Comparison of four hippurate hydrolysis methods for identification of thermophilic Campylobacter spp.

The test for hippurate hydrolysis is critical for separation of Campylobacter jejuni and C. coli strains. Glycine and benzoic acid are formed when hippurate is hydrolyzed by C. jejuni. The test used in most laboratories is one of several variations of the ninhydrin tube test described by Hwang and Ederer (M. Hwang and G. M. Ederer, J. Clin. Microbiol. 1:114-115, 1975) for detection of glycine. We evaluated three modifications of the Hwang and Ederer method and the gas-liquid chromatographic (GLC) method described by Kodaka et al. (H. Kodaka, G. L. Lombard, and V. R. Dowell, Jr., J. Clin. Microbiol. 16:962-964, 1982) for detecting benzoic acid. Campylobacter strains comprised 22 C. jejuni, 11 C. coli, and 8 C. laridis strains. The species identification of each strain was confirmed by DNA relatedness. All strains of C. jejuni were positive and all strains of C. coli and C. laridis were negative by the GLC method for detecting hippurate hydrolysis, whereas three strains of C. jejuni gave negative or variable results in the tube tests. The GLC method is more sensitive than the tube methods for detecting hippurate hydrolysis and should be used on cultures yielding variable or questionable test results.

Comparison of three reagents for detecting indole production by anaerobic bacteria in microtest systems.

Three reagents for detecting indole, Kovac, Ehrlich, and p-dimethylaminocinnamaldehyde (DMCA), were evaluated with commercial microtest systems for characterizing and identifying anaerobic bacteria. The DMCA reagent, the most sensitive of the three reagents, gave a positive reaction with 445 of 449 strains of various indole-producing anaerobic bacteria. There was 99.6% agreement between the results obtained with the DMCA in the microtest systems and results using the conventional tube test to detect indole by using xylene extraction and Ehrlich reagent. Ehrlich reagent detected indole in 163 of 176 (92.6%) indole-positive strains when the inoculum was overlaid with mineral oil before incubation. Kovac reagent was the least sensitive of the reagents tested. When the inoculum was overlaid with mineral oil, Kovac reagent detected only 80 of 108 (74.0%) of indole-positive strains. In addition to being the most sensitive reagent for detection indole, DMCA also allowed detection of indole derivatives (skatole, 3-indolepropionic acid, and 3-indolebutyric acid) produced by some clostridia.

Comparison of media in the Anaerobe-Tek and Presumpto plate systems and evaluation of the Anaerobe-Tek system for identification of commonly encountered anaerobes.

Using a variety of sporeforming and nonsporeforming anaerobic bacteria, we compared 10 differential agar media of the Anaerobe-Tek (A/T) system recently marketed by Flow Laboratories, Inc. (McLean, Va.) with 10 comparable media in Presumpto quadrant plates (Presumpto 1, 2, and 3) developed by the Centers for Disease Control Anaerobic Bacteria Branch. The A/T identification system was evaluated by comparing the species identity of anaerobes determined as recommended by the manufacturer's instruction manual with the identity of the strains obtained by the Centers for Disease Control Anaerobe Reference Laboratory by using conventional procedures. We also compared reactions obtained with the Presumpto plates with a chopped meat glucose broth culture as a source of inoculum with those obtained by using a turbid cell suspension from growth on blood agar as inoculum. The agreement of results for the 16 characteristics compared ranged from 92.8 to 100%. Comparison of test results obtained with 10 media in the Presumpto plate and A/T systems from the examination of 223 strains of anaerobes, representing 54 different taxa, showed the following agreement between A/T and CDC systems: catalase production, esculin hydrolysis, glucose fermentation, and lecithinase production (100%); inhibition of growth by bile agar (99.6%); lipase production (99%); DNase (98.7%); fermentation of lactose and mannitol (98.2%); starch hydrolysis (96.9%); gelatin hydrolysis (96.4%); and casein hydrolysis (94.6%). Of the 204 strains of common anaerobes tested with the A/T system, only 70% were correctly identified to the species level. However, several strains could have been identified correctly with the A/T system if data on certain other characteristics had been included in the A/T data base.

Gas-liquid chromatography technique for detection of hippurate hydrolysis and conversion of fumarate to succinate by microorganisms.

A gas-liquid chromatography technique which allows simultaneous detection of hippuric acid (N-benzoylglycine) hydrolysis and conversion of fumaric acid to succinic acid by microorganisms uses a new medium, hippurate-formate-fumarate broth, and a gas chromatograph equipped with a thermal conductivity detector. This technique gave more reproducible results than other tests used in the study for detecting hippurate hydrolysis and also gave consistent results in detecting succinic acid produced from utilization of fumaric acid.

Practical procedure for demonstrating bacterial flagella.

