Andrew W. Bruce

Oral use of Lactobacillus rhamnosus GR-1 and L. fermentum RC-14 significantly alters vaginal flora: randomized, placebo-controlled trial in 64 healthy women

Urogenital infections afflict an estimated one billion people each year. The size of this problem and the increased prevalence of multi-drug resistant pathogens make it imperative that alternative remedies be found. A randomized, placebo-controlled trial of 64 healthy women given daily oral capsules of Lactobacillus rhamnosus GR-1 and Lactobacillus fermentum RC-14 for 60 days showed no adverse effects. Microscopy analysis showed restoration from asymptomatic bacterial vaginosis microflora to a normal lactobacilli colonized microflora in 37% women during lactobacilli treatment compared to 13% on placebo (P=0.02). Lactobacilli were detected in more women in the lactobacilli-treated group than in the placebo group at 28 day (P=0.08) and 60 day (P=0.05) test points. Culture findings confirmed a significant increase in vaginal lactobacilli at day 28 and 60, a significant depletion in yeast at day 28 and a significant reduction in coliforms at day 28, 60 and 90 for lactobacilli-treated subjects versus controls. The combination of probiotic L. rhamnosus GR-1 and L. fermentum RC-14 is not only safe for daily use in healthy women, but it can reduce colonization of the vagina by potential pathogenic bacteria and yeast.

Probiotics to prevent urinary tract infections: the rationale and evidence

For over 30 years, urologists have recognized in females, that urinary pathogens almost always infect the host through ascension from the rectum, vagina to the urethra and bladder. Likewise, the Lactobacillus organisms that predominate in the vagina of healthy women, spread from the rectum and perineum and form a barrier in the vagina to bladder entry by uropathogens. The concept of artificially boosting the lactobacilli numbers through probiotic instillation has long been conceived, but only in recent years shown to be possible. Not all lactobacilli are effective, and to date clinical efficacy only exists for Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri B-54 and RC-14. These strains are only commercially available in Austria, and therefore for most urologists, while some probiotic organisms may reduce the recurrences of bladder cancer or oxaluria, no probiotics can be recommended widely to prevent UTI at present.

Selection of Lactobacillus Strains for Urogenital Probiotic Applications

Almost 20 years ago, it was shown that lactobacilli expressed properties that appeared to play a role in protecting the host from urogenital infection [1]. Since then, studies have shown that exogenously applied lactobacilli or indigenous lactobacilli, can reduce the risk of urinary tract infection (UTI) [2-4]. The investigations have focused upon the large clinical problem of lactobacilli depletion being associated with UTI, bacterial vaginosis (BV), and yeast vaginitis, which together cause an estimated one billion episodes of disease among women each year. The application of exogenous organisms, such as lactobacilli, to the host is termed probiotics, which is broadly defined as a living microorganism administered to promote the health of the host by treating or preventing disease. While many so-called probiotic products are available, the content and viability of most are unreliable [5, 6], leading to use of strains that do not colonize and therefore do not protect against recurrent UTI [7]. For probiotic applications to the urogenital tract to be successful, it is critical that a scientific basis be established for selection of strains. This should at least shed some light upon their mechanism of action in vivo. Several properties appear to be critical, namely the ability to colonize the host, to inhibit pathogen binding and growth, and to create a balanced flora that resists spermicidal killing [8-10].

Yogurt containing probiotic Lactobacillus rhamnosus GR-1 and L. reuteri RC-14 helps resolve moderate diarrhea and increases CD4 count in HIV/AIDS patients.

HIV/AIDS is changing the human landscape in sub-Saharan Africa. Relatively few patients receive antiretroviral therapy, and many suffer from debilitating diarrhea that affects their quality of life. Given the track record of probiotics to alleviate diarrhea, conventional yogurt fermented with Lactobacillus delbruekii var bulgaricus and Streptococcus thermophilus was supplemented with probiotic Lactobacillus rhamnosus GR-1 and L. reuteri RC-14. Twenty-four HIV/AIDS adult female patients (18 to 44 y) with clinical signs of moderate diarrhea, CD4 counts over 200, and not receiving antiretrovirals or dietary supplements, consumed either 100 mL supplemented or unsupplemented yogurt per day for 15 days. Hematologic profiles, CD4 cell counts, and quality of life was evaluated at baseline, 15 and 30 days postprobiotic-yogurt feeding. There was no significant alteration in the hematologic parameters of both groups before and after the probiotic-yogurt feeding. The probiotic yogurt group at baseline, 15 and 30 days had a mean WBC count of 5.8±0.76×109/L, 6.0±1.02×109/L, and 5.4±0.14×109/L, respectively. However, the mean CD4 cell count remained the same or increased at 15 and 30 days in 11/12 probiotic-treated subjects compared to 3/12 in the control. Diarrhea, flatulence, and nausea resolved in 12/12 probiotic-treated subjects within 2 days, compared to 2/12 receiving yogurt for 15 days. This is the first study to show the benefits of probiotic yogurt on quality of life of women in Nigeria with HIV/AIDS, and suggests that perhaps a simple fermented food can provide some relief in the management of the AIDS epidemic in Africa.

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.

