Intracellular bacterial communities of uropathogenic Escherichia coli in urinary tract pathogenesis

Urinary tract infections in young, healthy women frequently recur, despite their traditional classification as acute infections. Conventional wisdom dictates that uropathogens causing recurrent infections in such individuals come from the fecal or vaginal flora, in the same manner as the initial infection. However, recent studies of uropathogenic Escherichia coli have found that it can carry out a complex developmental program within the superficial epithelial cells of the mouse bladder, forming intracellular bacterial communities with many biofilm-like properties. These intracellular biofilms allow the bacteria to outlast a strong host immune response to establish a dormant reservoir of pathogens inside the bladder cells. Re-emergence of bacteria from this reservoir might be the source of recurrent infection.     

A fluorescent antibody staining technique to detect bacterial adherence to urinary tract epithelial cells

A fluorescent antibody technique has been devised to assess specifically the adherence of Escherichia coli in vitro to uroepithelial cells from healthy women and bacterial adherence in vivo to cells from women with symptomatic urinary tract infection. Similar values can be obtained using methylene blue as the bacterial stain, but this depends on the experience of the observer. The results indicate that E. coli adherence to uroepithelial cells is a factor in the infection process. We suggest that uroepithelial cells from patients with symptoms of a urinary tract infection whose urine has a low bacterial count (less than 10(3) cells/ml) could be examined for the presence of adherent uropathogens, which may be indicative of an infection. Although the fluorescent staining technique possibly would be expensive, the results would be specific and reliable. Other diagnostic and research applications suggest themselves as in studies of bacterial colonization of mucosal tissues or plastic catheters, where conventional light microscopy and radiolabelling methods are not effective.     

Effect of antibiotic treatment on receptivity of uroepithelial cells to uropathogens

The management of female patients with recurrent urinary tract infections still remains a problem, and long-term prophylactic or short intermittent courses of antibiotics are the standard forms of therapy. In this report, 10 patients were examined for the effects of long- and short-term treatment with trimethoprim-sulfamethoxazole (TMP-SMX) antibiotics on the receptivity of uroepithelial cells to bacterial adherence. The urine of all patients was sterile while on antibiotic therapy. Few bacteria were found adherent to the cells from adult patients (group 1, mean age 36 years) on long-term antibiotics, but the cells were highly receptive to uropathogens in vitro, especially for Escherichia coli expressing mannose-resistant adhesins. Controls of age-matched adult females were included and in vitro adherence levels were found to be higher for those women with a history of urinary tract infection compared with those with no past record of infection. In the second group, elderly patients (mean age 87 years) presented with bacteriuria, and their uroepithelial cells were found to be colonized by uropathogens to a significantly greater extent than their controls. The adherent population was reduced during 7-day TMP-SMX antibiotic treatment, but increased posttherapy, particularly in two patients who subsequently became reinfected. The in vitro results showed that uroepithelial cells retain their receptivity to uropathogenic adherence, both during and after treatment. Although antibiotics eradicate uropathogens from the urinary tract, patients remain susceptible to recolonization by uropathogens and are at risk of reinfection after completion of therapy.     

Lactobacillus inhibitor production against Escherichia coli and coaggregation ability with uropathogens

Previous investigations have shown that certain strains of lactobacilli can competitively exclude uropathogens from attaching to uroepithelial cells and from causing urinary tract infection in animals. The finding of an inhibitory effect produced by Lactobacillus casei ssp. rhamnosus GR-1 against the growth of uropathogens was investigated further using two Escherichia coli indicator strains Hu 734 and ATCC 25922. There were two phases to the inhibitor studies. The first one using an agar sandwich technique showed that the inhibitor activity was heat stable and inhibitory to the E. coli. The second phase showed that MRS broth provided optimum lactobacilli growth and inhibitor production. In addition, the inhibition was present under conditions buffering for acid and pH. The data indicated that the inhibitory effect was not due to bacteriophages or hydrogen peroxide. Strain GR-1 was found to coaggregate with E. coli ATCC 25922 in urine, a phenomenon that has not previously been reported for urogenital bacteria. An in vitro assay system was developed to study the coaggregation of various lactobacilli and uropathogens. The results demonstrated that highest coaggregation scores occurred after 4 h incubation at 37 degrees C with lactobacilli and two type-1 fimbriated E. coli strains. Of the nine lactobacilli strains tested, each was found to coaggregate with 2 or more of the 13 uropathogens. The dominance of inhibitor-producing lactobacilli on the urogenital epithelium and the ability of these organisms to interact closely with uropathogens would constitute an important host defense mechanism against infection.     

