R M McKinney

Legionella longbeachae Species Nova, Another Etiologic Agent of Human Pneumonia

A new species of bacteria that is an etiologic agent of human pneumonia has been isolated and characterized. Clinical symptoms of infection with this organism are not readily distinguishable from those caused by Legionella pneumophila infection. The organism was isolated from respiratory tract specimens from four patients. Two cases of infection apparently originated in California and one in Georgia, and a fourth was of unknown geographic origin. The name Legionella longbeachae species nova is proposed for this organism. The type strain of L. longbeachae is Long Beach 4 (= American Type Culture Collection 33462).

Nosocomial legionellosis, Paris, France. Evidence for transmission by potable water

During a five-week period in 1981, six cases of legionellosis due to Legionella pneumophila serogroup 1 were recognized in a hospital in Paris, France. Four cases were clearly nosocomial in origin. There was a direct association between development of disease and exposure to potable hot water (p = 0.003). The entire hot water system was contaminated with L. pneumophila serogroup 1; monoclonal antibody testing demonstrated that the case isolate and the potable water isolates belonged to the same subgroup. Although serogroup 1 was isolated from both the cooling tower and its drift, the cooling tower isolate was antigenically distant from the case isolate. In other nosocomial outbreaks of legionellosis, multiple sources have been found within the hospital environment, but an epidemiologic association of disease with potable water had not been shown. The significant association of cases with exposure to the potable hot water supply, and the identification of case and potable water isolates of the same subtype, suggest that the potable hot water was responsible for transmission of disease in this outbreak.

Recognition of a genus-wide antigen ofLegionella by a monoclonal antibody

A monoclonal antibody was produced against a cytoplasmic membrane protein that appears to be common to all species of the genusLegionella. The antibody was positive in polyacrylamide gel electrophoresis and Western blotting with extracts of all of 22 species type strains ofLegionella that were tested. The apparent molecular mass of the protein varied from 57.2 to 62.1 kilodaltons for the 23 species type strains ofLegionella. An enzyme-linked immunosorbent assay (ELISA) was developed with the monoclonal antibody to enable rapid screening of clinical and environmental isolates forLegionella. All of 23 species type strains ofLegionella that were tested were strongly positive with the monoclonal antibody in the ELISA. Among 27 other bacterial species and 84 strains that were tested, onlyBordetella ssp. andAcinetobacter lwoffii were cross-reactive in the ELISA. These two cross-reactive species are readily distinguishable fromLegionella by culture characteristics. The monoclonal antibody may also be useful in tests to detect the genus-wide antigen in body fluids of patients with legionellosis.

Monoclonal antibodies to Legionella pneumophila serogroup 1: possible applications in diagnostic tests and epidemiologic studies.

Monoclonal antibodies were produced against two strains of Legionella pneumophila serogroup 1. A panel of nine monoclonal antibodies were selected for their unique specificities observed in indirect fluorescent antibody tests with 130 strains of L. pneumophila serogroup 1. One monoclonal antibody was reactive with an antigen possessed by 128 of 130 strains. Two major subgroups were identified and 13 different antigen patterns were observed among the 130 serogroup 1 strains.

Evaluation of commercial fluorescein isothiocyanates used in fluorescent antibody studies.

Commercial preparations of fluorescein isothiocyanate (FITC) for immunofluorescence applications were obtained from 12 sources and examined for purity by quantitative infrared spectrophotometry and by labeling efficiency for bovine serum albumin (BSA). Quantitative photometric measurements were made of nonspecific staining (NSS) produced by conjugates prepared from the dyes. The purity of FITC from different sources was highly variable. The risk of NSS appears to increase as the purity of the dye decreases. In immunofluorescence applications it is desirable to use the purest FITC available in order to obtain conjugates with minimum NSS. It is recommended that 70% FITC, as determined by BSA labeling efficiency, be accepted as the minimum purity for immunofluorescence applications.