Adherence of cervical, vaginal and distal urethral normal microbial flora to human uroepithelial cells and the inhibition of adherence of gram-negative uropathogens by competitive exclusion

The adherence of the normal flora to human uroepithelial cells was examined using scanning and transmission electron microscopy and an in vitro adhesion assay. The normal flora were isolated from human cervical, vaginal and distal urethral surfaces. Human uroepithelial cells were obtained from healthy females (controls) and from female patients with recurrent urinary tract infection. The results indicate that: 1) there was no significant difference between the mean adherence of the normal flora to the uroepithelial cells from controls or patients, but there were quantitative differences between different bacterial species of the normal flora attaching to uroepithelial cells; 2) bacterial species isolated from the distal urethral and vaginal surfaces attached in larger numbers to uroepithelial cells from controls than from patients at day 10 of the menstrual cycle (peak value of adherence); 3) the adherence patterns of the cervical and vaginal microbial flora were bicyclic and appeared to be related to the human menstrual cycle; 4) complete or partial inhibition of adherence of several Gram-negative uropathogens was achieved by preincubating the uroepithelial cells with several bacterial species of the normal flora. Results from this study suggest that the normal flora of the urinary tract may play an important protective role against attachment of uropathogens to the surfaces of uroepithelial cells, and that the blocking capacity of these organisms may vary over the menstrual cycle.

Persistence of Lactobacillus fermentum RC-14 and Lactobacillus rhamnosus GR-1 but Not L. rhamnosus GG in the Human Vagina as Demonstrated by Randomly Amplified Polymorphic DNA

ABSTRACT Lactobacillus rhamnosus GR-1 and L. fermentum RC-14 are well-characterized probiotic strains with efficacy in the prevention and treatment of urogenital infections in women. The aim of the present study was to apply a molecular biology-based methodology for the detection of these strains and L. rhamnosus GG (a commercially available intestinal probiotic) in the human vagina in order to assess probiotic persistence at this site. Ten healthy women inserted vaginally a capsule containing either a combination of strains GR-1 and RC-14 or the GG strain for 3 consecutive nights. Vaginal swabs taken before and at various time points after probiotic insertion were analyzed, and the Lactobacillus flora was assessed by randomly amplified polymorphic DNA (RAPD) analysis. This method generated discrete DNA fingerprints for GR-1, RC-14, and GG and enabled successful detection of these strains in the vagina. Strain GR-1 and/or strain RC-14 was found to persist in the vaginal tract for up to 19 days after vaginal instillation, while L. rhamnosus GG was detectable for up to 5 days postadministration. In conclusion, the fates of probiotic L. rhamnosus and L. fermentum strains were successfully monitored in the human vagina by RAPD analysis. This technique provides molecular biology-based evidence that RC-14 and GR-1, strains selected as urogenital probiotics, persist in the human vagina and may be more suited to vaginal colonization than L. rhamnosus GG. This highlights the importance of proper selection of strains for urogenital probiotic applications.

Oral Administration of the Probiotic Combination Lactobacillus Rhamnosus GR-1 and L. Fermentum RC-14 for Human Intestinal Applications

Lactobacillus rhamnosus GR-1 and L. fermentum RC-14, previously characterized as urogenital probiotics were evaluated for human intestinal applications. RC-14 and GR-1 were tolerant to 0.3 and 0.5% (w/v) bile, respectively. Both strains were suspended in skim milk, stored as a frozen concentrate and administered in combination to five healthy women twice daily for 14 days. Faecal samples were analyzed and the Lactobacillus flora assessed by Randomly Amplified Polymorphic DNA (RAPD). Both strains were recovered from all subjects during the 14-day administration period and GR-1 was detected for at least 7 days post-administration in some individuals. No notable increases in serum IgG, IgA or IgM were observed and IL-2 and IL-4 were undetectable. Although IL6 and IFN-g levels increased slightly in some individuals, concentrations remained within normal ranges. Thus, L. rhamnosus GR-1 and L. fermentum RC-14 are bile tolerant and survive gastrointestinal transit without induction of systemic immune or inflammatory responses. These data together with the previously demonstrated probiotic properties of GR-1 and RC-14 make this strain combination potentially useful for human intestinal applications. r 2002 Elsevier Science Ltd. All rights reserved.

Nucleic Acid-Based Diagnosis of Bacterial Vaginosis and Improved Management Using Probiotic Lactobacilli

Bacterial vaginosis (BV) is a common condition in women that represents an imbalance of the vaginal microflora, lactobacilli depletion, and excess growth of mainly anaerobic Gram-negative pathogens. Diagnosis is made using a series of tests or a Gram stain of a vaginal smear. Treatment with antibiotics is quite effective, but recurrences are common. A study of 55 vaginal samples from 11 postmenopausal women showed the presence of BV by the Gram stain-based Nugent scoring system, and polymerase chain reaction-denaturing gradient gel electrophoresis showed that Bacteroides or Prevotella species were the most common isolates recovered (24 of 25), with Escherichia coli, Staphylococcus aureus, and Streptococcus agalactiae also found in some samples. In one case, only Gardnerella vaginalis was found. These findings illustrate that BV remains common even among otherwise healthy women, but it is not caused solely by either Gardnerella or Mobiluncus. Use of a FemExam system (Cooper Surgical, Shelton, CT), based upon elevated pH and trimethylamine levels, to screen vaginal smears from 59 healthy women showed poor correlation with the Gram stain method. A randomized, placebo-controlled trial of these subjects showed that the lactobacilli-dominant microbiota was restored in subjects with BV but not in controls, following 2 months of daily oral intake of Lactobacillus rhamnosus GR-1 and Lactobacillus fermentum RC-14. These studies show that nucleic acid-based methods are effective at identifying bacteria responsible for BV. If such methods could be used to develop a commercially available, self-use kit, women would be much better placed to take control of their own health, for example, using medicinal food or dietary supplement products such as the clinically proven probiotic strains L. rhamnosus GR-1 and L. fermentum RC-14.

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.