Marc W. Mittelman

Investigation of ciprofloxacin penetration into pseudomonas aeruginosa biofilms

Bacterial infections associated with indwelling medical devices often demonstrate an intrinsic resistance to antimicrobial therapies. In order to explore the possibility of transport limitation to biofilm bacteria as a contributing factor, the penetration of a fluoroquinolone antibiotic, ciprofloxacin, through Pseudomonas aeruginosa biofilms was investigated. Attenuated total reflection Fourier transform infrared (ATR/FT-IR) spectrometry was employed to monitor bacterial colonization of a germanium substratum, transport of ciprofloxacin to the biofilm-substratum interface, and interaction of biofilm components with the antibiotic in a flowing system. Transport of the antibiotic to the biofilm-substratum interface during the 21-min exposure to 100 micrograms/ml was found to be significantly impeded by the biofilm. Significant changes in IR bands of the biofilm in regions of the spectrum associated with RNA and DNA vibrational modes appeared following exposure to the antibiotic, indicating chemical modification of biofilm components. These results suggest that transport limitations may be an important factor in the antimicrobial resistance of biofilm bacteria and that ATR/FT-IR spectrometry may be used to follow the time course of antimicrobial action in biofilms in situ. Images

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.

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.

Effects of ciprofloxacin and protamine sulfate combinations against catheter-associated Pseudomonas aeruginosa biofilms.

Infection is a common complication associated with the use of transcutaneous and implanted medical devices. These infections are generally difficult to treat and frequently require removal of the biomaterial before the infection can be completely eradicated. The presence of a bacterial biofilm recalcitrant to treatment often mediates these infections. We studied the influence of a polycationic protein, protamine sulfate, on the efficacy of the fluoroquinolone ciprofloxacin against a clinical isolate of Pseudomonas aeruginosa. A P. aeruginosa biofilm was developed on 1-cm sections of red rubber catheter material and then treated with various combinations of protamine sulfate and ciprofloxacin. The present work demonstrated that ciprofloxacin in combination with protamine was more effective against biofilms than was ciprofloxacin alone. Protamine sulfate at 50 micrograms/ml combined with antibiotic at 0.5 microgram/ml reduced the number of viable cells by an average of 98.97%, while protamine sulfate at 50 micrograms/ml alone resulted in an average 107.8% increase and antibiotic alone resulted in an average 58.6% reduction after 24 h. Furthermore, protamine sulfate, in combination with ciprofloxacin, inhibited P. aeruginosa in a dose-dependent fashion. It was further observed that treatment with the combination of protamine sulfate and ciprofloxacin had a more drastic effect on planktonic organisms as compared with the P. aeruginosa biofilms; the MBC was reduced to < 0.05 microgram/ml in the presence of 25 micrograms of protamine sulfate per ml. These findings were substantiated by ultrastructure studies of treated cells using scanning and transmission electron microscopy. The synergism between ciprofloxacin and protamine sulfate significantly enhanced the efficacy of ciprofloxacin against planktonic and biofilm P. aeruginosa.

PCR-based technique for the detection of bacteria in semen and urine

Abstract Microorganisms associated with genito-urinary tract infections are often difficult to detect due to limitations associated with culture techniques. We have applied PCR-based detection of clinical isolates to complex sample matrices. Clinical isolates of Chlamydia trachomatis, Escherichia coli, Proteus vulgaris and Pseudomonas aeruginosa were grown, diluted and used to spike human urine and human semen. The urine and semen samples were centrifuged and the bacterial pellet was kept. A lysis buffer containing proteinase K, 8-methoxypsoralen and lauryl alcohol polyether was exposed to UV light to remove the bacterial DNA in the proteinase K, and was added to the bacterial pellet. After digestion, the proteinase K was destroyed and the lysates were subjected to 35 cycles of 16S rDNA amplification using a hot-starts technique and two primer pairs specific for eubacterial 16S rDNA: 8FPL-806R and 515FPL-13B. After amplification, the amplicons were cloned and sequenced to confirm amplification of the bacteria used to spiked the samples. We were able to detect as few as 10 5 bacteria per ml of urine or semen. Thirty unspiked semen samples were tested by PCR, and 17 were positive, including 10 samples negative by routine culture. The amplicons were cloned and sequenced for four PCR-positive/culture-negative semen samples: the 16S rDNA sequences obtained were mainly from strict anaerobes, including Prevotella spp. and Peptostrep-tococcus spp. Some 16S rDNA sequences were obtained that did not match any other 16S rDNA sequences available in various nucleic acid databases. Half of 14 unspiked urine sample tested were positive by PCR, including four samples negative by routine culture. Amplicons were cloned and sequenced for one urine sample showing only Lactobacillus spp. by routine culture, and sequences from Bacteroides ureolyticus, Clostridium spp., Corynebacterium urealyticum, Peptostrep-tococcus spp., and Lactobacillus acidophilus were found. These methods have great promise for the rapid detection of viable, but non-culturable bacteria in semen and urine. We are currently applying this technique for the detection of bacteria associated with idiopathic inflammatory conditions.

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.

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.

Effect of nutrient composition on the in vitro growth of urogenital lactobacilli and uropathogens

Previous clinical studies have shown that nutrients and probiotic agents can alter the composition of the vaginal flora. The present in vitro study has shown that uropathogens have a growth advantage over lactobacilli, but potentially there are natural substances that could be applied vaginally to stimulate lactobacilli growth to the detriment of the pathogens. When chemically defined medium representative of vaginal fluid at pH 5.5 was supplemented with skim milk, it acted as a better substrate for Lactobacillus rhamnosus GR-1 than for uropathogenic bacteria and Candida albicans. Lactobacillus MRS medium, even at pH 4.5, supports the growth of pathogens, but when supplemented with ascorbic acid or EDTA, Lactobacillus growth was significantly higher. When L. rhamnosus GR-1 was coincubated in a combined nutrient composition of vitamins and lactose, it survived better than Escherichia coli and Enterococcus faecalis. These in vitro results provide a basis for testing nutritional supplements to alter the urogenital flora in an attempt to enhance restoration and maintenance of a normal disease-free state.

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.

Histologic Studies of Intravesical Oxybutynin in the Rabbit

Intravesically applied oxybutynin has been reported to have no significant systemic anticholinergic side effects, with excellent efficacy in the treatment of neurogenic bladder dysfunction. Currently, the morphologic effects of intravesical oxybutynin on the local bladder tissue are not well established. It is the purpose of this study to address this issue in an animal model. Thirty-nine New Zealand White female rabbits were catheterized daily and intravesical solutions instilled for as long as 30 days. In part A of the study, the overall histologic effects of intravesical oxybutynin were examined by comparing oxybutynin with saline administration. Part B of this study compared the relative effects of crushed oxybutynin tablets and pure oxybutynin powder. The bladder histology and urine microbiological studies were analyzed in a blinded fashion. We found that the crushed oxybutynin tablets and saline administered intravesically produced similarly mild inflammation in the bladders (p < 0.05). When we compared the crushed oxybutynin tablets and pure oxybutynin powder, however, the crushed tablet group was found to have a mild eosinophilic infiltrate seen in 5 of 9 animals, which was not observed in any of the animals in the other groups (p = 0.029). Qualitative and quantitative analyses of the microbiological findings were not different among the different groups (p > 0.05). Our findings support the clinical use of intravesical oxybutynin as being safe for local tissue. However, consideration should be given to the use of the pure powdered form of oxybutynin, since the crushed oxybutynin tablets may lead to allergic reactions.