Evaluation of practical chromatographic procedures for identification of clinical isolates of mycobacteria.

After experimental conditions were established, 366 strains of mycobacteria belonging to 23 different species were studied for fatty acids, secondary alcohols, and mycolic acid cleavage products by capillary gas-liquid chromatography. Additionally, the mycolic acid pattern was studied by thin-layer chromatography. Capillary gas-liquid chromatography allowed direct identification of the following Mycobacterium spp.: M. kansasii, M. marinum, M. szulgai, M. xenopi, M. malmoense, and M. gordonae. The patterns of mycolic acid methyl esters recorded for the test strains of M. chelonae and M. agri may be of value in the identification of these species. Moreover, the combined use of the two chromatographic techniques provided precise identification of the M. tuberculosis complex, M. simiae, M. fallax, M. triviale, and M. chelonae-like organisms. A minimal set of biochemical tests is usually required to obtain identification to the species level when chromatographic procedures alone are not sufficient. Under the reported experimental conditions, thin-layer chromatography and capillary gas-liquid chromatography are rapid and very useful techniques for the identification of mycobacteria.     

High-performance liquid chromatography of corynomycolic acids as a tool in identification of Corynebacterium species and related organisms.

A high-performance liquid chromatography (HPLC) study of 307 strains of Corynebacterium species and related taxa revealed that strains classified as "Corynebacterium aquaticum"; "Corynebacterium asperum"; and Centers for Disease Control (CDC) groups 1, 2, A-3, A-4, A-5, B-1, B-3, E, F-2, and I-2 as well as some unidentified coryneforms do not contain any corynomycolic acids; therefore, they should not be included in the genus Corynebacterium. Such an HPLC method of identification permitted the correct assignment to the genus Rhodococcus of two unpigmented strains of coryneform bacteria whose mycolic acid profiles were comparable to those of Rhodococcus equi. Bacteria belonging to CDC groups ANF-1, ANF-3, F-1, G-1, G-2, and I-1, as well as some other Corynebacterium sp. strains, yielded corynomycolic acid HPLC patterns related to those of Corynebacterium species. Either similarities or differences were observed in the corynomycolic acid profiles of Corynebacterium species tested after culture on sheep blood agar and/or sheep blood agar supplemented with Tween 80, which demonstrated that identification at the species or group level is possible. However, Corynebacterium striatum and CDC group I-1 bacteria as well as CDC group G-1 and group G-2 bacteria had indistinguishable HPLC patterns. Conversely, some variations were observed within some species as Corynebacterium xerosis, C. striatum, and Corynebacterium minutissimum. The evaluation procedure of this HPLC method by mass spectrometry analysis of isolated eluted peaks revealed that analytical reverse-phase HPLC alone does not provide any structural information, since isomers with identical polarities coeluted as a single peak. Nevertheless, HPLC is a rapid and reliable method for identification of corynomycolic acid-containing bacteria in the clinical microbiological laboratory.     

Rapid method for the detection and identification of mycolic acids in aerobic actinomycetes and related bacteria.

A rapid method for the identification of lipids characteristic of the genera Corynebacterium, Mycobacterium, Nocardia, and the "rhodochrous group" has been developed. Modifications of previously described methods make this procedure suitable for use in the clinical laboratory. Thin-layer chromatography is used to demonstrate the presence of the lipid characteristic of Nocardia spp. (type A) in some corynebacteria, nocardias, and members of the "rhodochrous group." Precipitation in ether and ethanol is used to demonstrate the presence of mycobacterial mycolic acids. Since this procedure can be carried out in less than 2 days and the lipids are extracted from the same batch of cells grown for diaminopimelic acid and whole-cell sugar analyses, it can readily be added to the battery of tests performed in reference laboratories that deal with aerobic actinomycetes and related bacteria.     

Rapid identification of antibiotic-resistant corynebacteria with the API 20S system

The API 20S system (Analytab Products, Plainview, N.Y.) was evaluated for the rapid identification of multiply antibiotic-resistant aerobic diphtheroids. Sixty-eight clinical isolates of multiply resistant Centers for Disease Control group JK and group D2 corynebacteria had API 20S profiles which were clearly different from those of a number of strains of other Corynebacterium species which were tested. The API 20S system allowed more rapid identification of antibiotic-resistant diphtheroids than conventional biochemical tests. Its use for corynebacteria other than group JK and group D2 is not recommended at this time.     

Bloodstream infection caused by nontoxigenic Corynebacterium diphtheriae in an immunocompromised host in the United States

Corynebacterium species are well-known causes of catheter-related bloodstream infections. Toxigenic strains of Corynebacterium diphtheriae cause respiratory diphtheria. We report a bloodstream infection caused by a nontoxigenic strain of C. diphtheriae and discuss the epidemiology, possible sources of the infection, and the implications of rapid species identification of corynebacteria.     

