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J S Knapp

Publications and source records attributed to J S Knapp.

At least 91 records · Page 5Linked to original sources
PubMed1984

Rapid, inexpensive method for specific detection of microbial beta-lactamases by detection of fluorescent end products.

A rapid method was developed for specific detection of microbial beta-lactamases which uses ampicillin and cephalexin as substrates. The end products (open beta-lactam ring forms) generated after separately incubating either substrate with beta-lactamase-producing organisms initially were separated from the unhydrolyzed substrates by high-voltage electrophoresis at pH 2.1. The end products of both antibiotics were highly fluorescent and could be analyzed visually and semiquantitatively under a long-wave UV lamp. Application of 5 microliters of the same incubation mixture onto filter paper without subsequent electrophoretic separation also resulted in development of fluorescence after brief heating at 120 degrees C for 5 min. This spot test differentiates penicillinase activity from cephalosporinase activity and distinguishes between beta-lactamase and acylase activities, since the end products of acylase [the common side chain, D(-)-alpha-aminophenylacetic acid, and the intact beta-lactam nuclei, 6-aminopenicillanic acid and 7-aminodeacetoxycephalosporanic acid] are not fluorescent. This method was relatively rapid, inexpensive, and more sensitive than the chromogenic cephalosporin (nitrocefin) method when 21 strains of 7 gram-positive species and 77 strains of 29 gram-negative species of bacteria were tested.

Amidohydrolases↗
PubMed1983

DNA hybridization technique for the detection of Neisseria gonorrhoeae in men with urethritis.

A technique to detect Neisseria gonorrhoeae directly in clinical specimens was developed using a modified DNA-hybridization method. It uses the gonococcal cryptic plasmid as the radiolabeled probe, can detect as few as 100 colony-forming units of N gonorrhoeae or as little as 0.1 pg of purified gonococcal plasmid DNA, and is highly specific. This technique for differentiating between gonococcal and nongonococcal urethritis was evaluated in men with symptomatic urethritis in Seattle. Sixty-three (89%) of 71 who had cultures positive for N gonorrhoeae were also positive by DNA hybridization, and all 42 whose cultures were negative were also negative by DNA hybridization. Five of six isolates from patients who were positive by culture but negative by hybridization lacked the gonococcal cryptic plasmid and belonged to a unique auxo-type which requires proline, citrulline, and uracil for growth.

DNA, Bacterial↗
PubMed1983

Gonococcal sensitivity to fecal lipids can be mediated by an Mtr-independent mechanism.

Various factors affect the sensitivity of Neisseria gonorrhoeae to physiological levels of hydrophobic molecules. A total of 98 N. gonorrhoeae strains from rectal, cervical, and urethral cultures of homosexual men and heterosexual men and women were examined for their sensitivities to fecal lipids. Isolates were characterized according to cell envelope phenotype, auxotype, and protein I serogroup. Although cell envelope phenotype was an important factor in the resistance of this organism to fecal lipids (Mtr phenotype greater than wild type greater than Env phenotype), other factors were also of importance. AHU- strains (strains having a requirement for arginine, hypoxanthine, and uracil) uniformly exhibited a wild-type envelope phenotype but were as sensitive to fecal lipids as were Env strains. The protein I serogroup was not a factor in determining the sensitivity of wild-type envelope phenotype non-AHU- strains to fecal lipids. However, sexual preference and site of isolation were important factors. Wild-type envelope phenotype (non-AHU-) strains from homosexual men and heterosexual women were more resistant to fecal lipids than were similar isolates from heterosexual men. When these strains were compared by isolation site, it was observed that rectal isolates from homosexual men and heterosexual women were more resistant than were cervical isolates from heterosexual women or urethral isolates from heterosexual men. Urethral isolates from homosexual men were also more resistant to fecal lipids than were urethral isolates from heterosexual men. These data suggest that the host environment can select for increased resistance to hydrophobic molecules by an Mtr-independent mechanism. The basis for this Mtr-independent resistance is presently unknown, but it is likely that it involves an alteration of the target site(s) for fecal lipid inhibition.

Arginine↗
PubMed1983

Modified oxidation-fermentation medium for detection of acid production from carbohydrates by Neisseria spp. and Branhamella catarrhalis.

A modified oxidation-fermentation medium was developed as a practical medium for highly sensitive and specific detection of acid production from carbohydrates by Neisseria spp. and Branhamella catarrhalis. A total of 756 strains representing 17 Neisseria spp. and Branhamella catarrhalis were tested in this medium, in which the protein concentration was reduced relative to the carbohydrate concentration, phenol red was substituted for bromthymol blue at a low concentration, and the initial pH was adjusted to 7.2. Sugar utilization patterns were consistent with published results and with other cultural and biochemical characteristics for these species. The reactions obtained using this medium were qualitatively better and more reproducible than those obtained in cystine-Trypticase agar (BBL Microbiology Systems, Cockeysville, Md.) medium.

