concentration, liposome size, and the presence of poly(ethylene oxide) chains in the gel matrix or in the liposome membrane. Both neutral and charged liposomes displayed relatively high affinities for poly(ethylene glycol)gelatin gels, with only 10-15% release of initially sequestered liposomes while liposomes in which poly(ethylene glycol) was included within the membrane were not as well retained (approximately 65% release). The in vitro efflux of ciprofloxacin from liposomal gels immersed in serum was nearly complete after 24 h compared to 38% release of liposomal chlorhexidine after 6 days. The serum-induced destabilization of liposomal ciprofloxacin depended on the accessibility of serum components to gels as partly immersed gels retained approximately 50% of their load of drug after 24 h. In vivo experiments using a catheterized rabbit model of urinary tract infection revealed the absence of viable Escherichia coli on coated catheter surfaces in seven out of nine cases while all untreated catheter surfaces examined (n = 7) were contaminated.

Synthesis and characterization of a novel biodegradable antimicrobial polymer.

Bacterial infection is a frequent complication associated with the use of medical devices. In an effort to address this problem, antibacterial agents have been incorporated or applied directly onto the surfaces of numerous types of medical devices. This study assessed the feasibility of using a novel biodegradable polymer to release antibiotic drugs in response to inflammatory related enzymes. A model drug polymer was synthesized using 1,6-hexane diisocyanate (HDI), polycaprolactone diol (PCL), and a fluoroquinolone antibiotic, ciprofloxacin. Polymers were characterized by size-exclusion chromatography (SEC), and elemental analysis. Biodegradation studies were carried out by incubating the polymers with solutions of cholesterol esterase (CE) or phosphate buffer (pH 7.0) for 30 days at 37 degrees C. The degradation was assessed by high-performance liquid chromatography (HPLC), mass spectrometry (MS) and 14C radiolabel release. Subsequently, the activity of the released antibiotic was assessed against a clinical isolate of Pseudomonas aeruginosa. HPLC analysis showed the release of multiple degradation products which were identified, by tandem MS, to include ciprofloxacin and derivatives of ciprofloxacin. The microbiological assessment showed that the released ciprofloxacin possessed antimicrobial activity; 1 microg/ml was measured after 10 days. The results of this study suggest that these novel bioresponsive antimicrobial polymers or similar analogs show promise for use in the control of medical device associated infections.

Antibiotic hydrogel coated Foley catheters for prevention of urinary tract infection in a rabbit model.

PURPOSE: We developed an antibiotic liposome (ciprofloxacin liposome) containing hydrogel for external coating of silicone Foley catheters and evaluated its efficacy in a rabbit model. Our goal was to create a catheter that would hinder the development of catheter associated nosocomial urinary tract infections. MATERIALS AND METHODS: We inserted either an untreated, liposomal hydrogel coated or a liposome hydrogel with ciprofloxacin coated 10F silicone Foley catheter into New Zealand White rabbits. We challenged the system with 5x10(6) virulent Escherichia coli at the urethral meatus twice daily for 3 days. Urine cultures were evaluated twice daily for 7 days. When urine cultures became positive, the rabbits were sacrificed and urine, urethral catheter and urethral tissue were cultured. RESULTS: The time to bacteriuria detection in 50% of the specimens was double for hydrogel with ciprofloxacin coated catheters versus untreated and hydrogel coated catheters. A significant (p = 0.04) improvement in average time to positive urine culture from 3.5 to 5.3 days and a 30% decrease in the bacteriuria rate for hydrogel with ciprofloxacin coated catheters were noted compared to untreated catheters. CONCLUSIONS: A significant benefit was realized by coating the extraluminal catheter surface with a ciprofloxacin liposome impregnated hydrogel. We believe this procedure will provide a significant clinical advantage, while reducing health care costs substantially.

A liposomal hydrogel for the prevention of bacterial adhesion to catheters.

The adhesion of bacteria to medical implants and the subsequent development of a biofilm frequently results in the infection of surrounding tissue and may require removal of the device. We have developed a liposomal hydrogel system that significantly reduces bacterial adhesion to silicone catheter material. The system consists of a poly (ethylene glycol)-gelatin hydrogel in which liposomes containing the antibiotic ciprofloxacin are sequestered. A poly (ethylene glycol)-gelatin-liposome mixture was applied to a silicone surface that had been pre-treated with phenylazido-modified gelatin. Hydrogel cross-linking and attachment to surface-immobilized gelatin was accomplished through the formation of urethane bonds between gelatin and nitrophenyl carbonate-activated poly (ethylene glycol). Liposomal hydrogel-coated catheters were shown to have an initial ciprofloxacin content of 185+/-16 microg cm(-2). Ciprofloxacin was released over seven days with an average release rate of 1.9+/-0.2 microg cm(-2) h(-1) for the first 94 h. In vitro assays using a clinical isolate of Pseudomonas aeruginosa established the antimicrobial efficacy of the liposomal hydrogel. A modified Kirby-Bauer assay produced growth-inhibition zone diameters of 39+/-1 mm, while bacterial adhesion was completely inhibited on catheter surfaces throughout a seven-day in vitro adhesion assay. This new antimicrobial coating shows promise as a prophylactic and/or treatment for catheter-related infection.