A clinicopathological review of 34 cases of inflammatory breast disease showing an association between corynebacteria infection and granulomatous mastitis

AIM: Granulomatous mastitis is a rare condition of unknown aetiology. The great majority of cases has not been associated with bacterial pathogens if women with mammary tuberculosis are excluded. We noted that some women in Auckland with a histological diagnosis of granulomatous mastitis had both microbiological and histological evidence of corynebacteria infection and aimed to study this further. METHODS: Thirty-four women were reviewed who presented with inflammatory breast disease and had microbiological specimens from which corynebacteria were isolated and/or histological specimens containing coryneform bacteria. These 34 cases were compared with 28 controls with similar histology but no evidence of corynebacteria infection. RESULTS: Twenty-seven (79%) of the cases and 21 (75%) of the controls had histological and/or cytological evidence of suppurative granulomas. Fourteen of the 34 cases also had Gram-positive bacilli (GPB), recognisable as coryneform bacteria, in histological sections. In all cases the bacilli were confined to empty spaces, consistent with dissolved lipid, and were surrounded by neutrophils and, frequently, suppurative granulomas. Corynebacterium species were isolated from 52 of 116 microbiological specimens taken from the 34 cases. Forty of these 52 cultures were pure. Twenty-four of the cultures were further classified biochemically and using 16S rRNA gene sequencing. Twenty of the 24 were lipophilic Corynebacterium species and 14 were identified as Corynebacterium kroppenstedtii. The cases were more likely to present with fever or neutrophilia and more often formed sinuses than the controls but other clinical features were similar. Maori and Pacific Islanders accounted for 77% of the women across both groups. CONCLUSION: We suggest granulomatous mastitis can be associated with corynebacteria infection, particularly infection by C. kroppenstedtii. The significance of this finding, which has previously been described in only a single case report, is discussed.     

Identification of coryneform bacterial isolates by ribosomal DNA sequence analysis

Identification of coryneform bacteria to the species level is important in certain circumstances for differentiating contamination and/or colonization from infection, which influences decisions regarding clinical intervention. However, methods currently used in clinical microbiology laboratories for the species identification of coryneform bacteria are often inadequate. We evaluated the MicroSeq 500 16S bacterial sequencing kit (Perkin-Elmer Biosystems, Foster City, Calif.), which is designed to sequence the first 527 bp of the 16S rRNA gene for bacterial identification, by using 52 coryneform gram-positive bacilli from clinical specimens isolated from January through June 1993 at the Mayo Clinic. Compared to conventional and supplemented phenotypic methods, MicroSeq provided concordant results for identification to the genus level for all isolates. At the species level, MicroSeq provided concordant results for 27 of 42 (64.3%) Corynebacterium isolates and 5 of 6 (83.3%) Corynebacterium-related isolates, respectively. Within the Corynebacterium genus, MicroSeq gave identical species-level identifications for the clinically significant Corynebacterium diphtheriae (4 of 4) and Corynebacterium jeikeium (8 of 8), but it identified only 50.0% (15 of 30) of other species (P < 0.01). Four isolates from the genera Arthrobacter, Brevibacterium, and Microbacterium, which could not be identified to the species level by conventional methods, were assigned a species-level identification by MicroSeq. The total elapsed time for running a MicroSeq identification was 15.5 to 18.5 h. These data demonstrate that the MicroSeq 500 16S bacterial sequencing kit provides a potentially powerful method for the definitive identification of clinical coryneform bacterium isolates.     

Identification of nonlipophilic corynebacteria isolated from dairy cows with mastitis

Nonlipophilic corynebacteria associated with clinical and subclinical mastitis in dairy cows were found to belong to four species: Corynebacterium amycolatum, Corynebacterium ulcerans, Corynebacterium pseudotuberculosis, and Corynebacterium minutissimum. These species may easily be confused. However, clear-cut differences between C. ulcerans and C. pseudotuberculosis were found in their acid production from maltotriose and ethylene glycol, susceptibility to vibriostatic agent O129, and alkaline phosphatase. Absence of growth at 20 degrees C and lack of alpha-glucosidase and 4MU-alpha-D-glycoside hydrolysis activity differentiated C. amycolatum from C. pseudotuberculosis and C. ulcerans. The mastitis C. pseudotuberculosis strains differed from the biovar equi and ovis reference strains and from caprine field strains in their colony morphologies and in their reduced inhibitory activity on staphylococcal beta-hemolysin. C. amycolatum was the most frequently isolated nonlipophilic corynebacterium.     

