Mycolic acid analysis by high-performance liquid chromatography for identification of Mycobacterium species.

Mycobacterium tuberculosis is the etiologic agent of tuberculosis and can be accurately detected by laboratories using commercial genetic tests. Nontuberculosis mycobacteria (NTM) causing other mycobacterioses can be difficult to identify. The identification processes are confounded by an increasing diversity of newly characterized NTM species. The ubiquitous nature of NTM, combined with their potential to be opportunistic pathogens in immunocompromised as well as nonimmunodeficient patients, further complicates the problem of their identification. Since clinical case management varies depending on the etiologic agent, laboratories must identify the species in a timely manner. However, only a few identification methods can detect the species diversity within the Mycobacterium genus. Over the last decade, high-performance liquid chromatography analysis of the mycolic acids has become an accepted method for identification of mycobacteria. In this review, we assess its development and usefulness as an identification technique for Mycobacterium species.

Characterization of an SAV organism and proposal of Mycobacterium triplex sp. nov.

Polyphasic taxonomic methods were employed to characterize a new species of slowly growing, nonpigmented mycobacteria. We propose the name Mycobacterium triplex sp. nov. for this new taxon. Conventional identification testing demonstrated a group of similar organisms that were geographically widespread in the United States. Commercially available nucleic-acid probes specific for the Mycobacterium avium complex were unreactive for these strains. High-performance liquid chromatography analysis of the mycolic acids revealed mycolate profiles that closely resembled Mycobacterium simiae. Comparative 16S rRNA sequence data confirmed the phylogenetic relationship of the strains with the slowly growing mycobacteria. Representative-type strains have been deposited in the American Type Culture Collection as strain ATCC 700071 [corrected].

Improved rapid identification of mycobacteria by combining solid-phase extraction with high-performance liquid chromatography analysis of BACTEC cultures.

Identification of mycobacteria from BACTEC 12B cultures is achieved in 7 to 21 days by reverse-phase high-performance liquid chromatography (HPLC) using a UV spectrophotometer to detect nonpolar p-bromophenylacyl mycolic acid derivatives. However, cultures grown in BACTEC and other liquid media seldom contain sufficient mycolic acids to permit reliable identification under usual HPLC assay conditions, so the sample size must be increased. Unfortunately, samples prepared from cultures in liquid media such as BACTEC cultures also contain large amounts of extraneous polar and strongly nonpolar contaminants that interfere with the analysis and hasten deterioration of the HPLC column. The contaminants were removed from 10 samples simultaneously by solid-phase extraction (SPE), i.e., by passing the crude suspension containing the mycolic acid derivatives into disposable 500-mg tC18 SPE columns in place of the usual final filtration step used to prepare specimens for HPLC. Fifteen milliliters of 20% (vol/vol) dichloromethane in methanol was passed through the columns (< 3 ml/min) to wash through the undesired contaminants and bind the mycolic acid derivatives. The mycolates were quantitatively eluted in 3 ml of dichloromethane for analysis by HPLC. Treating a panel of 31 strains of frequently isolated mycobacteria by SPE reduced the content of contaminants by 89.3 to 99.9% without altering the chromatographic patterns compared with the same strains grown on conventional solid media and processed without SPE. Peak heights of mycolates prepared from BACTEC cultures were increased from < or = 6 to > or = 25 absorbance milliunits with SPE, sufficient for reliable interpretation by visual inspection of chromatograms obtained with a UV detector. Also, removal of the contaminants improved column longevity.

The susceptibility of S-layer-positive and S-layer-negative Aeromonas strains to complement-mediated lysis.

Forty strains of Aeromonas hydrophila and Aeromonas veronii recovered from invasive and non-invasive infections were tested for their susceptibility to complement-mediated lysis by 65% pooled human serum (PHS). Based upon the results of this assay, two major populations could be defined. The first group (n = 20) consisted of serogroup O:11 strains, all of which possessed a paracrystalline surface layer (S layer); all of these strains were refractory to the bactericidal activity of 65% PHS with the exception of A. hydrophila strain AH-121, which was composed of mixed subpopulations of serum-susceptible and serum-resistant clones. A second collection of isolates (n = 20), all of which were S-layer-negative, contained a subgroup of strains (n = 7) that were highly susceptible to complement-mediated lysis, showing a greater than 100-fold reduction of viable progeny within 30 min of exposure to 65% PHS. Serum-resistant strains from both groups could not be lysed by exposure of bacterial cells to polyclonal somatic or whole cell antisera or to 30 micrograms ml-1 of polymyxin B nonapeptide prior to challenge with 65% PHS. Analysis of selected serum-resistant and serum-susceptible strains from both groups showed that all isolates activated the complement pathway and most bound C3b to the cell surface, indicating that the inability of complement to lyse serum-resistant strains was related to a defect in the terminal portions of the complement pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Aeromonas species in septicemia: laboratory characteristics and clinical observations.

