Rapid identification of Actinomycetaceae and related bacteria.

Identification of new isolates belonging to the family Actinomycetaceae requires extensive numbers of biochemical tests, supplemented with gas-liquid chromatography determination of fermentation end products and, often, analysis of cell wall composition. This paper describes the results of the testing of 162 strains of Actinomycetaceae and related taxa for 20 different enzymatic activities including phosphatases, esterases, aminopeptidases, and glycosidases. The results of all tests were read after 4 h of incubation. The results obtained in the study provide significant new information on the biochemical properties of these groups of bacteria. An identification scheme based upon 13 selected tests, which allow the identification of these groups of bacteria within 4 h, is proposed.     

Sequence-based identification of aerobic actinomycetes

We investigated the utility of 500-bp 16S rRNA gene sequencing for identifying clinically significant species of aerobic actinomycetes. A total of 28 reference strains and 71 clinical isolates that included members of the genera Streptomyces, Gordonia, and Tsukamurella and 10 taxa of Nocardia were studied. Methods of nonsequencing analyses included growth and biochemical analysis, PCR-restriction enzyme analysis of the 439-bp Telenti fragment of the 65 hsp gene, susceptibility testing, and, for selected isolates, high-performance liquid chromatography. Many of the isolates were included in prior taxonomic studies. Sequencing of Nocardia species revealed that members of the group were generally most closely related to the American Type Culture Collection (ATCC) type strains. However, the sequences of Nocardia transvalensis, N. otitidiscaviarum, and N. nova isolates were highly variable; and it is likely that each of these species contains multiple species. We propose that these three species be designated complexes until they are more taxonomically defined. The sequences of several taxa did not match any recognized species. Among other aerobic actinomycetes, each group most closely resembled the associated reference strain, but with some divergence. The study demonstrates the ability of partial 16S rRNA gene sequencing to identify members of the aerobic actinomycetes, but the study also shows that a high degree of sequence divergence exists within many species and that many taxa within the Nocardia spp. are unnamed at present. A major unresolved issue is the type strain of N. asteroides, as the present one (ATCC 19247), chosen before the availability of molecular analysis, does not represent any of the common taxa associated with clinical nocardiosis.     

Rapid gas-chromatographic method for identification of metabolic products of anaerobic bacteria.

The volatile fatty acids, alcohols, nonvolatile fatty acids, and ketones produced by anaerobes can be separated and identified by using a single type of gas-chromatographic column. A rapid and simple procedure is described.     

Rapid screening method for detection of bacteria in platelet concentrates

Public awareness has long focused on the risks of the transmission of viral agents through blood product transfusion. This risk, however, pales in comparison to the less publicized danger associated with the transfusion of blood products contaminated with bacteria, in particular, platelet concentrates. Up to 1,000 cases of clinical sepsis after the transfusion of platelet concentrates are reported annually in the United States. The condition is characterized by acute reaction symptoms and the rapid onset of septicemia and carries a 20 to 40% mortality rate. The urgent need for a method for the routine screening of platelet concentrates to improve patient safety has long been recognized. We describe the development of a rapid and highly sensitive method for screening for bacteria in platelet concentrates for transfusion. No culture period is required; and the entire procedure, from the time of sampling to the time that the final result is obtained, takes less than 90 min. The method involves three basic stages: the selective removal of platelets by filtration following activation with a monoclonal antibody, DNA-specific fluorescent labeling of bacteria, and concentration of the bacteria on a membrane surface for enumeration by solid-phase cytometry. The method offers a universal means of detection of live, nondividing, or dead gram-negative and gram-positive bacteria in complex cellular blood products. The sensitivity is higher than those of the culture-based methods available at present, with a detection limit of 10 to 10(2) CFU/ml, depending upon the bacterial strain.     

Rapid method for detection of adherent bacteria on Foley urinary catheters.

An accurate, rapid, and inexpensive method was developed for detecting and enumerating bacteria adherent to Foley urinary catheters based on malachite green staining of acridine orange-prestained specimens. This method has proven to be quick and reliable and will find application in quantitative studies of biomaterial-related sepsis.     

Use of the BioMerieux ID 32C yeast identification system for identification of aerobic actinomycetes of medical importance.

The BioMerieux ID 32C Yeast Identification System was examined to determine its usefulness as a rapid method for the identification of medically important aerobic actinomycetes. More than 290 strains were tested by this method and the results were compared to those obtained by conventional methods. It was found that aerobic actinomycetes could be differentiated to species level in 7 days by the ID 32C system.     

Fatty and mycolic acids of Mycobacterium malmoense.

The fatty acids and mycolic acids of 16 clinical isolates of Mycobacterium malmoense were studied by gas chromatography and thin-layer chromatography. All strains contained 2-methyleicosanoic and 2,4,6-trimethyltetracosanoic acids and alpha-, alpha'-, and keto-mycolic acids. The reported findings suggest that lipid analysis is a very useful approach in the species identification of M. malmoense.     