Acids↗
PubMed1982

Epidemiology of penicillinase-producing Neisseria gonorrhoeae infections: analysis by auxotyping and serogrouping.

Auxotyping and serogrouping by coagglutination were used to characterize penicillinase-producing Neisseria gonorrhoeae and penicillinase-negative isolates from the state of Washington, Shreveport (Louisiana), and the Far East. Fifty-four of 75 penicillinase-producing isolates (72 per cent) from Washington required proline for growth and were serogroup W-l (Pro-1), the predominant type of penicillinase-producing strains in the Philippines; none of 86 penicillinase-negative isolates from Washington was Pro-1 (P less than 0.0001). All 38 penicillinase-producing isolates from Shreveport required proline and were serogroup W-11 (Pro-11); five of 26 penicillinase-negative isolates (19 per cent) from Shreveport were also Pro-11 (P less than 0.0001) but had antigenic specificities within serogroup W-ll that distinguished them from the penicillinase-producing isolates. We conclude that the Washington and Shreveport outbreaks resulted from the spread of imported strains rather than transmission of penicillinase-encoding plasmids to indigenous gonococci. The Shreveport outbreak involved a single strain of penicillinase-producing N, gonorrhoeae and probably originated from a common source, whereas several types were involved in the multiple-source Washington outbreak, indicating repeated introduction of new strains.

Disease Outbreaks↗
PubMed1982

Serology of Neisseria gonorrhoeae: W-antigen serogrouping by coagglutination and protein I serotyping by enzyme-linked immunosorbent assay both detect protein I antigens.

A total of 224 strains were serogrouped by coagglutination (COA) and serotyped by protein I enzyme-linked immunosorbent assay (ELISA). Of these strains, 61 were from patients with disseminated gonococcal infection, 21 were from patients with pelvic inflammatory disease, and 115 were from patients with uncomplicated gonococcal infection in Singapore, the Philippines, and Denmark. Twenty-seven were laboratory reference strains. Of the patient strains, 102 belonged to COA serogroup WI, and all of the 100 strains that typed with protein I serotypes 1, 2, or 3 were in this group. Most of the strains of gonococci from the 61 patients with disseminated gonococcal infection were within this group (COA WI, 53 or 87%; protein I serotypes 1, 2, or 3, 51 or 84%). All 46 strains that were protein I serotypes 4 through 7 were also COA serogroup WII. Protein I serotypes 8 and 9 accounted for 49 (25%) of the 197 patient strains. Twenty-eight of these strains typed as COA serogroup WII, 20 typed as serogroup WIII, and 1 typed as serogroups WII and WIII. COA W serogrouping and protein I ELISA both appeared to detect antigens on the protein I molecule of the outer membrane of Neisseria gonorrhoeae. Protein I serotyping, which uses unboiled organisms, may generally recognize more variable and surface-exposed antigenic determinants. In contrast, COA W serogrouping, which uses boiled organisms, may recognize less exposed shared antigenic determinants in addition to variable protein I antigenic determinants. Both methods may prove useful for further studies of the epidemiology and pathogenesis of gonorrhea.

Agglutination Tests↗
PubMed1982

Serological classification of Neisseria gonorrhoeae with monoclonal antibodies.

Hybrid cells producing monoclonal antibodies against antigens of Neisseria gonorrhoeae were obtained by the polyethylene glycol-mediated fusion of mouse myeloma cells and lymphocytes from mice immunized with gonococcal protein I or outer membrane proteins. From four fusions, 16 phenotypically stable, independently cloned hybrid cell lines were selected for continued study. Each of the cell lines produced a characteristically different monoclonal antibody which reacted in immunoprecipitation assays with a unique antigenic determinant on protein I of the outer membrane complex of the bacteria. In antibody binding, immunofluorescence, and coagglutination assays these antibodies each reacted with a restricted group of N. gonorrhoeae strains. None of the monoclonal antibodies reacted with 17 other different species of Neisseria or with Branhamella catarrhalis. When tested on 34 N. gonorrhoeae reference serotyping strains, the monoclonal antibodies demonstrated serological relationships between the strains which paralleled those observed with conventional polyvalent antisera. These antibodies now provide standardized reagents for the rapid and precise serological characterization of many strains of N. gonorrhoeae.

Animals↗
PubMed1982

Gonococcal strains from homosexual men have outer membranes with reduced permeability to hydrophobic molecules.