Structure and functional characteristics of bacterial biofilms in fluid processing operations.

Bacterial biofilms create a number of serious problems for industrial fluid processing operations. Mechanical blockages, impedance of heat transfer processes, and biodeterioration of the components of metallic and polymeric systems result in billions of dollars in losses each year. Product spoilage and possible risks to public health are also consequences of biofilm-mediated contamination. Fundamentally, these biofouling activities can be described in terms of the physicochemical properties that are associated with bacterial metabolism and biofilm development. Treatment of biofouling is also complicated by the unique structural attributes of biofilms: extracellular polymeric substances create diffusional barriers to antimicrobial agents, protecting labile cellular targets from both oxidizing and nonoxidizing compounds. The mechanisms associated with the initial events of bacterial adhesion to engineered surfaces and subsequent fouling of biofilm formation are poorly understood. However, studies of bacterial biofilm architecture have been greatly facilitated by the application of confocal laser microscopy, scanning or transmission electron microscopy, and Fourier transform infrared spectroscopy. This paper reviews the genesis of biofilm formation and describes the influence of structure on biofouling activities in industrial fluid handling systems.

Distribution of free and liposomal cefoxitin in plasma and peritoneal fluid in a porcine intra-abdominal sepsis model.

The plasma and peritoneal fluid pharmacokinetic parameters obtained after the intravenous administration of free and liposomal cefoxitin were studied in a porcine model of intraabdominal sepsis. No prior assumptions were made to predict the number of compartments pertaining to drug clearance from the administration of either cefoxitin formulation. The experimental data obtained were applied to fit mathematical models of multiexponential drug clearance and the pharmacokinetic data were found to best fit a two-compartment open model. Liposomal encapsulation significantly altered the plasma drug distribution pattern resulting in changes in the magnitude of a number of pharmacokinetic parameters examined. The mean post-distributive half-life of liposomal cefoxitin was substantially longer than that of free cefoxitin by at least 3 times. The peritoneal cavity appeared to provide a reservoir for the initial distributive phase of rapid drug clearance from the plasma compartment followed by a less-rapid post-distributive phase. The cumulative drug level, as determined by the area under the concentration curve (AUC) as a function of time, in the plasma of animals treated with liposomal cefoxitin was about 3-4 fold as high as that of animals treated with free cefoxitin. The differences in pharmacokinetic parameters appeared to account for the improved therapeutic efficacy of liposomal cefoxitin in this animal model.

Investigation of interactions between antimicrobial agents and bacterial biofilms using attenuated total reflection Fourier transform infrared spectroscopy.

Biomaterial-centred infections are often difficult to treat. An impaired immune response, acute inflammatory reactions and the presence of recalcitrant attached microorganisms are all contributing factors. A brief review of the role of attached bacteria in biomaterial-centred infections is presented. Two major hypotheses which may explain the recalcitrance of biofilms to antimicrobial agents are discussed. The analytical capabilities of attenuated total reflection Fourier transform infrared (ATR/FTIR) spectroscopy for providing information on both transport of an antimicrobial agent to bacteria embedded in the biofilm and interactions between an antimicrobial agent and biofilm components are described.

Bacterial cell size and surface charge characteristics relevant to filter validation studies.

There are two recognized mechanisms whereby organisms are retained by liquid filters; namely, sieve-retention and adsorption. The efficiency of each may be influenced by the organism, suspending milieu, and by the filtration conditions. Validations of sterilizing filtrations require the use of organism suspensions in product-specific media. However, where the product is bactericidal to the challenge organism(s), surrogate solutions may be required. The ideal surrogate solution would minimize adsorptive retention, ensuring that the sterilizing action of the filter under consideration is the consequence of sieve-retention. This review explores the impact that various physicochemical factors may have on bacterial cell size and cell surface characteristics. An understanding of interactions among challenge bacteria, suspending fluid, and filter medium is essential for the development of surrogate solutions that provide a "worst case" mileu for filter validation studies or a "placebo," non-inhibitory challenge solution.

Detection of eubacteria in interstitial cystitis by 16S rDNA amplification.