Cystic neutrophilic granulomatous mastitis: A clinicopathologic study of a distinct entity with supporting evidence of a role for Corynebacterium-targeted therapy

Cystic neutrophilic granulomatous mastitis (CNGM) is a distinct histopathologic entity characterized by neutrophilic and granulomatous inflammation surrounding clear cystic spaces. Rare gram-positive bacilli are sometimes identified within these cystic spaces. Studies in the literature have identified these gram-positive bacilli to be Corynebacterium species. We describe the clinicopathologic features of 7 cases of CNGM, including a case with evidence of Corynebacterium amycolatum. Patients were young to middle aged parous women ranging in age from 28 to 53 years (median age: 41 years). Gram-positive bacilli were identified in 4 cases, all within cystic spaces. Microbial culture from a 41-year old Hispanic woman grew Corynebacterium species on multiple occasions and Corynebacterium amycolatum was identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) on two separate occasions. Antibiotic susceptibility testing performed both times showed resistance to multiple antibiotics and susceptibility to vancomycin. Follow-up of all patients (range 3–12 months, median 6 months) showed a widely variable clinical course and varying response to a variety of treatment modalities. Five of the seven CNGM patients were parous, reproductive-aged Hispanic women who were born outside of the United States. Our findings further support the association of CNGM with corynebacteria and gram-positive bacilli. Furthermore, this study shows that Corynebacterium amycolatum, a nonlipophilic and multidrug-resistant corynebacterium can be associated with CNGM, hence the need for targeted antibiotic therapy. We propose identifying corynebacteria to the species level and performing antibiotic susceptibility testing in patients with CNGM because of the varied susceptibility testing profile that has been reported among different species of corynebacteria.     

High-performance liquid chromatography of mycolic acids as a tool in the identification of Corynebacterium, Nocardia, Rhodococcus, and Mycobacterium species

High-performance liquid chromatography of bromophenacyl esters of mycolic acid was used as an aid to assign a particular organism to one of four mycolic acid-containing genera. A gradient elution system, with methanol and chloroform, was used to distinguish representative mycolic acid patterns for the genera Corynebacterium, Rhodococcus, Nocardia, and Mycobacterium.     

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.     

Multicenter evaluation of the Vitek 2 anaerobe and Corynebacterium identification card

The new anaerobe and Corynebacterium (ANC) identification card for Vitek 2 was compared with a 16S rRNA gene sequencing (16S) reference method for accuracy in the identification of corynebacteria and anaerobic species. Testing was performed on a Vitek 2 XL system with modified software at three clinical trial laboratories. Reproducibility was determined with nine ATCC quality control strains that were tested 20 times over a minimum of 10 days at all three sites. A challenge set of 50 well-characterized strains and 365 recent fresh and frozen clinical isolates were included in the study. The expected positive and negative biochemical well reactions were also evaluated for substrate reproducibility. All strains were tested with the ANC card, and clinical isolates were saved for 16S rRNA gene sequencing. All reproducibility tests yielded expected results within a 95% confidence interval, except for that with Corynebacterium striatum ATCC 6940, for which identification failed at one trial site. For the challenge isolates, there was 98% correct identification, 5% low discrimination, and 2% incorrect identification, and 0% were unidentified. For clinical strains, there was 95.1% correct identification, 4.9% low discrimination, and 4.6% incorrect identification, and 0.3% were unidentified. The 4.6% (17/365) of clinical isolates that were incorrectly identified consisted of 14 isolates that were correct at the genus level and three that were incorrect at the genus level. The new ANC card met all performance criteria within a 95% confidence interval compared to the identification performance by 16S rRNA gene sequencing.     

Mycolic acids analysis from various species mycobacterium by high pressure liquid chromatography (HPLC)

HPLC is the most useful method to analyze various species of mycobacteria by using mycolic acids. The purpose was to prepare a library containing chromatographic patterns of mycolic acids derived from reference species Mycobacterium, which had been cultivated in standard conditions. 28 reference strains (27 ones from American Type Culture Collection and one cultivated from the vaccine M. bovis BCG) were used. The analysis of mycolic acids involved chromatographic separation of their bromophenacyl derivatives according to Centers for Disease Control recommendation. Mycolic acids profiles formed by HLPC were reproducible for all reference species in this study. Standard deviation of relative retention time of every peak did not exceed 2.5%. The species included into M. tuberculosis complex beyond M. bovis BCG shared the same mycolic acids pattern. HPLC is the only mean to distinguish M. tuberculosis from M. bovis BCG. The other studied stains had species specific patterns which differed from M. tuberculosis complex and M. bovis BCG. The prepared library comprising 28 reference elution profiles of mycolic acids from known mycobacteria species can be applied in diagnostic procedure of tuberculosis and mycobacteriosis.     

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.     

