Gerald J. Domingue

Dormant Microbes in Interstitial Cystitis

Interstitial cystitis (IC) is an inflammatory disease of the urinary bladder that has no known etiology. A microbial association with this disease has not been supported since routine cultures of urine from IC patients are usually negative. However, we have demonstrated the presence of bacterial 16S rRNA genes in bladder biopsies from 29% of patients with IC, but not from control patients with other urological diseases. The ability to identify the presence of bacterial DNA in these patients was accomplished using a sensitive and specific nested PCR method capable of amplifying 16S rRNA genes from a wide variety of bacterial genera. Cloning and sequencing of 16S rRNA gene fragments amplified from bladder tissue of IC patients showed that these genes were derived from genera representing Gram-negative bacteria. In addition to the molecular data, a novel finding of 0.22 micron. filterable forms has been isolated in culture from the biopsy tissue of 14 of 14 IC patients and from 1 of 15 controls. The forms contain nucleic acids and resemble cell wall-deficient bacteria in gross morphology; however, their swirled myelin-like ultrastructure is unusual and suggests a heretofore unclassified microbe. These results demonstrate for the first time an association of Gram-negative bacterial DNA and filterable forms with affected bladder tissue from patients with IC.

Pathogenic Significance of P-Fimbriated Escherichia Coli in Urinary Tract Infections

The over-all aim of this study was to determine the pathogenic significance, and bacteriological and serological characteristics of P-fimbriated organisms isolated from a general population of patients with bacteriuria. A P-receptor specific particle agglutination test was used to identify P-fimbriated bacteria among 2,010 isolates from male and female patients with bacteriuria (age range infancy to 91 years). Of the 2,010 isolates 206 (10.2 per cent) were positive for P-fimbriae by the P-receptor specific particle agglutination test. Only Escherichia coli was found to be P-fimbriated, with an incidence of 21.5 per cent among 956 Escherichia coli isolates. The critical characteristic of pyelonephritic strains of Escherichia coli was P-fimbriation. In cases of nonobstructive acute pyelonephritis 100 per cent of the infecting bacteria were P-fimbriated. The data indicated clearly that the serotype, biotype, presence of type 1 fimbriae (mannose sensitive), undefined mannose-resistant adhesions, hemolysin production and motility of P-fimbriated Escherichia coli were clinically unimportant differential strain characteristics and not indicative of the virulence of P-fimbriated Escherichia coli within clinical syndromes. Isogenic P-fimbriated Escherichia coli strains were isolated from noncompromised patients in all clinical categories, that is pyelonephritis, asymptomatic bacteriuria and cystitis. A variety of bacterial strains appears to be capable of causing acute pyelonephritis in the presence of obstructive uropathic conditions, regardless of P-fimbriation. Therefore, P-fimbriation becomes a noncritical factor in compromised patients. The P-receptor specific particle agglutination test is a simple and rapid method to determine whether bacteria are P-fimbriated and may be an important screening method to identify those bacteria isolated from individuals at risk for nonobstructive acute pyelonephritis.

Culture of seminal fluid in a fertility clinic.

To determine whether the culture of seminal fluid in a fertility clinic is of importance, bacterial cultures were obtained in a consecutive series of 96 patients. Routine bacteriologic cultures were performed within 1 hour of collection. Ureaplasma urealyticum (T-mycoplasma) cultures were also obtained in the last 31 of the patients in this series. Of these 96 patients, 11% showed significant bacterial growth (greater than 10(4) colonies/ml) in the semen specimens, 8% in those patients with normal semen analyses and 14% in those with abnormal analyses. Fifty-eight per cent of semen specimens were positive for U. urealyticum. In reference to normal and abnormal semen analyses the distribution was the same regardless of the presence or absence of U. urealyticum. Antibiotic treatment resulted in minor changes in motility and morphology in a few patients despite conversion to a negative culture. Cultures were also coordinated with microscopic urinalysis and the presence of white blood cells or bacteria in stained smears of semen. There were no significant differences between groups with positive or negative cultures. Trichomonas vaginalis was not seen.