We retrospectively analyzed clinical and epidemiological data on and laboratory characteristics of 53 cases of aeromonas septicemia. Only four Aeromonas genomospecies (species defined by DNA relatedness) were associated with the 53 cases, with Aeromonas hydrophila (sensu stricto) predominating (47%). Nearly 60% of all Aeromonas isolates from blood fell into one of four somatic groups: serogroups O:11, O:16, O:18, and O:34. Unlike Aeromonas-associated gastroenteritis, septicemia did not peak in frequency during the warmer months but rather was most common in January through March, when approximately 40% of cases occurred. In vitro tests of the pathogenicity of 20 selected blood isolates of Aeromonas indicated that resistance to complement-mediated lysis, elevated levels of protease and hemolysin activity, and the ability to elaborate siderophores correlated with higher virulence. Species and serogroup designations also correlated with the degree of virulence. Susceptibility studies of 50 strains indicated that A. hydrophila was the most drug-resistant species and that Aeromonas veronii was the most susceptible. Susceptibility to first- and second-generation cephalosporins and carbenicillin was species-associated.

Curvilinear-gradient high-performance liquid chromatography for identification of mycobacteria.

Over a 1-year period, 502 mycobacterial cultures submitted to the Microbial Diseases Laboratory were identified by high-performance liquid chromatography (HPLC) in parallel with standard biochemical methods. Identification by HPLC using a curvilinear gradient was achieved by comparing the chromatograms of the unknown cultures to chromatograms for known reference strains, together with calculation of peak height or peak area ratios, as necessary. The overall agreement between HPLC and biochemical identification was 97.2%. In addition, 7 of 12 cultures of Mycobacterium bovis were identified by HPLC as the BCG strain. Of 111 cultures biochemically identified as members of the M. avium complex (MAC), 108 were confirmed as MAC by DNA probe and 106 were confirmed by HPLC. Of the latter 106, 58 probe-positive strains were identified as M. avium, 38 were identified as M. intracellulare, and 10 were identified as Mycobacterium sp. strain "X" by HPLC. Of the remaining five nonchromogenic cultures, four had MAC-like chromatograms that did not match any in our library sufficiently to permit definitive identification. Of the latter four, two were confirmed as MAC strains by DNA probe and two were not. The last of the cultures biochemically identified as MAC (1 of 111) was a mixture of MAC and non-MAC strains. Overall, only 2 of 502 cultures yielded results by HPLC that differed from those obtained by standard biochemical methods. The HPLC result was confirmed in both cases by an independent national reference laboratory. In the 12 instances in which HPLC did not provide identification, the chromatograms were either uninterpretable or did not match available reference chromatograms. These findings show that the identification obtained by HPLC concurs well with that obtained by both the standard biochemical methods and the DNA probes. Thus, identification by HPLC provides mycobacteriology laboratories with a reproducible and specific method for accurate and timely identification of most medically important mycobacteria.

Mycobacterium avium and Mycobacterium intracellulare chromatotypes defined by curvilinear gradient HPLC of mycolic acids.

Seventy-nine strains of Mycobacterium avium complex bacteria (MAC), previously characterized by genetic probe analysis, were assayed using two methods of reverse phase high-performance liquid chromatography (HPLC) that employed curvilinear gradients. Although different in column length and cycle time, the methods produced equivalent results, yielding seven distinct chromatographic patterns (chromatotypes) of M. avium and M. intracellulare based on the ratio of mycolate concentrations in the late vs. the middle of three peak clusters (L:M ratio). The M. avium strains (n = 36) were assigned to chromatotypes 1 through 4 (L:M ratios less than 3), and the M. intracellulare strains (n = 25) to chromatotypes 5 through 7 (L:M ratios greater than 4). Of 18 Mycobacterium 'X' strains, seven resembled M. avium, seven others resembled M. intracellulare, and four were intermediate between M. avium and M. intracellulare.

Mycobacterium avium and Mycobacterium intracellulare infections in patients with and without AIDS.

