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.     

Isolation of Corynebacterium tuscaniae sp. nov. from blood cultures of a patient with endocarditis

A strain of an unknown coryneform bacterium was repeatedly isolated in pure culture from the blood of a patient affected by endocarditis. Comparative 16S rRNA gene sequence analysis revealed that this isolate represented a new subline within the genus Corynebacterium. This new taxon can be identified by the presence of corynomycolic acids and its enzymatic activities and fermentation of sugars. Acid production from glucose and maltose, pyrazinamidase and alkaline phoshatase activities, and hippurate hydrolysis were the most characteristic phenotypic features of the bacterium. On the basis of both phenotypic and phylogenetic evidence, it is proposed that this isolate be classified as a novel species, Corynebacterium tuscaniae sp. nov. The type strain, ISS-5309, has been deposited in the American Type Culture Collection (ATCC BAA-1141) and in the Culture Collection of the University of G?teborg (CCUG 51321).     

Detection of mycoloylglycerol by thin-layer chromatography as a tool for the rapid inclusion of corynebacteria of clinical origin in the genus Corynebacterium

A chemotaxonomic study of some corynebacteria isolated from clinical samples revealed characteristic thin-layer chromatographic patterns for meso-diaminopimelic acid containing species included in the genera Corynebacterium, Dermabacter and Brevibacterium. Notably, a specific compound was consistently detected in mycolic acid containing species of the genus Corynebacterium. This compound was composed by glycerol and mycolic acids and structural analyses carried out by fast atom bombardment mass spectrometry in C. minutissimum confirmed its identification as mycoloylglycerol. The chain length of mycoloyl groups in this molecule ranged from 28 to 34 carbon atoms, being mono-, di- or triunsaturated. Detection of mycoloylglycerol by thin-layer chromatography may be thus useful for the rapid inclusion of a great variety of corynebacteria of clinical origin in the genus Corynebacterium in laboratories employing chromatographic techniques as an adjunct for the identification of these microorganisms.     

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.     

rpoB gene sequencing for identification of Corynebacterium species

The genus Corynebacterium is a heterogeneous group of species comprising human and animal pathogens and environmental bacteria. It is defined on the basis of several phenotypic characters and the results of DNA-DNA relatedness and, more recently, 16S rRNA gene sequencing. However, the 16S rRNA gene is not polymorphic enough to ensure reliable phylogenetic studies and needs to be completely sequenced for accurate identification. The almost complete rpoB sequences of 56 Corynebacterium species were determined by both PCR and genome walking methods. In all cases the percent similarities between different species were lower than those observed by 16S rRNA gene sequencing, even for those species with degrees of high similarity. Several clusters supported by high bootstrap values were identified. In order to propose a method for strain identification which does not require sequencing of the complete rpoB sequence (approximately 3,500 bp), we identified an area with a high degree of polymorphism, bordered by conserved sequences that can be used as universal primers for PCR amplification and sequencing. The sequence of this fragment (434 to 452 bp) allows accurate species identification and may be used in the future for routine sequence-based identification of Corynebacterium species.     

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.     

Nonspecific immunostimulant activities of synthetic trehalose-6,6''-diesters (lower homologs of cord factor).

Mycobacterial cord factors (6,6'-diesters of trehalose with mycolic acids ranging from C80 to C90) have been shown to protect mice effectively against infection with Klebsiella pneumoniae or with Listeria monocytogenes. Our present findings indicate that the low-molecular-weight cord factor of Corynebacterium diphtheriae (with corynomycolic acids ranging from C28 PTO C36) is equally active. Moreover, its synthetic analog (with synthetic C32 mycolic acid) has the same activity. Two lower synthetic 6,6'-diesters of trehalose with C22 acids, which are described here for the first time, as well as dipalmitate and a dioleate of sucrose, were found inactive. The synthetic C76 trehalose diesters, which are capable of enhancing nonspecific resistance to infection, increase the immune response in mice, even when injected in metabolizable oil. They induce in the injected paws an inflammatory process weaker and more transient than the natural cord factor.     

Multiple-genome comparison reveals new loci for Mycobacterium species identification

To identify loci useful for species identification and to enhance our understanding of the population structure and genetic variability of the genus Mycobacterium, we conducted a multiple-genome comparison of a total of 27 sequenced genomes in the suborder of Corynebacterineae (18 from the Mycobacterium genus, 7 from the Corynebacterium genus, 1 each from the Nocardia and Rhodococcus genera). Our study revealed 26 informative loci for species identification in Mycobacterium. The sequences from these loci were used in a phylogenetic analysis to infer the evolutionary relations of the 18 mycobacterial genomes. Among the loci that we identified, rpoBC, dnaK, and hsp65 were amplified from 29 ATCC reference strains and 17 clinical isolates and sequenced. The phylogenetic trees generated from these loci show similar topologies. The newly identified dnaK locus is more discriminatory and more robust than the widely used hsp65 locus. The length-variable rpoBC locus is the first intergenic locus between two protein-encoding genes being used for mycobacterial species identification. A multilocus sequence analysis system including the rpoBC, dnaK, and hsp65 loci is a robust tool for accurate identification of Mycobacterium species.     