Bacterial infection in prostatodynia

PURPOSE We investigated a possible bacterial etiology for prostatodynia. MATERIALS AND METHODS We evaluated segmented urine specimens from 22 patients and 16 controls by bacteriological localization studies. Immunological studies were performed on patient and control sera. RESULTS Nine patients had positive cultures from prostatic secretions. When compared to controls, this novel finding was statistically significant (p < 0.025). Coagulase-negative staphylococci were the most common isolates (68%). No humoral (IgG) immune differences were found between patients and controls. CONCLUSIONS In a subset of prostatodynia patients bacteria may have an etiological role. Antibiotic treatment demonstrated clinical efficacy.

Outer Membrane Proteins of E. Coli in the Host-Pathogen Interaction in Urinary Tract Infection

Outer membrane protein patterns (Omp) of Escherichia coli obtained directly from the urine of bacteriuric patients without passage on artificial culture media (ACM) were studied by polyacrylamide gel electrophoresis (SDS-PAGE) in an effort to determine whether in vivo conditions of growth affected the expression of these bacterial surface structures. Seventeen strains studied showed two distinct Omp patterns: one protein band appeared at the level of porin proteins (40 kDa) in both patterns, but Omp A protein was at the level of 36 kDa in the first pattern and a new protein was observed at 21.5 kDa in the second pattern suggesting that it is a fragment of Omp A. High molecular weight proteins were also observed in most of the strains and this finding was related to lack of free iron when the same strains were grown under iron restricted conditions in vitro. The same strains grown in pooled urine from normal females showed the first pattern mentioned above. Comparative growth on ACM of urinary strains and E. coli strains isolated from blood, feces and wounds showed an increase in the number of porins expressed (from 1 to 2 or 3, with some variability observed between strains). Differences in osmolality between pooled urine and ACM used, plus in vitro studies varying the osmolality of culture media, showed that osmolality accounted for differences in the number of porins expressed: porin expression decreased in urine the ACM of high osmolality, suggesting that the same phenomena occurred in vivo. It is concluded that host factors including low availability of iron and high osmolality present in the urinary tract influence the expression of several E. coli surface proteins. These proteins may relate to the ability of E. coli to colonize and invade the urinary tract by regulating the physiologic and/or metabolic state of the bacterial cell favoring survival of the organism in a hostile environment. Specific immune responses directed against porins could influence the outcome of this host-parasite interaction.

Cell Wall Deficient Bacteria as a Cause of Idiopathic Hematuria

Idiopathic hematuria in the absence of bacteriuria is a medical challenge. Routine cultures of catheterized bladder and endoscopically obtained ureteral urine specimens from a 22-year-old woman with a 6-week history of hematuria showed no growth after 24 to 48 hours of incubation. However, bacterial variants were grown on enriched media. Colonies were typical cell wall deficient/defective bacteria. Phase and electron microscopy of cystoscopic urine specimens obtained by retrograde ureteral catheterization as well as phase microscopy of the cultures revealed the classic morphology of these organisms. When the variant cultures were subcultured the organisms reverted to their related walled forms, that is Streptococcus agalactiae and Staphylococcus haemolyticus. Because the colonies of these organisms showed various patterns of biochemical reactivity, each phenotype was tested against 15 antimicrobials. Collectively, all biotypes had a common susceptibility to only nitrofurantoin. The patient was treated with nitrofurantoin for 6 weeks. Four days after initiation of therapy she had complete remission of hematuria. During the next 3 years she remained well and free of hematuria.

Vasectomy in rhesus monkeys I. Surgical techniques and gross observations

Vasectomy and vaso-occlusion techniques were used in 47 male rhesus monkeys to maximize and minimize the amount of sperm allowed to escape from the vas into surrounding tissues for up to seventy-two weeks postoperatively. Body weight changes and blood clinical data indicated that the general health of all the monkeys remained good. Normal seasonal changes in body weights and testicular volumes suggested that there were no disturbances to the endocrine system and that the monkeys remained responsive to seasonal environmental stimuli. Vasectomy appears to cause no short-term deleterious effects in the rhesus monkeys, based on observations made during the seventy-two weeks that these monkeys were study after vasectomy. This conclusion agrees with the findings of other investigators.