Loci designated penA, penB, and mtr contribute additively to penicillin G resistance in Neisseria gonorrhoeae; the mtr locus also confers resistance to hydrophobic dyes, detergents, and antibiotics, env mutations suppress the phenotypic expression of mtr and penB and are responsible for increased sensitivity to various hydrophobic molecules. We postulated that the host environment is important in the selection of gonococcal strains with these particular outer membrane phenotypes. Thus, mtr strains should predominate in environments that are high in hydrophobic molecules. To test this hypothesis we determined the outer membrane phenotype of 152 strains of N. gonorrhoeae. Rectal and urethral isolates from 58 homosexual men, urethral isolates from 55 heterosexual men, and cervical and rectal isolates from 39 heterosexual women were used in this study. Strains from 43 of the homosexual men were matched with those from heterosexual men with respect to auxotype and year and season of isolation. Cell envelope phenotype was determined for each strain on the basis of its resistance to various hydrophobic compounds. The identity of mtr strains was confirmed by genetic transformation. Among the matched pairs, mtr strains were significantly more prevalent among isolates from homosexual men than among those from heterosexual men (P = 0.03). Serogrouping by coagglutination demonstrated that 17 of 19 mtr strains versus 76 of 131 non-mtr strains belonged to coagglutination group WII (P = 0.01). Coagglutination group WII strains were also associated with homosexuality (P = 0.02). Gonococci were also tested for resistance to fecal lipids, mtr strains were more resistant to growth inhibition by fecal lipids than were non-mtr strains.

Bacterial Outer Membrane Proteins↗
PubMed1982

Rapid method for simultaneous detection of the arginine dihydrolase system and amino acid decarboxylases in microorganisms.

A specific procedure has been developed for the detection of the first two enzymes involved in the arginine dihydrolase system and the detection of the decarboxylases of arginine, glutamic acid, histidine, lysine, ornithine, phenylalanine, tryptophan, and tyrosine. A loopful of growth of each organism from dihydrolase-decarboxylase induction agar medium (or broth) was washed and incubated separately with 0.2-ml samples of three test media supplemented with different amino acids. Each spent test medium was dansylated, and the dansyl derivatives were separated by two-dimensional thin-layer chromatography on polyamide sheets. The end products (citrulline, ornithine, gamma-amino-n-butyric acid, and amines) produced during incubation were estimated by comparing the fluorescent intensities of end products from the spent test media and of the corresponding parent amino acids from test medium controls after thin-layer chromatography. The method is reproducible, requiring incubation of an organism in three test media for 1 h for simultaneous detection of the first two enzymes involved in the arginine dihydrolase system and of eight amino acid decarboxylases. This method has been successfully applied to gram-positive and gram-negative microorganisms and also to Mycoplasmatales. It could simplify and improve the accuracy of the corresponding biochemical tests performed in clinical laboratories for the identification and differentiation of microorganisms, and it may prove particularly useful for the differentiation of species of Pseudomonas and Mycoplasma.

Bacteria↗
PubMed1981

Neisseria gonorrhoeae strains inhibited by vancomycin in selective media and correlation with auxotype.

Strains of Neisseria gonorrhoeae that failed to grow on Thayer-Martin (T-M) and Martin-Lewis (M-L) media accounted for 2.0% of isolates at the University of Colorado Hospital and its Venereal Disease Clinic. A total of 31 inhibited and 31 control strains were compared by agar dilution testing for their susceptibilities to 13 antimicrobial agents used for treatment or in selective media. All 62 isolates were resistant to lincomycin, colistin, nystatin, amphotericin B, trimethoprim lactate, polymyxin B, and anisomycin. Vancomycin was the inhibitory antibiotic for N. gonorrhoeae in both T-M and M-L media. The vancomycin-inhibited strains were also significantly more sensitive to penicillin and ampicillin than were the control strains (P less than 0.001). The presence of the other antibiotics in selective media did not affect the minimum inhibitory concentrations of vancomycin for gonococci. All 31 inhibited strains were sensitive to 8.0 micrograms of vancomycin per ml, and 26 of these were sensitive to 2.0 microgram/ml. Decreasing the size of inoculum of gonococci results in greater inhibition by any given concentration of vancomycin. The vancomycin-sensitive strains contained significantly more arginine- hypoxanthine-, and uracil-requiring auxotypes (28 out of 31) than did the control strains (9 out of 31). As with T-M medium, some strains of gonococci will be missed when M-L medium with 4.0 micrograms of vancomycin per ml is the only medium used for the diagnosis of gonorrhea. This may be of particular importance in the confirmation of disseminated infection with Arg- Hyx- Ura- auxotypes of N. gonorrhoeae when cultures of blood, joint fluid, or skin lesions are negative.