Lactobacillus rhamnosus L60, a potential probiotic isolated from the human vagina

The vagina has been increasingly viewed as an "ecosystem" whose normal microflora help protect it from invading pathogens, including those that cause urinary tract infections and sexually transmitted diseases. We tested new strains of lactobacilli as potential probiotics for maintenance of urogenital tract health, as well as prevention and therapy of urogenital infections. A strain of lactobacilli isolated from the vagina of nonpregnant, healthy, premenopausal women was identified as Lactobacillus rhamnosus L60 by 16S rDNA sequence homology. L60 was evaluated for antimicrobial activity, in vitro antibiotic resistance, autoaggregation, surface hydrophobicity, co-aggregation with other bacterial species, hydrogen peroxide (H(2)O(2)) production, and bacterial adherence. It displayed a wide spectrum of antimicrobial activity against urogenital pathogens, and resistance to antibiotics commonly prescribed for infections caused by these pathogens. L60 produced H(2)O(2), adhered to vaginal epithelial cells, co-aggregated with Escherichia coli and Candida albicans, and displayed self-aggregation. In view of these characteristics, L60 is considered a potential probiotic, and will be further evaluated for preventive and therapeutic application locally in the vaginal tract.     

Pathogenesis of urinary tract infection in the elderly: the role of bacterial adherence to uroepithelial cells

The in vitro adherence of nine strains of Gram-negative bacteria to uroepithelial cells from 24 women patients (>65 years) was significantly higher than to cells from 24 premenopausal women (18–40 years). Uroepithelial cells from patients with a history of previous urinary tract infection (UTI) were marginally more receptive to attachment of uropathogens than cells from women without a history of UTI, but this was not statistically significant. Serum from four elderly women with asymptomatic bacteriuria was used to stabilize samples for electron-microscopic examination, which showed the presence of fibrous glycocalyx material surrounding the bacteria and attached to the uroepithelial cells. Eighty uropathogenic isolates from elderly and premenopausal women were found to express adhesins, to produce urease and hemolysins, and to ferment sucrose, salicin, and dulcitol. These results suggest that the increased receptivity of uroepithelial cells to bacterial attachment may be a predisposing factor in the onset of UTI in the hospitalized and domiciliary elderly population.     

Activity of cecropin P1 and FA-LL-37 against urogenital microflora

Two mammalian antimicrobial peptides, FA-LL-37 and cecropin P1, were tested for activity against six uropathogens and five Lactobacillus strains by broth microdilution assay. Both peptides inhibited Escherichia coli at 25 microM (FA-LL-39), and 1.56 microM (cecropin P1), Pseudomonas aeruginosa (12.5 microM, and 25 microM), and Klebsiella pneumoniae, (50 microM, and 1.56 microM), but not Enterococcus faecalis and Staphylococcus epidermidis. FA-LL-37 acted bacteriocidally against E. coli and bacteriostatically against the other two Gram-negative organisms. Cecropin P1 was bacteriocidal to all susceptible bacteria. Lactobacilli were resistant to both peptides, with the exception of poultry isolate Lactobacillus fermentum B-54, which was susceptible to FA-LL-37 at 100 microM. The differential activities of these peptides toward Gram-negative uropathogens versus urogenital lactobacilli demonstrate their potential as a topical treatment for urinary tract infections. In addition, production of such peptides in vivo could be a natural mechanism to aid in the maintenance of the lactobacilli-dominated urogenital flora at the expense of pathogens.     

Local and diffuse bacterial adherence on uroepithelial cells

The concept of local and diffuse adherence has been described for enteropathogenicEscherichia coli. In the present study, similar findings are reported for bacterial adherence to uroepithelial cells from patients with acute urinary tract infection and following incubation in an in vitro adherence assay. A population of cells were seen with few or no bacteria attached; others had localized areas of adherent organisms, while some cells were heavily colonized in a diffuse manner. These patterns were noted in vitro for anEscherichia coli strain and aLactobacillus casei strain, which possess different adhesins, therefore indicating that the adherence patterns were probably due to epithelial cell differences. The light microscopy and scanning electron microscopy observations illustrate that bacterial adherence to uroepithelial cells occurs in localized and diffuse distributions. The results indicate that there are differences in uroepithelial cell receptivity for bacterial attachment. The availability of cells receptive to uropathogens and indigenous flora, such as lactobacilli, is probably one of several factors that influence the pathogenesis of urinary tract infections.     