Formic Acid-based direct, on-plate testing of yeast and corynebacterium species by bruker biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry

An on-plate testing method using formic acid was evaluated on the Bruker Biotyper matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry system using 90 yeast and 78 Corynebacterium species isolates, and 95.6 and 81.1% of yeast and 96.1 and 92.3% of Corynebacterium isolates were correctly identified to the genus and species levels, respectively. The on-plate method using formic acid yielded identification percentages similar to those for the conventional but more laborious tube-based extraction.     

Phospholipase D activity of Corynebacterium pseudotuberculosis (Corynebacterium ovis) and Corynebacterium ulcerans, a distinctive marker within the genus Corynebacterium.

A search has been made for corynebacterial phospholipase D, "ovis toxin," a sphingomyelinase (phosphatidylcholine phosphohydrolase, EC 3.1.4.4), among a wide variety of corynebacteria. Phospholipase D activity has been found in strains exhibiting the biochemical properties characteristic of Corynebacterium pseudotuberculosis or of Corynebacterium ulcerans and in no other species of Corynebacterium. Methods for the assay of phospholipase D as a sphingomyelinase and methods for screening for phospholipase D in the presence of Corynebacterium equi on washed sheep blood agar are discussed.     

Analysis of mycolic acid cleavage products and cellular fatty acids of Mycobacterium species by capillary gas chromatography.

After growth and experimental conditions were established, the mycolic acid cleavage products, constituent fatty acids, and alcohols of representative strains of Mycobacterium tuberculosis, M. smegmatis, M. fortuitum complex, M. kansasii, M. gordonae, and M. avium complex were determined by capillary gas chromatography. Reproducible cleavage of mycolic acid methyl esters to tetracosanoic (24:0) or hexacosanoic (26:0) acid methyl esters was achieved by heating the sample in a high-temperature muffle furnace. The major constituent fatty acids in all species were hexadecanoic (16:0) and octadecenoic (18:1 omega 9-c, oleic) acids. With the exception of M. gordonae, 10-methyloctadecanoic acid was found in all species; moreover, M. gordonae was the only species tested which contained 2-methyltetradecanoic acid. M. kansasii was characterized by the presence of 2,4-dimethyltetradecanoic acid, M. avium complex by 2-eicosanol, and M. tuberculosis by 26:0 mycolic acid cleavage product. The mycolic acid cleavage product in the other five species tested was 24:0. Although a limited number of strains and species were tested, preliminary results indicate that this gas chromatographic method can be used to characterize mycobacterial cultures by their mycolic acid cleavage products and constituent fatty acid and alcohol content.     

Coryneform bacteria in infectious diseases: clinical and laboratory aspects.

Coryneform isolates from clinical specimens frequently cannot be identified by either reference laboratories or research laboratories. Many of these organisms are skin flora that belong to a large number of taxonomic groups, only 40% of which are in the genus Corynebacterium. This review provides an update on clinical presentations, microbiological features, and pathogenic mechanisms of infections with nondiphtheria Corynebacterium species and other pleomorphic gram-positive rods. The early literature is also reviewed for a few coryneforms, especially those whose roles as pathogens are controversial. Recognition of newly emerging opportunistic coryneforms is dependent on sound identification schemes which cannot be developed until cell wall analyses and nucleic acid studies have defined the taxonomic groups and all of the reference strains within each taxon have been shown by molecular methods to be authentic members. Only then can reliable batteries of biochemical tests be selected for distinguishing each taxon.     

Identification of major slowly growing pathogenic mycobacteria and Mycobacterium gordonae by high-performance liquid chromatography of their mycolic acids.

A rapid, reverse-phase high-performance liquid chromatography method was used to detect rho-bromophenacyl mycolic acid ester patterns for strains of four major pathogenic Mycobacterium species and for the most commonly encountered saprophytic species, Mycobacterium gordonae. Mycobacteria in low numbers (2.5 X 10(6) CFU) were detected and identified to the species level. Standard chromatographic patterns characteristic of each species were established. Simple pattern recognition enabled rapid identification of M. tuberculosis, M. kansasii, M. avium, M. intracellulare, and M. gordonae.     

Contribution of high-performance liquid chromatography to the identification of some Corynebacterium species by comparison of their corynomycolic acid patterns.

Reverse-phase high-performance liquid chromatography of corynomycolic acids provided a specific pattern for each Corynebacterium species studied. These data suggest that a fast and reproducible procedure is now available for bacteriological identification at the genus and at the species level of corynomycolic-acid-containing bacteria. Mass spectrometry analysis of post-column collected fractions provided the order of elution of some corynomycolic acids and isomers and showed the high specificity of the chromatographic assay which could be used for the routine bacteriological identification of some species belonging to the genus Corynebacterium.