We developed a simple, rapid method for demonstrating bacterial flagella with Ryu staining solution that gave satisfactory results for numerous motile and nonmotile bacteria. Two major advantages of this method are that the staining solution, ready for use, is stable at ambient temperature indefinitely and that microscopic examination of bacteria in the stained drop preparations can be performed rapidly.

Gelatin agar medium for detecting gelatinase production by anaerobic bacteria.

A new medium, Lombard-Dowell gelatin agar, was developed for detecting gelatinase activity by anaerobic bacteria. The medium contained: Trypticase (BBL Microbiology Systems), 5.0 g; yeast extract (Difco Laboratories), 5 g; sodium chloride, 2.5 g; sodium sulfite, 0.1 g; L-tryptophan, 0.2 g; L-cystine, 0.4 g; hemin, 10.0 mg; vitamin K1, 10.0 mg; agar, 20.0 g; D-glucose, 1.0 g; gelatin, 4.0 g; and distilled water to 1 liter. The pH was adjusted to 7.5. The medium was dispensed in 100- by 15-mm quadrant plastic dishes (5 ml per quadrant). To test for gelatinase activity, we inoculated the medium with a young enriched thioglycolate or chopped meat glucose broth culture or a turbid cell suspension in Lombard-Dowell broth, using a sterile cotton swab, and incubated it under anaerobic conditions for 48 h at 35 degrees C. The quadrants were then flooded with Frazier solution, and clear zones around the bacterial growth were recorded as positive for gelatinase activity. The new medium was tested with a variety of anaerobic bacteria, and the results were compared with data obtained with the conventional technique for detecting gelatinase activity. Overall, there was satisfactory agreement between the two tests in the detection of gelatinase activity, but the Lombard-Dowell gelatin agar tests was more rapid and somewhat more sensitive than the conventional test.

Atypical toxin variant of Clostridium botulinum type B associated with infant botulism.

An atypical toxin variant of Clostridium botulinum (strain 657) was isolated from the feces of a 6-week-old female infant whose symptoms and clinical history were consistent with infant botulism. Toxin detected in the feces and the toxin produced by isolates from the feces and from two rectal swabs could be neutralized by type B botulinal antitoxin only at very high ratios of of antitoxin to toxin in the neutralization mixture. One international unit of type B antitoxin neutralized only about 10 lethal doses of 657 toxin as compared with approximately 10,000 lethal doses of conventional type B toxin from the Beans strain. Antitoxin prepared against 657 toxin was 10 times more effective against the conventional toxin than against the homologous toxin. Toxoid-antitoxin-binding studies indicate that both 657 toxin and type B toxin are heterogeneous and that both toxins may contain the same molecular variants, but that the proportions of the variants are different in each.

Clinical features of types A and B food-borne botulism.

Medical records of 55 patients with type A and type B food-borne botulism reported to the Centers for Disease Control during 2 years were reviewed to assess the clinical features and severity of illness, diagnostic test results, nature of complications, amd causes of death. Some patients had features not usually associated with botulism including paresthesia (14%), asymmetric extremely weakness (17%), asymmetric ptosis (8%), slightly elevated cerebrospinal fluid protein values (14%), and positive responses to edrophonium chloride(26%). Several observation suggest that type A was more severe than type B disease. Although the case-fatality ratio was not significantly greater, patients with type A disease saw a physician earlier in the course of illness, were more likely to need ventilatory support, and were hospitalized longer. Patients who died were older than those who survived. Deaths within the first 2 weeks resulted from failure to recognized the severity of the disease or from pulmonary or systemic infection whereas the three late deaths were related to respirator malfunction.

Immunochemical evidence for multiple serotypes of Bacteroides fragilis.

An immunochemical comparison of outer membrane antigens obtained from five select and biochemically defined strains indicated that there are several serotypes of Bacteroides fragilis. Each strain was serologically defined by individual or by combinations of determinant groups composed of carbohydrates in the form of polysaccharides or glycoproteins. The carbohydrate constituents were tentatively identified as glucose, galactose, fucose, rhamnose, glucosamine, galactosamine, and traces of mannose. Strains were observed to have minor qualitative and major quantitative variations in carbohydrate composition.

Serological study of trichloroacetic acid extracts of Bacteroides fragilis.

Immunodiffusion techniques were used on trichloroacetic acid extracts from 10 strains of Bacteroides fragilis in detecting precipitating antibodies against this species in immune rabbit sera. Species and even strain specificities were observed in these precipitin reactions. Multiple antigens were detected in the extracts from some strains, whereas only one precipitin band per extract developed during agar-gel diffusion tests of others. The antigen extracts were found to be both heat stable and resistant to hydrolysis by alpha-chymotrypsin. Four serological patterns were demonstrated in homologous and heterologous reactions with the B. fragilis. antigen-antibody systems used. The results showed that some strains were serologically distinct from others, indicating that the strains tested are of more than one serotype.