Matrix-assisted laser-desorption/ionization BIOTYPER: experience in the routine of a University hospital

Matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) is positioned at the forefront of bacterial identification in the future. Its performance needed to be evaluated in a routine Bacteriology laboratory to determine its true benefits. A prospective study was carried out in the Bacteriology laboratory of the Pellegrin University Hospital in Bordeaux, France, from April to May 2009. Bacterial isolates from clinical samples were identified by conventional phenotypic bacteriological methods [Phoenix (Becton-Dickinson) or API strips (bioMérieux)] and in parallel with a mass spectrometer (Ultraflex III TOF/TOF and the biotyper database from Bruker Daltonics). In case of a discrepancy between these results at the genus level, a 16S rRNA and/or rpoB gene sequencing was performed. Of the 1013 bacteria tested, 837 (82.6%) were correctly identified at the species level by MALDI-TOF mass spectrometry (MS) without extraction and 189 after extraction, i.e. 986 (97.3%) were correctly identified at the species level by MALDI-TOF MS, vs. 945 (93.2%) by phenotypic methods. Indeed, the extraction step was necessary for only 15% of the isolates. These results were even better when considering the genus, reaching almost 99% with MALDI-TOF MS and 98% with phenotypic methods. The performance of MALDI-TOF MS is very attractive considering its efficiency and rapidity, and the technique constitutes a precious tool for bacteriological identification in a routine laboratory.     

Species identities and antimicrobial susceptibilities of corynebacteria isolated from various clinical sources

Over a 14-month period, 415 clinical isolates of coryneform gram-positive rods were recovered from various sources and identified to the species level according to recent identification schemes.Corynebacterium urealyticum, Corynebacterium striatum, Corynebacterium amycolatum, andCorynebacterium jeikeium predominated, accounting for 63% of all isolates.Corynebacterium accolens, Corynebacterium striatum, Corynebacterium argentoratense, Corynebacterium propinquum andCorynebacterium pseudodiphtheriticum were mostly recovered from the respiratory tract, whereasCorynebacterium afermentans, CDC group G, andCorynebacterium jeikeium were mainly isolated from blood. None of the isolates was identified asCorynebacterium diphtheriae orCorynebacterium xerosis. Ampicillin resistance was detected inCorynebacterium jeikeium (96%) andCorynebacterium urealyticum (99%) and varied amongCorynebacterium amycolatum (56%) and CDC group G (26%). These data emphasize the need for an accurate identification of coryneform organisms at the species level and for antimicrobial susceptibility testing of these organisms.     

Identification of non-diphtheriae corynebacterium by use of matrix-assisted laser desorption ionization-time of flight mass spectrometry

We evaluated the Bruker Biotyper matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry for identification of 92 clinical isolates of Corynebacterium species in comparison to identification using rpoB or 16S rRNA gene sequencing. Eighty isolates (87%) yielded a score of ≥1.700, and all of these were correctly identified to the species level with the exception of Corynebacterium aurimucosum being misidentified as the closely related Corynebacterium minutissimum.     

Phenotypic and phylogenetic characterization of a new Corynebacterium species from dogs: description of Corynebacterium auriscanis sp. nov.

Six strains of a previously undescribed catalase-positive coryneform bacterium isolated from clinical specimens from dogs were characterized by phenotypic and molecular genetic methods. Biochemical and chemotaxonomic studies revealed that the unknown bacterium belonged to the genus Corynebacterium sensu stricto. Comparative 16S rRNA gene sequencing showed that the six strains were genealogically highly related and constitute a new subline within the genus Corynebacterium; this subline is close to but distinct from C. falsenii, C. jeikeium, and C. urealyticum. The unknown bacterium from dogs was distinguished from all currently validated Corynebacterium species by phenotypic tests including electrophoretic analysis of whole-cell proteins. On the basis of phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be classified as a new species, Corynebacterium auriscanis. The type strain of C. auriscanis is CCUG 39938(T).     

Prosthetic valve endocarditis caused by Corynebacterium diphtheriae in a patient with pemphigus vulgaris.