Anti-Bacterial Agents↗
PubMed1980

Gonococcal salpingitis is less likely to recur with Neisseria gonorrhoeae of the same principal outer membrane protein antigenic type.

If protective immunity were to develop following an episode of gonococcal pelvic inflammatory disease (salpingitis), PID should recur with organisms bearing antigens that do not react with these immune mechanisms. To test this hypothesis, gonococci from 15 women with gonococcal PID, who experienced 19 subsequent episodes of gonococcal infection, were serotyped for their principal outer membrane protein (Protein I) antigens. Of nine cases in which the initial and subsequent infections involved the same Protein I serotype, none was associated with recurrence of PID. Of 10 cases in which the initial and subsequent infections involved different Protein I types, five were associated with recurrent PID (p = 0.02). These data suggest that an episode of gonococcal PID produces some immunity to repeated episodes of salpingitis with the same Protein I serotype, while not preventing reinfection with the same Protein I serotype. The immune response to Protein I antigen may thus provide serotype-specific protection against gonococcal salpingitis.

Antigens, Bacterial↗
PubMed1978

Phenotypic and epidemiologic correlates of auxotype in Neisseria gonorrhoeae.

Previous studies from Seattle, Wash., suggested that strains of Neisseria gonorrhoeae which require arginine, hypoxanthine, and uracil (Arg-Hyx-Ura- auxotype) are unifomly highly susceptible to penicillin G, are relatively resistant to complement-dependent killing by heated, pooled human serum, and are associated with disseminated gonococcal infection. For further study of the epidemiology of these strains and for analysis of the susceptibility to penicillin, serum sensitivity, and the nutritional requirements of gonococcal isolates from other cities, a survey was made of urethral and cervical strains isolated in 1972--1974 from 50 randomly selected pateit-s with uncomplicated gonorrhea from each of nine cities. Arg-Hyx-Ura-strains represented greater than 50% of isolates from Seattle and Des Moines, Iowa, 22% of isolates from Denver, Colo., and Dayton, Ohio, and less than or equal to 12% of the isolates from Boston, Mass., Newark, N.J., Norfork, Va., Miami, Fla., and Oakland, Calif. Arg-Hyx-Ura- strains were recovered from 42% of white and 9% of black patients (P less than 0.001), and clincis with the highest incidences of these strains had the highest proportion of white patients among those with gonorrhea. Arg-Hyx-Ura- strains were all susceptible to less than or equal to 0.125 microgram of penicillin G/ml and were more resistant than strains with other auxotypes to killing by heat-inactivated human serum plus complement (P less than 0.001).

Arginine↗
PubMed1977

Asymptomatic gonorrhea in men: caused by gonococci with unique nutritional requirements.

In a retrospective case-control study, gonococci with nutritional requirements for arginine, hypoxanthine, and uracil were recovered from 24 of 25 men with asymptomatic gonorrhea and 10 of 25 men with symptomatic gonorrhea (P = .0001). These strains represent a smaller proportion of gonococcal isolate from blacks than from whites. Asymptomatic urethral infection is important in the epidemiology of gonorrhea, particularly among whites.

Arginine↗
PubMed1977

Immunologic classification of Neisseria gonorrhoeae with micro-immunofluorescence.

A reproducible immunologic classification of Neisseria gonorrhoeae strains has been achieved by the micro-immunofluorescence (Micro-IF)3 method by using formalinized whole organisms as test antigens and mouse antisera prepared by i.v. immunization with the whole organisms as antibody. Immunologic differences among Neisseria species were also distinct in this test system. Immunologic differences among gonococcal strains were not influenced by gonococcal colony type. Classification of gonococci was facilitated by use of antisera absorbed with an antigenically unique gonococcus strain. Of 180 gonococcal strains, 175 could be classified into three immunotypes: A, B, and C. Each type was further divided into subtypes designated A1, A2, A3, B1, B2, B3, C1, and C2. Minor antigenic differences still exist within each subtype. The two gonococcal isolates from each of 17 pairs of sexual contacts fell into the same subtype. Seventy-one of 73 isolates which required arginine, hypoxanthine, and uracil for growth (Arg-Hyx-Ura-) and seven of 107 other auxotypes belonged to subtypes A2 and A3. Marked geographical differences in distribution of gonococcal immunotypes were observed among those available for testing. Subtypes A2 and A3 were predominant in Seattle whereas types B and C were predominant in Southeast Asia. The only Arg-Hyx-Ura- isolates not belonging to subtypes A2 or A3 were the only two that were serum sensitive. This Micro-IF immunotyping appears potentially useful for future immunologic, epidemiologic, and genetic studies of N. gonorrhoeae.

Asia, Southeastern↗