Use of Lactobacillus to prevent infection by pathogenic bacteria

This review focuses on the use and potential of Lactobacillus to prevent infections of the urogenital and intestinal tracts. The presence and dominance of Lactobacillus in the vagina is associated with a reduced risk of bacterial vaginosis and urinary tract infections. The mechanisms appear to involve anti-adhesion factors, by-products such as hydrogen peroxide and bacteriocins lethal to pathogens, and perhaps immune modulation or signaling effects. The instillation of Lactobacillus GR-1 and B-54 or RC-14 strains into the vagina has been shown to reduce the risk of urinary tract infections, and improve the maintenance of a normal flora. Ingestion of these strains into the gut has also been shown to modify the vaginal flora to a more healthy state. In addition, these strains inhibit the growth of intestinal, as well as urogenital pathogens, colonize the gut and protect against infections as shown in mice. Other probiotic strains, such as Lactobacillus GG, have been shown to prevent and treat gastroenteritis caused by rotavirus and bacteria. Given that lactobacilli are not the dominant commensals in a gut which comprises around 10(10) organisms, much work is still needed to define the mechanisms whereby GR-1, RC-14, GG and other strains contribute to health restoration and maintenance. Such critically important studies will require the medical science community to show a willingness to turn away from pharmaceutical remedies as the only solution to health and disease.     

Purification and characterization of a surface-binding protein from Lactobacillus fermentum RC-14 that inhibits adhesion of Enterococcus faecalis 1131

Lactobacilli have been shown to be important in the maintenance of the healthy urogenital flora. One strain, Lactobacillus fermentum RC-14, releases surface-active components which can inhibit adhesion of uropathogenic bacteria. Using a quantitative method for determining inhibition of adhesion, a protein with high anti-adhesive properties against Enterococcus faecalis 1131 was purified. The N-terminal sequence of the 29-kDa protein was identical to that of a collagen-binding protein from Lactobacillus reuteri NCIB 11951, and exhibited close homology with a basic surface protein from L. fermentum BR11. The results suggest that this anti-adhesive cell surface protein of Lactobacillus could protect against uropathogens by preventing their adhesion. the Federation of European Microbiological Societies.     

The presence of bacteria and yeast like fungi in specimens from surgical patients

The aim of this study was to determine the incidence of Candida spp. strains in specimens obtained from surgically treated patients as well as to analyze the accompanying bacterial flora, both aerobic and anaerobic. The material came from two groups of patients. In the first group consisting of patients operated for colon and rectum carcinoma, the samples included peritoneal fluid, colon or rectum bioptates, pus, blood, and wound swabs. In the other group, biopsy material and smears from post operation wounds were taken from patients who underwent a surgical treatment of larynx carcinoma. Altogether, 282 various clinical specimens from 165 patients were analysed, and 41 Candida spp. strains were isolated: 39 strains of C. albicans and 2 strains of C. tropicalis. In 20 out of 41 specimens infected with Candida spp. (48.8%) the co-infection with bacterial aerobic flora was found. In 10 cases (24.4%), the fungi were isolated together with aerobic and anaerobic bacterial flora, whereas in 2 specimens (4.9%) the anaerobes and Candida albicans were diagnosed. The remaining 9 samples showed only the presence of Candida spp. (21.9%). From among aerobic bacterial flora Enterococcus spp. strains (n = 17) and Gram negative rods from Enterobacteriaceae family (n = 13) were the most frequently isolated. The bacterial strains of Streptococcus spp. (n = 5), Pseudomonas spp. (n = 3), Staphylococcus spp. and Corynebacterium spp. (2 strains, both) were identified more rarely. Bacteroides spp. were the most frequent members of bacterial anaerobic flora (n = 10). Other isolated anaerobic bacteria were classified as Fusobacterium spp. or Peptostreptococcus spp. (1 strain each). E. coli and Enterococcus spp. strains of aerobic bacterial flora were more frequently isolated together with Candida spp. CONCLUSIONS: (i) Mixed bacterial flora was found to predominate in the clinical material from the patients after surgery. (ii) Candida spp. were most frequently found together with aerobic bacterial flora.     