Vasectomy in rhesus monkeys II. Failure to demonstrate humoral and cellular immune responses specific for sperm

Abstract A study designed to determine whether humoral antibodies and a cell-mediated immune response to sperm antigen(s) in rhesus monkeys after unilateral and bilateral vas ligations has revealed negative findings. Kibrick agglutination, indirect hemagglutination, and sperm immobilization tests on pre- and postvasectomy sera obtained at timed intervals of one to one hundred and two weeks indicated that significant sperm agglutinating, hemagglutinating, and immobilizing antibodies did not develop in these animals. Also, lymphocyte transformation studies showed that a significant cellular antibody response to sperm antigen(s) did not develop in vasectomized animals. Furthermore, sera from rhesus monkeys injected with autologous and homologous washed sperm in Freunds adjuvant failed to demonstrate a positive reaction in agglutination and immobilization tests. On the other hand, immunization of a female rhesus monkey with washed sperm in Freunds adjuvant produced high titer sperm agglutinating and immobilizing antibodies. Therefore, it has not been possible to repeat or contribute supporting data to literature reports concerning the presence of antibodies to sperm in vasectomized rhesus monkeys. It is further suggested that a number of antigens unrelated to sperm might react with sera obtained from vasectomized animals and lead to positive findings in sperm agglutination and immobilization tests. As a result, erroneous conclusions may be drawn about the development of antibodies specific for sperm in vasectomized rhesus monkeys. Definitive conclusions concerning the development of specific antibodies to sperm antigens after vasectomy will be possible only when sperm antigens have been isolated, purified, and chemically and immunologically characterized for use in serologic tests.

Effects of immunization with ethanol-soluble enterobacterial common antigen on in vivo bacterial clearance and hematogenous pyelonephritis.

Representatives of all genera within the Family Enterobacteriaceae share a common antigen (CA) which was described by Kunin, Beard and Halmagyi in 1962 (1) and which could be demonstrated by the indirect hemagglutination test. It is difficult to correlate these initial findings with some of the work which followed, for in their attempts to purify and characterize chemically this CA, the final product obtained failed to modify erythrocytes for hemagglutination. Subsequent work by Whang and Neter (2) showed that antigenic material in the heat-killed supernatant material derived from various enteric bacteria could be attached to erythrocytes which there-by became agglutinable in the presence of homologous as well as heterologous anti-CA sera. Furthermore, hemolysis could be demonstrated in the presence of complement, but neither bacterial agglutination nor precipitation could be shown in the presence of antibodies versus CA. Additional studies showed that the O antigen interfered with the antigenicity of CA and this led to ethanol fractionation to separate O from CA, which renders CA antigenic (3). Studies on the biological significance of this complex antigen-antibody system via phagocytic experimentation revealed that CA antibodies opsonize enteric bacteria and also modified latex particles coated with this antigen (4, 5), suggesting that antibodies against CA might play a role in protection. Gorzyn-ski, Ambrus and Neter (6) showed that passive immunization resulted in slight transient protection of mice against experimental Salmonella infection. Domingue et al. (7) reported that immunization with the heat-killed supernatant CA followed by ethanol-soluble CA protected rabbits against experimental hematogenous and retrograde pyelonephritis. Although specificity of protective activity was shown in the pyelonephritic experiments, the vaccine (heat-killed supernatant) contained endotoxin. Because of the nonspecific protective action of endotoxin, it was therefore deemed necessary to evaluate the role of immunization with the ethanol-soluble fraction CA only, and unrelated to priming with the supernatant material derived from boiling bacterial cells for 1 hr.

ANTIBODIES TO ESCHERICHIA COLI 06 PORINS CROSS-REACT WITH URINARY PATHOGENS

Antibodies to partially purified E. coli 06 35-40 KDa porin trimers recognized the reactive epitopes in the intact porin surface molecule present in various wild-type, heterologous, urinary pathogens. The presence of lipopolysaccharide in the membrane did not shield the antibody binding sites. The reactivity was shown to be specific for porins since LPS-absorbed porin antisera reacted with porins on immunoblots and showed no reactivity with LPS. Additionally, the cross-reactions were abolished by absorption of the porin antisera with E. coli 06 containing porin trimers. These data strengthen the rationale for exploring the enhancement of immunoprotection by monoclonal antibodies to specific immunoreactive antigens in the porin molecule.