A genetic probe (Gen-Probe) was used to evaluate potential epidemiologic and susceptibility differences of Mycobacterium avium complex (MAC) strains isolated from 154 patients with and without the acquired immunodeficiency syndrome (AIDS). Genetic analysis revealed that 98% of the 45 patients with AIDS harbored only M. avium regardless of the anatomic or geographic source of the isolate; in contrast, approximately 40% of MAC isolates recovered from 109 patients without AIDS were M. intracellulare. Most M. intracellulare of respiratory origin recovered from patients without AIDS were involved in infectious processes. When 95 MAC isolates (M. avium, n = 53; M. intracellulare, n = 42) were evaluated for in vitro susceptibility to primary or secondary antimycobacterial drugs, significant differences were noted. M. intracellulare was more susceptible to streptomycin, rifampin, and ethambutol than M. avium; the converse was true for ethionamide. The results of this study suggest potentially important differences in disease spectrum and in vitro susceptibility profile for M. avium and M. intracellulare.

Quality control of individual components used in Middlebrook 7H10 medium for mycobacterial susceptibility testing.

The acceptability of different lots of commercial components which constitute our basal medium for susceptibility testing of mycobacteria was evaluated. The basal medium consisted of Middlebrook 7H10 agar supplemented with 10% oleic acid-albumin-dextrose-catalase and 0.5% glycerol. Studies were performed by using three separate microbiologic assays, and results were compared with parallel tests on previously standardized and acceptable lots of media. Components were rejected if comparison with standardized medium showed a major change in growth support or susceptibility status of any reference strain to any antimicrobial agent tested. Of the components tested in such a manner, 7 of 23 (30%) lots of 10% oleic acid-albumin-dextrose-catalase, 2 of 13 (15%) lots of Middlebrook 7H10 agar, and 0 of 5 lots of glycerol were found to be unacceptable. This study demonstrates that individual lots of components of this basal medium may vary significantly in their suitability for susceptibility testing, and failure to detect such variation may dramatically affect susceptibility profiles.

Histamine production by Klebsiella pneumoniae and an incident of scombroid fish poisoning.

A histamine-producing strain of Klebsiella pneumoniae was isolated from a sample of tuna sashimi implicated in an outbreak of scombroid fish poisoning. None of the other nine gram-negative bacterial strains isolated from the tuna sashimi was capable of equivalent histamine production. Bacterial histamine production was monitored in a tuna fish infusion broth (TFIB), and the implicated K. pneumoniae was capable of producing 442 mg of histamine per 100 g of tuna in TFIB in 7 h under controlled incubation conditions. Only 12 of 50 other K. pneumoniae strains, representing 5 distinct biochemical types, which had been originally isolated from foods, were able to produce such levels of histamine in TFIB. No correlation was found between histamine production and other biochemical characteristics or antibiotic resistance. Of the 12 histamine-producing strains, 11 belonged to type 2, which is characterized as indole negative with positive reactions in the urea and Voges-Proskauer tests. However, only 50% of the type 2 strains examined produced high levels of histamine in TFIB. Additionally, the implicated K. pneumoniae strain and one other strain belonged to type 1, which is characterized by positive reactions in the indole, urea, and Voges-Proskauer tests.

Scombroid poisoning. Report of an outbreak.

An outbreak of scombroid poisoning occurred in San Francisco in the fall of 1977. The vehicle was sashimi prepared from spoiled tuna fish. Prompt public health measures prevented further consumption of the implicated food. Laboratory studies showed the presence in the tuna of bacterial species capable of producing large amounts of histamine, a substance strongly implicated in scombroid poisoning. Chemical analysis showed that histamine is very unevenly distributed in the flesh of spoiling tuna, therefore accounting for the sometimes random occurrence of disease among people eating the same food at the same table.

Comparison of miniaturized multitest systems with conventional methodology for identification of Enterobacteriaceae from foods.

Four miniaturized multiple test systems were compared with tube methodology used to identify Enterobacteriaceae encountered in foods. Identification aids supplied with each system were used to assign names to isolates at the species level. For the 129 strains tested, the Minitek system demonstrated a 96.9 percent agreement with reactions in tubed media. The Inolex, Analytab, and PathoTec test systems exhibited 94.3, 93.8, and 92.7 percent agreement, respectively. Analytab identified 96.1 percent of the isolates to the species level, whereas the Minitek, PathoTec, and Inolex systems were able to identify 78.3, 32.6, and 27.1 percent, respectively. The results indicate that the Analytab and Minitek systems are acceptable substitutes for the tube methodology routinely employed in identifying enterics from foods. Although the PathoTec system might be used to screen isolates for their identity, neither the presently available PathoTec nor the Inolex systems should be substituted for current methodology when definitive identification of foodborne organisms is required.