OBJECTIVE: To determine what role non-culturable microorganisms play in the etiology of interstitial cystitis (IC). MATERIALS AND METHODS: Thirty patients fulfilling NIH criteria for the diagnosis of interstitial cystitis and sixteen control patients with culture negative urine gave written informed consent and underwent bladder biopsy. Polymerase chain reaction (PCR) using two sets of universal primers for bacterial 16S rDNA was performed on urine from the cystoscope and on a cold cup bladder biopsy specimen. Of the PCR positive bladder biopsies, three patients with interstitial cystitis and three controls were randomly selected and cloned. Ten clones from each were sequenced and putative taxonomic assignments made. RESULTS: 12/26 (46%) IC and 5/12 (42%) control urine specimens and 16/30 (53%) and 9/15 (60%) bladder biopsies were PCR positive, respectively. The bacterial populations in the two patient groups tested appeared to be different based upon analysis of the 16S rRNA sequences. CONCLUSIONS: Both IC and control patients had non-culturable bacteria in their bladders. A random sampling of the two populations revealed that the bacterial populations are different, suggesting a possible link between one or more bacterial species and IC.

Stone formation after augmentation cystoplasty: the role of intestinal mucus.

PURPOSE: We evaluated the role of mucus in urine after bladder augmentation and hypothesize that mucus acts as a possible etiological factor in stone formation. MATERIALS AND METHODS: Mucus was collected via centrifugation from the 24-hour urine specimens of 8 stone forming and 10 nonstone forming patients who were randomly selected from our augmentation population. The mucus and stones were lyophilized, and then analyzed via scanning electron microscopy and energy dispersive x-ray spectrometry for calcium, phosphate, magnesium and sodium. The 24-hour urine collections were also analyzed to determine any metabolic differences between the 2 groups. RESULTS: Scanning electron microscopy and energy dispersive x-ray spectrometry spectra showed increased calcium, phosphate, and magnesium, and significantly higher (p < 0.05) calcium-to-phosphate ratios in the mucus of stone versus nonstone forming patients. Of the 8 stones examined all had viscous fluid (mucus) centers rich in calcium, phosphate and magnesium. Calcium-to-phosphate ratios in the corresponding mucus recovered from stone centers were similarly high. Urinary citrate levels were low in both groups, and calcium, phosphate and magnesium were within normal ranges. CONCLUSIONS: Mucus appears to have an important role in the genesis of bladder stones after augmentation, possibly acting as a nidus. Metabolic changes following augmentation were similar in stone and nonstone forming populations. Our data suggest that mucous calcium-to-phosphate ratios may be predictive of future stone formation. Furthermore, there may be a benefit in instituting more aggressive measures aimed at clearing mucus from the bladder.

Effects of metronidazole on Porphyromonas gingivalis biofilms.

Subgingival bacteria exist within a biofilm consisting of cells and extracellular matrix which may afford organisms protection from both antibiotics and components of the host immune system. MIC values for planktonic Porphyromonas gingivalis treated with metronidazole were compared with those obtained for the same strain in biofilms associated with hydroxyapatite (HA) surfaces. The treated biofilms were examined for growth and studied by scanning electron microscopy. A broth assay resulted in an MIC of 0.125 microgram/ml for metronidazole against P. gingivalis, P. gingivalis biofilms exhibited growth after treatment with 20 micrograms/ml metronidazole, which was 160 times the MIC for planktonic organisms. The results of this study indicate that biofilm-associated P. gingivalis may be resistant to metronidazole at concentrations which are usually attained by systemic administration.

Polymerase chain reaction-based detection of bacteria in semen.

OBJECTIVE: To determine if the presently used bacterial detection techniques provide accurate and complete profiles of microorganisms found in human semen. DESIGN: Routine bacterial cultures and molecular biology techniques using polymerase chain reaction (PCR), with a universal eubacterial primer, cloning, then sequence analysis were used to detect bacteria (culturable or nonculturable) in the semen. SETTING: University and hospital-based research laboratory. PATIENTS: Thirty infertile men and nine semen donors, all with no symptoms of a urinary tract infection, donated semen for the study. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Detection of bacteria using routine cultures and molecular biology techniques. RESULTS: Using PCR, we found > 10(4) bacteria/mL in the semen of 66% of the infertile asymptomatic men and 66% of the semen donors. This contrasts with our routine culture results which detected "significant" bacteriospermia in only 27% of the infertile men and in none of the preselected semen donors. From four of these semen specimens, DNA sequence analysis identified an average of nine different bacterial species per specimen, with close to 90% of the species being anaerobes. CONCLUSIONS: These data indicate that the present microbiologic detection methods underestimate the incidence of significant bacteriospermia, particularly anaerobic bacteria. The molecular biologic methods should help researchers confirm or refute the role of infection in male infertility.

Biocompatibility of silver-coated peritoneal dialysis catheter in a porcine model.

