Virginia L. Thomas

Antibody-Coated Bacteria in the Urine and the Site of Urinary-Tract Infection

Abstract An immunofluorescence test for the detection of antibody-coated bacteria in urinary sediments of patients with urinary-tract infections was studied for its predictive value in determining the site of infection. Antibody-coated bacteria were observed in urine specimens from 34 of 35 patients with pyelonephritis; they were not observed in urines from 19 of 20 patients with cystitis. Most of the patients (20 of 28) with antibody-coated bacteria in the urine had high serum antibody titers against their own infecting bacteria. These results suggest that the immunofluorescence test can be useful in distinguishing infection of the kidney from infection of the bladder. (N Engl J Med 290:588–590, 1974)

The Treatment of Urinary Tract Infections in Women with Diabetes Mellitus

Forty-five women with diabetes mellitus and urinary tract infections have been followed an average of 34 mo on treatment protocols based on localization of infection as determined by the presence or absence of antibody-coated bacteria (ACB). Treatment was usually, but not exclusively, trimethoprim-sulfamethoxazole. Two weeks of oral therapy was equally efficacious to 6 wk of treatment in asymptomatic women with antibody-coated bacteria (ACB)-positive infection in eradicating bacteriuria. Recurrences in all groups were predominantly reinfections with differing serotypes or species of microorganisms. The sustained remission rate (fractional extraction) after initial treatment was similar to other reported groups, but possibly less efficacious with recurrences. Suppressive therapy with trimethoprim-sulfamethoxazole for repeated recurrences effectively prevented infection but provided no posttreatment benefit. A high prevalence of underlying structural genitourinary tract abnormalities, usually detectable on pelvic examination, and which were not direct consequences of diabetes mellitus, were possible contributing factors to recurrent infection in this patient group. Progressive elevation in serum creatinine in seven patients with initial ACB-positive infections appeared to relate more closely to diabetic nephropathy rather than chronic pyelonephritis. ACB-positivity correlated well with elevated serum antibody titers and the presence of underlying anatomic abnormalities, but ACB categorization did not lead to improved therapeutic strategy or outcome and hence was of limited clinical usefulness.

A dual fluorescence technique for visualization of Staphylococcus epidermidis biofilm using scanning confocal laser microscopy.

A new dual fluorescence technique is described which, when combined with scanning confocal laser microscopy (SCLM), can be used to visualize the components of biofilm produced byStaphylococcus epidermidis. Chemostat cultures of RP62A (a well-characterized slime-producing strain ofS. epidermidis) were used to produce mature biofilm on polyvinylcholoride (PVC) disks immobilized in a modified Robbins device using a ‘seed’ and ‘feed’ model system. Serial horizontal and vertical optical thin sections, as well as three-dimensional computer reconstructions, were obtained onin situ biofilm using the dual fluorescence procedure. Bacteria were visualized by green autofluorescence excited at 488 nm with an Argon laser. Cell-associated and exocellular matrix material (slime) was visualized by red fluorescence excited at 568 nm with a Krypton laser after interaction of the biofilm with Texas Red-labeled wheat germ agglutinin which is a slime-specific lectin marker. Structural analysis revealed that the cocci grew in slime-embedded cell clusters forming distinct conical-shaped microcolonies. Interspersed open channels served to connect the bulk liquid with the deepest layers of the mature, hydrated biofilm which increased overall surface area and likely facilitated the exchange of nutrients and waste products throughout the biofilm. The combined dual fluorescence technique and SCLM is potentially useful as a specific noninvasive tool for studying the effect of antimicrobial agents on the process of biofilm formation and for the characterization of the architecture ofS. epidermidis biofilm formedin vivo andin vitro on medical grade virgin or modified inert polymer surfaces.

Enzyme-Linked Lectinsorbent Assay Measures N-Acetyl-D-Glucosamine in Matrix of Biofilm Produced by Staphylococcus epidermidis

Abstract. An enzyme-linked lectinsorbent assay (ELLA) was developed for quantification of in situ biofilm produced by Staphylococcus epidermidis in polystyrene 96-well tissue culture plates with phosphatase-labeled wheat germ agglutinin (WGA) as a specific probe for the GlcNAcβ-1,4n component of exocellular matrix material (EMM) that is responsible for intercellular adhesion and accumulation. The ELLA and the modified Christensen dye assay were used to test 13 laboratory strains of coagulase-negative staphylococci and 10 clinical isolates of S. epidermidis. Four biofilm-positive laboratory strains of S. epidermidis were positive by both tests, and six biofilm-negative strains were negative by both. One strain of S. hemolyticus was positive by the ELLA only. Two of the 10 clinical isolates of S. epidermidis were positive by both assays, two were negative by both, and the remaining were positive by the ELLA only. The ELLA was objective, reproducible, specific, sensitive, and useful for screening strains for their capacity to adhere to plastic, produce EMM, and form biofilm.

Lectin-biotin assay for slime present in in situ biofilm produced by Staphylococcus epidermidis using transmission electron microscopy (TEM).

A lectin-biotin assay was developed for use in the specific detection of slime produced byStaphylococcus epidermidis RP62A and M187sp11 grown in a chemically defined medium. Mature biofilm was formed on polyvinylchloride (PVC) disks using a combined chemostat-modified Robbins device (MRD) model system. Specimens fixedin situ were: 1) stained with ruthenium red; 2) reacted overnight with biotin-labeled lectins (WGA, succinyl-WGA, Con A, or APA) followed by treatment with gold-labeled extravidin; or 3) reacted with antibodies againstS. epidermidis RP62A capsular polysaccharide/adhesin (PS/A) using an immunogold procedure. WGA and succinyl-WGA (S-WGA), which specifically bindN-acetylglucosamine, were shown by TEM to react only with slime, both cell-associated and exocellular. In contrast, Con A, APA and anti-PS/A reacted with the bacterial cell surface but did not react with slime. These results indicate the usefulness of WGA lectin as a specific marker for detection of the presence and distribution of slime matrix material inS. epidermidis biofilm.