Comparative evaluation of three chromogenic agars for detection and rapid identification of aerobic Gram-negative bacteria in the normal intestinal microflora

Objective  To compare three different chromogenic agars and MacConkey agar for the detection of aerobic Gram-negative bacteria in the normal intestinal microflora and to assess the accuracy of the chromogenic agars for the direct identification of Escherichia coli .
Methods  A total of 164 Gram-negative clinical isolates ( E. coli , Proteus , Klebsiella , Enterobacter , Morganella and Pseudomonas species) and 30 stool specimens were inoculated in parallel on four media: Chromagar E. coli /Coliform, Chromogenic urinary tract infection UTI medium, CHROMagar Orientation and MacConkey agar. All colonies that differed by color and/or morphology were selected for further identification by VITEK 1 and/or API 20E from each medium.
Results  On E. coli /Coliform agar five out of 32 (16%) E. coli strains failed to produce the color as described by the manufacturer. No remarkable discrepancies were found for the other clinical isolates. There was no significant difference in detection rate (DR) of aerobic Gram-negative bacteria in stool specimens between the different chromogenic agars and MacConkey agar. The overall DR was about 84%, and varied from 100% for monomicrobial specimens to 33% for polymicrobial specimens. The positive predictive values (PPV) for the direct identification of E. coli on Chromagar E. coli /Coliform, Chromogenic UTI medium and CHROMagar Orientation were 1.00, 0.93 and 0.93, respectively. The negative predictive values (NPV) were 0.53, 0.68 and 0.69, respectively.
Conclusion  Chromogenic UTI medium and CHROMagar Orientation are the preferred media because of the higher NPV. The high PPV of these agars allows accurate and rapid identification of E. coli .     

Fatty acid characterization of rapidly growing pathogenic aerobic actinomycetes as a means of identification.

The fatty acid compositions of 39 type strains and 529 clinical or reference strains of pathogenic aerobic actinomycetes were analyzed after standardized culture by using the Microbial Identification System (MIS). Library entries for each type strain were created by using the MIS Library Generation Software, and the fatty acid profiles of clinical and reference strains were compared to these library entries. The bacteria separated into two large groups based upon major amounts of branched-chain or of saturated or monounsaturated straight-chain fatty acids. Identification of isolates was possible by using only the type strains for comparison, but fatty acid heterogeneity occurred within most species.     

Rapid identification of cell wall components as a guide to the classification of aerobic coryneform bacteria from human skin.

In a survey of over 1000 isolates of aerobic skin coryneforms from a wide variety of sources, chromatographic methods were used to identify the major cell-wall components in whole-cell hydrolysates. Most of the skin isolates-like members of the genus Corynebacterium--possessed meso-diaminopimelic acid and arabinose. However, substantial numbers of coryneforms apparently resident on the skin did not have this pattern; the sites from which they were isolated suggested that some were derived from the environment whilst others (possessing meso-DAP and galactose but not arabinose as the major wall components) were members of the resident skin flora.     

Rapid and simple method for purification of nucleic acids.

We have developed a simple, rapid, and reliable protocol for the small-scale purification of DNA and RNA from, e.g., human serum and urine. The method is based on the lysing and nuclease-inactivating properties of the chaotropic agent guanidinium thiocyanate together with the nucleic acid-binding properties of silica particles or diatoms in the presence of this agent. By using size-fractionated silica particles, nucleic acids (covalently closed circular, relaxed circular, and linear double-stranded DNA; single-stranded DNA; and rRNA) could be purified from 12 different specimens in less than 1 h and were recovered in the initial reaction vessel. Purified DNA (although significantly sheared) was a good substrate for restriction endonucleases and DNA ligase and was recovered with high yields (usually over 50%) from the picogram to the microgram level. Copurified rRNA was recovered almost undegraded. Substituting size-fractionated silica particles for diatoms (the fossilized cell walls of unicellular algae) allowed for the purification of microgram amounts of genomic DNA, plasmid DNA, and rRNA from cell-rich sources, as exemplified for pathogenic gram-negative bacteria. In this paper, we show representative experiments illustrating some characteristics of the procedure which may have wide application in clinical microbiology.     

Rapid method for identification and enumeration of oral Actinomyces.

Serotype-specific antisera prepared against whole cells of Actinomyces viscosus, A. naeslundii, and A. israeli were labeled with fluorescein dye and used to detect and quantitate antigenically related microorganisms in human dental plaque. By relating the DNA content of the dental plaque microflora to the number of Actinomyces present in the plaque samples, a reproducible method was developed for specifically enumerating five serotypic representatives of this genus found in human plaque.     

Rapid identification of bacteria by PCR-single-strand conformation polymorphism.