Quantitative PCR assays for mouse enteric flora reveal strain-dependent differences in composition that are influenced by the microenvironment

The mammalian gastrointestinal (GI) tract is inhabited by over a hundred species of symbiotic bacteria. Differences among individuals in the composition of the GI flora may contribute to variation in in vivo experimental analyses and disease susceptibility. To investigate potential interindividual differences in GI flora composition, we developed real-time quantitative PCR-based assays for the detection of the eight members of the Altered Schaedler Flora (ASF) as representative members of different bacterial niches within the mammalian GI tract. Quantitative and reproducible strain-specific variations in the numbers of the ASF members were observed across 23 different barrier-housed inbred mouse strains, suggesting that the ASF assays can be used as sentinels for changes in GI flora composition. A significant cage effect was also detected. Isogenic mice that cohabited at weaning, whether from the same or different litters, showed little variation in ASF profiles. Conversely, litters split among different cages at weaning showed divergence in ASF profiles after three weeks. Individual ASF profiles, once established, were highly stable over time in the absence of environmental perturbation. Furthermore, cohabitation of different inbred strains maintained most of the interstrain variation in the GI flora, supporting a role of host genetics in determining GI flora composition.     

老年上呼吸道微生态研究

目的 通过研究健康老年机体上呼吸道微生态菌群构成,了解老龄人群上呼吸道优势菌群种类及数量,为从微生态学角度预防呼吸道感染的发生提供参考。方法 自沈阳市选取年龄为65~70岁健康老人为研究对象,无菌咽拭法采集咽后壁粘膜表面标本,对菌群进行定量分析、定性鉴定。结果 老年人群咽后壁需氧菌群与厌氧菌群数量之比为2.65∶1,检出率较高的需氧菌及厌氧菌包括唾液链球菌、口腔链球菌、缓症链球菌、微黄奈瑟菌、灰色奈瑟菌、麻疹孪生球菌等,检出率较高的菌构成比也较高。结论 健康老年人群中检出率和构成比都较高的需氧菌和厌氧菌共同构成老年人上呼吸道口咽部优势菌群,在维持呼吸系统微生态平衡中起重要作用,可作为上呼吸道微生态学研究的重要指标菌。     

Menstrual Hygiene Practices,WASH Access and the Risk of Urogenital Infection in Women from Odisha,India

Menstrual hygiene management (MHM) practices vary worldwide and depend on the individual’s socioeconomic status, personal preferences, local traditions and beliefs, and access to water and sanitation resources. MHM practices can be particularly unhygienic and inconvenient for girls and women in poorer settings. Little is known about whether unhygienic MHM practices increase a woman’s exposure to urogenital infections, such as bacterial vaginosis (BV) and urinary tract infection (UTI). This study aimed to determine the association of MHM practices with urogenital infections, controlling for environmental drivers. A hospital-based case-control study was conducted on 486 women at Odisha, India. Cases and controls were recruited using a syndromic approach. Vaginal swabs were collected from all the participants and tested for BV status using Amsel’s criteria. Urine samples were cultured to assess UTI status. Socioeconomic status, clinical symptoms and reproductive history, and MHM and water and sanitation practices were obtained by standardised questionnaire. A total of 486 women were recruited to the study, 228 symptomatic cases and 258 asymptomatic controls. Women who used reusable absorbent pads were more likely to have symptoms of urogenital infection (AdjOR=2.3, 95%CI1.5-3.4) or to be diagnosed with at least one urogenital infection (BV or UTI) (AdjOR=2.8, 95%CI1.7-4.5), than women using disposable pads. Increased wealth and space for personal hygiene in the household were protective for BV (AdjOR=0.5, 95%CI0.3-0.9 and AdjOR=0.6, 95%CI0.3-0.9 respectively). Lower education of the participants was the only factor associated with UTI after adjusting for all the confounders (AdjOR=3.1, 95%CI1.2-7.9). Interventions that ensure women have access to private facilities with water for MHM and that educate women about safer, low-cost MHM materials could reduce urogenital disease among women. Further studies of the effects of specific practices for managing hygienically reusable pads and studies to explore other pathogenic reproductive tract infections are needed.     

Characteristic male urine microbiomes associate with asymptomatic sexually transmitted infection

Background

The microbiome of the male urogenital tract is poorly described but it has been suggested that bacterial colonization of the male urethra might impact risk of sexually transmitted infection (STI). Previous cultivation-dependent studies showed that a variety of non-pathogenic bacteria colonize the urethra but did not thoroughly characterize these microbiomes or establish links between the compositions of urethral microbiomes and STI.