Calcium- and mucin-binding proteins of staphylococci

The association of staphylococci with the mucus gel that overlays the mucosa of the respiratory tract may lead to clearance of cocci or, in certain conditions such as cystic fibrosis (CF), to colonization. In the present study, a quantitative radioassay was used to study the effect of Ca2+, which is elevated in CF sputa, on the adhesion of 3H-labelled Staphylococcus aureus to submaxillary gland mucin immobilized in MaxiSorp 96-well, break-apart modules. Ca2+ significantly enhanced the adhesion of S. aureus (five strains) and Staphylococcus epidermidis (four strains). The reaction was specific because adhesion was not enhanced in the presence of Mg2+, Ca(2+) + EGTA (a Ca2+ chelator) or protamine and was not attributable to hydrophobicity of the test strains. Staphylococcal adhesion was significantly (P < or = 0.005) blocked in the presence of highly sialated and sulphated reagents, which suggests that Ca2+ binds to the sialic acid and sulphate residues of immobilized mucin. The Ca(2+)-binding sites on the surface of S. aureus were trypsin-sensitive; in addition, 125I-labelled solubilized S. aureus surface proteins reacted with immobilized mucin in a direct binding assay, and the reaction was significantly enhanced by Ca2+. Autoradiography demonstrated that 45Ca bound directly to two polypeptides (M(r) 170,000 and 150,000) of solubilized staphylococcal surface proteins separated by SDS-PAGE, and that 125I-labelled mucin bound directly to three staphylococcal polypeptides (M(r) 40,000, 35,000, and 29,000). These results suggest that S. aureus adhesion to mucin is mediated by at least two mechanisms: via Ca(2+)-binding surface proteins in the presence of Ca2+ and via mucin-binding surface proteins unrelated to Ca2+.

Immunoglobulin levels and antibody-coated bacteria in urines from patients with urinary tract infections.

Summary The presence of antibody-coated bacteria in urines from patients with urinary tract infections has previously been reported to correlate with renal infection as opposed to bladder infection. Urine specimens from 12 patients with pyelonephritis and 12 patients with cystitis were studied to determine whether the antibody coating the bacteria is associated with elevated urine levels of total protein or of particular classes of immunoglobulins. The classes of antibody bound to the infecting bacteria in urines from the patients with pyelonephritis were compared to the levels of unbound antibody in the urine. Each specimen was found to contain antibody-coated bacteria, but not all of the specimens had elevated levels of total protein or immunoglobulins. Thus, the occurrence of antibody-coated bacteria in pyelonephritis did not depend on marked elevations of total urinary protein or immunoglobulins. Studies of patients with cystitis showed that immunoglobulins and protein present in the urines, even in elevated quantities, did not react with the infecting bacteria in patients with bladder infections, as each of these patients had negative FA tests for antibody-coated bacteria.

A comparison of two indirect methods for localizing the site of urinary tract infection: Beta glucuronidase levels and the presence of antibody-coated bacteria☆

A comparison of two of the indirect methods used for localization of the site of urinary tract infection was made for 218 obstetric patients. There was no correlation between urinary beta glucuronidase activity and the presence of a positive fluorescent antibody test. The ranges of beta glucuronidase activity were so variable that this test could not differentiate between the presence of renal, bladder, or absence of infection.

Relationships Between Human Blood Groups, Bacterial Pathogens, and Urinary Tract Infections

Blood groups of 137 patients with acute pyelonephritis and chronic upper tract infection, cystitis, and asymptomatic bacteriuria were compared with those of a normal uninfected control population. In addition, the identified uropathogens were categorized according to the patients blood group. There was a significant association between the diagnosis of chronic upper tract infection and blood group B as compared with controls (p = <0.05, χ2). Analysis of the bacterial isolates showed that more patients with blood group B had infections with Pseudomonas sp., Klebsiella pneumoniae, and Proteus sp. than was expected; and fewer patients with blood group A had infections with Psuedomonas than predicted (p = <0.05, χ2). There was an increased number of patients in blood group AB with infections caused by Escherichia coli and Klebsiella pneumoniae. These results suggest that an individuals blood group may be a significant factor in the host–response to bacterial invasion and influence the development of infection with certain gram-negative bacilli.

In Vivo Transfer of an R-Plasmid in a Urinary Tract Infection Model

AbstractWith the increased use of antibiotics in urinary tract infections, the appearance of resistant organisms. We demonstrated transfer of an R-factor in vitro at a transfer frequency rate of 0.5 to 1.0 × 10−7 and in vivo rate of 5 × 10−2. When urine was used instead of nutrient broth as the medium for in vitro transfer, recombinants were not recovered. Transfer of R-plasmids between place in the intestine and possibly elsewhere, depending on a number of factors. This mechanism is thought to be responsible for hospital and community outbreaks of infections with resistant organisms. We demonstrated transfer of an R-factor in vitro at a transfer frequency rate of 0.5 to 1.0 × 10−7 and in vivo rate of 5 × 10−2. When urine was used instead of nutrient broth as the medium for in vitro transfer, recombinants were not recovered. Transfer of R-plasmids between bacterial strains is thought to be the primary mechanism by which antibiotic resistance has flourished in bacterial populations and the finding of trans...