A new molecular biological approach for the identification of bacteria is described. This approach employs PCR of bacterial cell lysates with conserved primers located in the 16S rRNA sequence flanking a variable region, and analysis of the amplified product was based on the principle of single-strand conformation polymorphism (SSCP). The PCR product was denatured and separated on a nondenaturing polyacrylamide gel. SSCP patterns were detected by silver staining the nucleic acids. The mobility of the single-stranded DNA is sequence dependent and could be used to identify the unknown bacteria. Feasibility of the technique was demonstrated for a broad panel of gram-negative and gram-positive bacteria. We tested over 100 strains of bacteria representing 15 genera and 40 species. With the use of only two primer sets, P11P-P13P and ER10-ER11, we were capable to discriminate the tested species at the genus and species levels. Species-specific patterns were obtained for, e.g., Clostridium spp., Listeria spp., Pseudomonas spp., and Enterobacter spp. PCR-SSCP is a sensitive technique; e.g., the sensitivity obtained for Escherichia coli cells was 30 CFU. This technique is a simple and rapid method for the detection and identification of a wide spectrum of bacteria by whole-cell-based PCR amplification with the use of conserved primers and identification by nondenaturing gel electrophoresis.     

Rapid method for presumptive identification of Corynebacterium jeikeium.

Corynebacterium jeikeium causes systemic infections, particularly in immunocompromised hosts. A minitube assay has been developed for the presumptive identification of C. jeikeium. With our rapid sucrose-urea test and conventional biochemical tests, sixty isolates of gram-positive, catalase-positive bacilli were identified in our laboratory. Results indicated that our assay has a sensitivity of 100% and a specificity of 90%.     

Rapid method for the detection of DNase of campylobacters.

A rapid agar diffusion method for the detection of DNase production of Campylobacter jejuni, C. coli, and C. pylori was developed. A strong pink zone indicating DNA hydrolysis was seen around the wells after 20 to 24 h of aerobic incubation at 37 degrees C. Pretreatment of cells with polymyxin B, which releases the cell-associated DNase, both shortened the time needed to read positive results to 8 h and increased the zone size.     

Rapid method for detecting beta-lactamase producing bacteria in clinical specimens.

The use of liquid media to detect the production of beta-lactamase by beta-lactamase producing organisms has been compared with the conventional method of inoculation on to agar media. Pharyngeal cultures were obtained from 162 children treated with penicillin for acute tonsillitis. beta-lactamase producing organisms were detected within 72 h in 80 (49%) of the specimens inoculated on to agar media, while beta-lactamase production was found in 76 (47%) of the specimens after their incubation in liquid media for 24 h. Twenty one of the cultures were positive only after anaerobic incubation while in liquid media while nine were positive only after aerobic incubation. Incubation in liquid media enabled detection of beta-lactamase activity in 53 of the 76 (70%) specimens within 12 h.     

High-performance liquid chromatography analysis of mycolic acids as an aid in laboratory identification of Rhodococcus and Nocardia species.

High-performance liquid chromatography analysis of the p-bromophenacyl esters of mycolic acids from whole organisms gave chromatographic patterns that were useful in differentiation of Rhodococcus and Nocardia species. Rhodococcus equi, R. erythropolis, and R. rhodochrous contained more-polar mycolic acids and were easily separated from the less-polar mycolic acid-containing species of R. sputi, R. bronchialis, R. corallinus, R. rubropertinctus, and R. terrae. The less-polar mycolic acid-containing Rhodococcus species showed chromatographic patterns that partially overlapped (in elution times) the patterns of Nocardia asteroides, N. otitidiscaviarum, and N. brasiliensis, but the larger number of peaks in the last species made separation between the genera possible. Distinct chromatographic patterns were found for most species, except for R. equi strains that showed two different patterns. Strains of R. rubropertinctus and R. terrae appeared identical. N. asteroides and N. otitidiscaviarum showed similar mycolic acid patterns.     

Rapid antigen detection assay for identification of Chlamydia trachomatis infection.

A rapid antigen detection test was compared with direct fluorescent-antibody staining and with tissue culture isolation for the detection of Chlamydia trachomatis infections in 507 women. The sensitivities observed were 75, 76, and 84%, respectively, with specificities of > 99%.     

Routine analysis of short-chain fatty acids for anaerobic bacteria identification using capillary electrophoresis and indirect ultraviolet detection

The diagnosis of anaerobes can be difficult to perform, using classical biochemical tests. Characterization of metabolic end-products such as short-chain fatty acids (SCFA) was often used because of their reproducible biosynthesis. Despite this, SCFA are difficult to study using gas chromatography, due to their high volatility. Furthermore, the treatment of the samples are long and fastidious. Capillary electrophoresis and indirect UV detection (CE-indirect UV) is a well-known analytical method to study inorganic or organic anions. In this work, we validate the analysis of SCFA using CE-indirect UV detection. To do this, we studied the culture media of 98 anaerobic strains for the detection and quantitation of the following acids: succinic, pyruvic, acetic, lactic, propionic, 2-hydroxybutyric, butyric, 2-hydroxyvaleric, isovaleric, isocaproic, and 3-phenylpropionic. We verified that the CE-indirect UV detection analysis of SCFA for taxonomical data can be used as a mean for rapid identification for the study of anaerobes.