Methodology/Findings

Here, we used 16S rRNA PCR and sequencing to identify bacteria in urine specimens collected from men who lacked symptoms of urethral inflammation but who differed in status for STI. All of the urine samples contained multiple bacterial genera and many contained taxa that colonize the human vagina. Uncultivated bacteria associated with female genital tract pathology were abundant in specimens from men who had STI.

Conclusions

Urine microbiomes from men with STI were dominated by fastidious, anaerobic and uncultivated bacteria. The same taxa were rare in STI negative individuals. Our findings suggest that the composition of male urine microbiomes is related to STI.     

Dissemination of yeasts after gastrointestinal inoculation in antibiotic-treated mice

Mice pretreated with antibiotics were inoculated intragastrically with different yeast isolates to determine whether the resulting disruption of the normal flora ecology would allow certain fungi to colonize and disseminate from the gastrointestinal tract. Antibiotic treatment decreased the total population levels of the indigenous bacterial flora, and predisposed mice to gastrointestinal overgrowth and subsequent dissemination by Candida albicans, C. parapsilosis, C. pseudotropicalis, C. tropicalis, and Torulopsis glabrata. A clinical isolate of Rhodotorula rubra, on the other hand, was unable to maintain a stable population in the gut of similar mice and could not be isolated from systemic organs. Control animals not receiving antibiotic therapy, challenged with C. albicans, showed significantly lower gut population levels of yeasts, and Candida organisms could not be grown from visceral organs. It is suggested that suppression of fungi within the gastrointestinal tract by members of the normal bacterial flora may be an important mechanism whereby fungi are confined to the alimentary tract.     

Intravaginal instillation of lactobacilli for prevention of recurrent urinary tract infections

Urinary tract infections remain a common problem, particularly in the female population. New methods are required to manage recurrent cystitis, and extensive research to date has suggested that restoration of the lactobacilli flora of the urogenital tract may prevent these infections. In this study, five females suffering from recurrent urinary tract infections were treated twice weekly with intravaginal and perineal implantation of Lactobacillus casei GR-1. These organisms colonized the epithelium and prevented the emergence of coliform bacteria in most instances, but did not appear to affect enterococcal colonization. In vitro studies showed that L. casei GR-1 inhibited the growth of the coliforms but did not inhibit enterococci. Each of the five patients had infection-free periods ranging from 4 weeks to 6 months. The treatment was well tolerated, had no side effects, led to an improved well-being, and was preferred to antibiotic treatment by all of the patients. These human studies, albeit of a limited nature, are the first to examine the potential for lactobacillus therapy in the prevention of urinary tract infections. The results show that lactobacilli therapy, using carefully selected organisms to treat patients who are closely followed, may be effective in the prevention of recurrent urinary tract infections.     

31例非特异性阴道炎病人阴道微生物群的研究

本文对31例非特异性阴道炎病人和31例正常人阴道微生物群进行定性、定量分析.结果表明,阴道炎病人的阴道乳杆菌的平均浓度明显低于正常人,而肠杆菌科、葡萄球菌、棒状杆菌、类杆菌属、支原体却明显增多,阴道炎病人未发现特异性病原体,菌群失调可能是其发病因素。乳杆菌为阴道正常优势菌,对改善阴道的微环境,防止条件致病菌引起的内源性感染具有重要的生理作用。     

Dynamics of vaginal bacterial communities in women developing bacterial vaginosis, candidiasis, or no infection, analyzed by PCR-denaturing gradient gel electrophoresis and real-time PCR

The microbial flora of the vagina plays a major role in preventing genital infections, including bacterial vaginosis (BV) and candidiasis (CA). An integrated approach based on PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR was used to study the structure and dynamics of bacterial communities in vaginal fluids of healthy women and patients developing BV and CA. Universal eubacterial primers and Lactobacillus genus-specific primers, both targeted at 16S rRNA genes, were used in DGGE and real-time PCR analysis, respectively. The DGGE profiles revealed that the vaginal flora was dominated by Lactobacillus species under healthy conditions, whereas several potentially pathogenic bacteria were present in the flora of women with BV. Lactobacilli were the predominant bacterial population in the vagina for patients affected by CA, but changes in the composition of Lactobacillus species were observed. Real-time PCR analysis allowed the quantitative estimation of variations in lactobacilli associated with BV and CA diseases. A statistically significant decrease in the relative abundance of lactobacilli was found in vaginal fluids of patients with BV compared to the relative abundance of lactobacilli in the vaginal fluids of healthy women and patients with CA.