Rapid differentiation of the major causative agents of bacterial meningitis by use of frequency-pulsed electron capture gas-liquid chromatography: analysis of amines

Abstract

The major causative agents of bacterial meningitis (Haemophilus influenzae serogroup B, Neisseria meningitidis serogroups B and C, Klebsiella pneumoniae, Steptococcus pneumoniae, and two types of Escherichia coli) were cultured in a chemically defined medium, and selected strains were further studied in Todd-Hewitt medium. After acidic extraction of the spent media with chloroform, a basic extraction was made with chloroform to obtain amines. A third extraction was performed on re-acidified Todd-Hewitt medium with ethyl ether to obtain hydroxyacids. The extracts were derivatized with heptafluorobutyric anhydride-ethanol to form electron-capturing derivatives, and the derivatives were analyzed on a frequency-pulsed electron capture gas-liquid chromatograph (FPEC-GLC) equipped with a PEP-2 computer. The data obtained from the study showed that amines were produced by these organisms that formed characteristic patterns. Different serotypes of K. pneumoniae and the two serogroups of N. meningitidis produced different types of FPEC-GLC profiles within serotypes. E. coli produced several hydroxy acids on Todd-Hewitt medium that made it unique among the organisms studied. The methods used are practical and the techniques have potential for use in clinical laboratories and hospitals as a valuable aid for the rapid identification of the major causative agents of bacterial meningitis.

Selective procedures for detecting femtomole quantities of tuberculostearic acid in serum and cerebrospinal fluid by frequency-pulsed electron capture gas-liquid chromatography

“…For carboxylic acids, the acidic chloroform extract was derivatized with trichloroethanol (TCE) as previously described (1). The samples were also analyzed for amines and hydroxy acids as previously described (2,(6)(7)(8).…”

“…The bromination procedure was done as follows. (i) The TCE-derivatized sample in xylene-ethanol (50:50) was evaporated with clean, dry air (1,7) to about 25 ,ul, and 100 pul of a 50:50 CHCl3-CCL4 solution was added. (ii) A 25-pul portion of a solution of 5% bromine in CCl4 was added, and the reaction mixture was permitted to sit for 5 min.…”

Rapid differentiation of rocky mountain spotted fever from chickenpox, measles, and enterovirus infections and bacterial meningitis by frequency-pulsed electron capture gas-liquid chromatographic analysis of sera

“…Next, the residual basic aqueous phase was acidified to about pH 2 with H2SO4 and extracted with 20 ml of ethyl ether (Fisher reagent grade stabilized with butylated hydroxytoluene) to obtain hydroxy acids. The acidic chloroform extracts were derivatized with trichloroethanol-heptafluorobutyric anhydride (TCE-HFBA) to form TCE esters of carboxylic acids and HFBA esters of alcohols as described (1,6). The basic chloroform extracts containing amines and the acidic ethyl ether extracts containing hydi-oxy acids were derivatized with HFBA pyridine-ethanol to form amides and esters, respectively, as described (2, 7).…”

“…Recent studies have shown that frequencypulsed electron capture gas-liquid chromatography (FPEC-GLC) of body fluids (pleural fluids and cerebrospinal fluids) is a promising tech-nique for classifying various types of pleural effusions (3) and for distinguishing among tuberculous, cryptococcal, and viral meningitis (5,8). FPEC-GLC has also been used in spent-media analysis to distinguish organisms at the species and strain levels (6,7).…”

“…Recent studies have shown that frequencypulsed electron capture gas-liquid chromatography (FPEC-GLC) of body fluids (pleural fluids and cerebrospinal fluids) is a promising tech-nique for classifying various types of pleural effusions (3) and for distinguishing among tuberculous, cryptococcal, and viral meningitis (5,8). FPEC-GLC has also been used in spent-media analysis to distinguish organisms at the species and strain levels (6,7). The volatile components (carboxylic acids, hydroxy acids, alcohols, and amines) present in the diseased body fluids and spent media originate as a result of metabolites produced by the infecting agent, as a result of specific host response to the cause or causative agent producing the disease, as a result of metabolic changes induced in the host by the disease, or by a combination of all of these factors.…”