OBJECTIVE: Previous studies have shown that silver formulations coated onto implantable materials retard bacterial colonization and reduce the incidence of catheter-related infections. The objective of this study was to assess the histologic effects of sputter-coated silver/ silicone implants on host tissue. DESIGN: Sputter silver-coated silicone peritoneal dialysis catheter segments with and without Dacron cuffs were implanted in the subcutaneous fat and muscle in 4 pigs. Noncoated implants served as controls. The specimens were retrieved at 1, 2, 3, 4, 7, 8, 9, 10, 12, and 27 weeks. EXPERIMENTAL ANIMALS: Four 6-week-old male Yorkshire-Landrace pigs (5-6 kg) were used. MAIN OUTCOME MEASURES: Histologic parameters evaluated included the degree of inflammation, the number of giant cells, the extent of silver particulate inclusions, and the thickness of the capsules. All specimens were evaluated by a single blinded pathologist. Microbiologic analyses were also performed. RESULTS: The silver-coated catheters were associated with less inflammation than were the noncoated catheters, both in fat and muscle (p = 0.04). The number of giant cells was also lower around the silver-coated than the non-coated catheters, which were implanted in subcutaneous fat (p < 0.05). Particulate inclusions compatible with silver or silver oxide were observed only in tissue around silver-coated implants (p < 0.0001). The thickness of the capsules and the extent of the inflammatory zones were not significantly different. There was no evidence of infection-related changes. CONCLUSIONS: These data suggest that the sputter silver coating does not act as a significant tissue irritant.

Liposomal cefoxitin in a porcine model of intra-abdominal sepsis: hemodynamic changes.

The effects of free versus liposomal cefoxitin on various physiological parameters in a porcine model of Gram-negative intra-abdominal sepsis were evaluated. Four different doses of Escherichia coli inoculum mixed with sterile pig feces were used (10(8), 10(9), 10(10), and 10(11) cfu/animal), and the most consistent hemodynamic changes were observed with an inoculum of approximately 10(11) bacteria/20 kg animal. Two treatment groups were established as follows: free cefoxitin (n = 9) and liposomal cefoxitin (n = 9). All animals were maintained under anesthesia for the duration of the study, and then euthanized 24 h following intra-abdominal inoculation. The inoculated and nontreated animals showed increases in heart rate, mean pulmonary arterial pressure, systemic and pulmonary vascular resistance, and decreases in mean systemic arterial pressure and cardiac index. These changes were significant (p < .05) compared with a control group injected with normal saline. Liposomal cefoxitin-treated animals showed significantly lower decreases in mean systemic arterial pressure and increases in heart rate (p < .05) compared with both the inoculated nontreated and free cefoxitin-treated groups. Both liposomal and free cefoxitin significantly modulated the mean pulmonary arterial pressure compared with the inoculated nontreated animals (p < .05). Acidosis that developed during intra-abdominal infection diminished 6 h following the first dose of liposomal cefoxitin (p < .05). The results of these experiments demonstrate that liposomal cefoxitin exerts a beneficial modulation of some of the hemodynamic disturbances during intra-abdominal Gram-negative sepsis.

Liposomal cefoxitin in a porcine model of intra-abdominal sepsis: bactericidal efficacy.

The bactericidal effect of free versus liposomal cefoxitin was evaluated in the major reticuloendothelial organs in a porcine model of intra-abdominal sepsis. Yorkshire Landrace pigs were inoculated with 3.2 x 10(10) (n = 5) or 1.4 x 10(11) (n = 7) cfu of Escherichia coli mixed in sterile feces/animal. Two treatment groups inoculated with 1.4 x 10(11) cfu were established: free cefoxitin (n = 9) and liposomal cefoxitin (n = 9). All animals were maintained under anesthesia and euthanized after 24 h. The number of E. coli recovered in the liver, lungs, and spleen was significantly affected by inoculum size (p < .05). The liver had significantly higher numbers of bacteria (p < .05) compared with the other organs, regardless of the inoculum size. The liver and the lung of the liposomal cefoxitin-treated group showed significantly lower numbers of E. coli (5.0 x 10(4) and 6.3 x 10(2), respectively) compared with the untreated (liver, 6.3 x 10(7); lung, 2.0 x 10(6)) and free cefoxitin (liver, 5.0 x 10(6); lung, 7.9 x 10(4))-treated groups (p < .05). At 2 h following the injection of free and liposomal cefoxitin, the decrease of E. coli in peritoneal fluid compared with the nontreated septic group was significant (p < .05). No growth was observed from blood cultures taken 24 h after sepsis induction. All control experiments yielded negative cultures. The results of these experiments demonstrated that liposomal cefoxitin exerts an enhanced bactericidal effect in liver and lungs during Gram-negative sepsis.