Alternative procedures for identification of anaerobic bacteria.

Recognizing the need for practical inexpensive procedures which allow identification of anaerobes commonly encountered in clinical laboratories, a number of investigators have in recent years proposed various alternatives to the expensive, time-consuming techniques presently recommended by reference anaerobe laboratories. Some of these techniques are potentially good alternatives. In this paper we review some of the more promising approaches and present a detailed description of two commercial microbiochemical systems for characterization of anaerobes. When used in conjunction with other available tests, the microsystems are useful and convenient methods for identification of anaerobic bacteria at a reasonable cost.

Fluorescent-antibody studies on selected strains of Bacteroides fragilis subspecies fragilis.

Antisera against seven strains of Bacteroides fragilis subspecies fragilis were produced from dense suspensions of whole cells. These sera exhibited high agglutination titers with homologous antigens. Reciprocal cross-reactions in agglutination tests with each immunizing strain yielded lower titers. Both the indirect and direct fluorescent-antibody techniques were used to evaluate these reagents in the serological identification of 24 defined strains of B. fragilis subspecies fragilis. Subspecies and even strain specificities were noted with particular antisera. A pooled antiserum and conjugate were prepared and studied. Study results showed that specific and high-titered antisera against strains within this subspecies can be produced by the methods described herein and that possibly more than one serotype exists within the seven strains studied. The development of more antibody pools will be necessary to encompass a wider antigenic coverage before the fluorescent-antibody technique can be relied upon altogether for serologically identifying isolates of B. fragilis subspecies fragilis. Test data showed that the indirect method of fluorescent-antibody staining with whole antiserum is an excellent means of identifying strains of this organism.

Coproexamination for botulinal toxin and clostridium botulinum. A new procedure for laboratory diagnosis of botulism.

Stool or serum specimens or both from 318 persons pertaining to 165 botulism investigations over a three-year period were examined. Botulinal toxin was detected in stools of 19 of 56 patients and in sera of 20 of 60 patients with clinical botulism; it was not detected in specimens from 246 persons with an illness other than botulism or well contacts of patients. Clostridium botulinum was identified in stools of 36 of 60 clinical botulism patients and in four of 27 asymptomatic contacts of patients with botulism victims, but not in stools of 65 persons not associated with confirmed botulism. When stool and serum samples were examined, confirmatory evidence was obtained for 72.9% of the botulism cases. Detection of botulinal toxin or C botulinum in the stool of a persons should be considered evidence supporting the clinical diagnosis of botulism.

Cellular fatty acids of Peptococcus variabilis and Peptostreptococcus anaerobius.

Cellular fatty acids of Peptococcus variables and Peptostreptococcus anaerobius were identified by gas chromatography, mass spectrometry, and associated analytical techniques. Iso- and anteiso-branched-chain acids were major components in both species.

Type A botulism from commercially canned beef stew.

Two of three persons who ate lunch together became ill with symptoms characteristic of botulism. One died before botulism was suspected and before specimens could be collected for laboratory testing, but a serum specimen from the other patient, who survived, yielded botulinal toxin, type A. The third person remained asymptomatic, but Clostridium botulinum type A was cultured from his stool. The three persons had shared two canned foods: home-canned green beans and commercially canned beef stew. The green beans were initially assumed to be the cause of the outbreak. However, the empty stew can was recovered from the garbage, and washings from the can yielded C botulinum, type A, and its toxin.

Modification of the Minitek Miniaturized Differentiation System for characterization of anaerobic bacteria.

The Minitek Miniaturized System (BBL) was modified for characterization of anaerobic bacteria. The modified system and the conventional Center for Disease Control method were used to test a variety of anaerobic bacteria, and results were compared. Tests performed by both techniques were indole and H2S production, esculin hydrolysis, nitrate reduction, and fermentation of glucose, mannitol, lactose, sucrose, maltose, salicin, glycerol, xylose, arabinose, mannose, rhamnose, and trehalose. The manufacturer's recommended procedure for the Minitek system was modified by using a new suspension medium (Lombard-Dowell broth) and an inoculum equivalent to the density of a McFarland no. 5 nephelometer standard. The Minitek results, recorded after 48 h, agreed satisfactorily with the conventional test results, usually recorded after 5 to 7 days of incubation. In the examination of 80 strains representing 22 different species or subspecies of anaerobic bacteria, with 16 biochemical tests performed in triplicate, 93.8% of the Minitek test results agreed with those of the corresponding conventional tests. Only tests for indole, H2S, and nitrate reduction gave less than 90% agreement. It was concluded that the modified Minitek system is a suitable substitute for the more expensive and time-consuming conventional procedure for determining carbohydrate fermentation and esculin hydrolysis by anaerobes. This system, when used in conjunction with other tests, can effectively aid in the definitive identification of commonly isolated anaerobes.