Preparation and testing of a polyvalent conjugate for direct fluorescent-antibody detection ofLegionella pneumophila

A polyvalent conjugate forLegionella pneumophila, the Legionnaires’ disease bacterium, was prepared by combining monospecific antibodies for the four recognized serogroups ofL. pneumophila. Pure cultures ofL. pneumophila and other bacteria representing 18 genera and 50 species of heterologous organisms were used in evaluating the reagent. A total of 358 specimens from patients suspected of having Legionnaires’ disease also were tested. The results show the practicality and advantages of using a polyvalentL. pneumophila conjugate for screening clinical specimens.

Improved immunoadsorption procedure with anion-exchange bacterial cell columns.

Bacterial cell columns for immunoadsorption were prepared with Streptococcus cells and triethylaminoethyl cellulose (Cellex-T) matrix material as a model system. Good column flow properties and satisfactory retention of the cells were obtained with ratios as high as 2 ml of packed cells/3 g dry weight of cellulose. Anion-exchange fractionation of whole serum by the Cellex-T was prevented by using 0.25 M NaCl in the developing buffer. Antibodies were adsorbed directly from whole serum and recovered in high yield by desorption at pH 2.3. Pre-exposing bacterial cells to formalin and washing them with acetone was necessary to ensure that they remained on the columns. One strain of Streptococcus salivarius (SS 908) was satisfactorily retained on a column only after cells were labeled with fluorescein isothiocyanate and washed with acetone. The means by which Cellex-T retains bacterial cells appears to be a combination of electronic attraction and physical entrapment.

Characterization of fluorescein isothiocyanate dyes by infrared and thin-layer chromatography methods

Abstract Many commercial FITC products still leave much to be desired. Two of the seven commercial FITC products were found to contain appreciable amounts of a xanthene derivative (II), which results from improper reduction of the precursor nitrofluorescein. The presence of a number of other impurities was demonstrated. The analytical tests in this report should prove useful both to those who want to establish the purity of a product with which they plan to label and to those seeking to synthesize a purer product. The most fruitful combination of techniques is that of the BSA labeling test and TLC. The former measures the protein labeling capacity, while the latter indicates the number of nonvolatile organic impurities in a given sample. The quantitative IR measurement of isothiocyanate content measures total isothiocyanate, including at least one impurity that does not label protein.

Soluble antigen extracts used as blocking agents to obtain specificity in serotyping of Streptococcus mutans

Specific immunofluorescent (IF) conjugates were prepared for Streptococcus mutans serotypes d and g. Serotype specificity was obtained by blocking the cross-reacting antibodies with soluble antigen extracted from cells of the cross-reacting strains. Soluble antigen extracts are particularly useful in obtaining specificity with quantities of conjugates that are too small to be efficiently subjected to adsorption procedures. They also can be used advantageously to block traces of cross-reactivity that frequently remain with conjugates that have been subjected to conventional adsorption procedures. S. mutans isolates from dental plaque samples were identified as belonging to serotypes d or g on the basis of IF staining with conjugates that were made type-specific by blocking the cross-reacting antibodies with appropriate soluble antigen extracts.

Reaction paths to pure 5- and 6-aminofluorescein. Pitfalls encountered in conventional synthesis procedures

Previously unrecognized pitfalls in the published procedure for the preparation of 5- and 6-isothiocyanatofluoresceins were examined. Hydrolysis of 5-nitrofluorescein diacetate with hot ethanol saturated with sodium hydroxide gave varying amounts of 5,5″-azoxydifluorescein in addition to 5-nitrofluorescein. The use of methanol and sodium hydroxide was found to give pure 5- and 6-nitrofluorescein from the corresponding diacetate. Further, reduction of 5-nitrofluorescein with hydrogen and W-2 Raney nickel gave a mixture of 5-aminofluorescein and 9-(4-amino-2-carboxyphenyl)-3,6-dihydroxyxanthene. A sodium sulphide–sodium hydrosulphide reduction system reduced 5-nitrofluorescein cleanly to the amine without reductive lactone cleavage. Synthesis and characterization of 5,5″-azoxydifluorescein, 9-(4-amino-2-carboxyphenyl)-3,6-dihydroxyxanthene, and related fluorescein and xanthene derivatives are described.