The clinical and bacteriological findings in a case of prosthetic aortic valve endocarditis caused by Corynebacterium diphtheriae are presented. The patient died despite adequate medical therapy. This appears to be the first report of endocarditis caused by this species in a prosthetic aortic valve.     

Corynebacterium nigricans sp. nov.: proposed name for a black-pigmented Corynebacterium species recovered from the human female urogenital tract

Six independent isolates of an unusual black-pigmented Corynebacterium species (strains CN-1, CN-2, CN-3415, W70124, 91-0032, and 92-0360) were recovered from the human female urogenital tract. Four of the six source patients had complications of pregnancy, including spontaneous abortion, preterm labor, and low amniotic fluid volume at the time of the pathogen isolation. One isolate was recovered from a vaginal ulcer. All six strains yielded black-pigmented colonies on sheep blood agar, chocolate agar, and colistin-nalidixic acid agar after 24 to 48 h of incubation at 35 degrees C. The dry, adherent colonies pitted the agar surface. The cells were coccobacillary to rod-shaped, catalase positive, nonmotile, and nonlipophilic. Only five of six isolates were available for characterization. Biochemical and chemotaxonomic studies revealed that the strains belong to the genus Corynebacterium but differ from known corynebacterial species. Comparative 16S rRNA gene sequence analysis showed that the strains are closely related and form a new subline within the genus Corynebacterium. We propose the name Corynebacterium nigricans sp. nov. for this group of coryneforms. The type strain of Corynebacterium nigricans is CN-1. It is deposited in the American Type Culture Collection (assigned strain number ATCC 700975) and in the Institute Pasteur collection (assigned strain number CIP 107346).     

Evaluation of five test-kits —API,AuxoTab, Enterotube,PathoTec and R/B— for identification ofEnterobacteriaceae

Five test-kits—API, AuxoTab, Enterotube, PathoTec and R/B-have been compared for accuracy in individual tests and for identification on the genus or species level with conventional biochemical tests on 329 strains belonging toEnterobacteriaceae. The API system was found to be the most reliable followed by Enterotube, R/B, PathoTec and AuxoTab. The API system was used in parallel with the standard biochemical testing in the routine bacteriological work for identification of aerobic gramnegative fermentative rods from clinical specimens during a 10 weeks period. 2351 of 2404 strains were identified with this system and the system was found to be very reliable.     

Evaluation of the rapid CORYNE identification system for Corynebacterium species and other coryneforms.

The Rapid CORYNE system for identification of aerobic, nonsporeforming, gram-positive rods was evaluated according to the manufacturer's instructions with 177 organisms. After inoculation with a heavy suspension of growth, strips containing 20 cupules were incubated for 24 h, reagents were added, and the results of 21 biochemical reactions were recorded as numerical profiles. The strains consisted of pathogenic species of the genus Corynebacterium, primarily C. diphtheriae (n = 29), opportunistic species of Corynebacterium including C. jeikeium (n = 75), recognized species of non-corynebacteria such as Gardnerella and Arcanobacterium (n = 51), and Centers for Disease Control (CDC) coryneform groups (n = 22). Results from single tests read after 24 h yielded correct identifications to species level with no additional tests for 26 (89.7%) of the pathogenic species; 64 (85.3%) of the opportunistic organisms; 51 (100%) of the non-corynebacteria, and 8 (36.4%) of the CDC coryneform groups. Supplemental tests produced the correct identification for three additional pathogenic isolates (100% total) and four additional isolates from the opportunistic species (90.6% total). Twelve of the 15 isolates not identified by the system were in the CDC coryneform groups. Four of the six misidentified and one of the unidentified isolates were C. matruchotii, which was not included in the data base. The system is an excellent rapid alternative to conventional biochemical tests.     

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.     

Carbohydrate patterns,cellular lipoquinones,fatty acids and phospholipids of the genus Pasteurella sensu stricto

The carbohydrate patterns, isoprenoid quinones, fatty acids and phospholipids of the species of the genus Pasteurella sensu stricto were investigated to evaluate their taxonomic significance and their applicability for the identification of these bacteria. Forty-six representative strains of the 11 species of Pasteurella were examined. The data obtained indicated that the carbohydrate patters are species or subspecies specific and may, therefore, become an important and useful diagnostic tool. Fatty acids and phospholipids showed a feature characteristic of the members of the genus and the isoprenoid quinones exhibited a mostly genus-specific feature with remarkable quantitative differences.     

Isolation, identification and clinical significance of species of the Flavobacterium genus

Strictly aerobic Gram negative bacteria are found more and more often in human pathological specimens and in the environment. Amongst these bacteria, Flavobacterium poses problems of bacteriological diagnosis as their precise taxonomy is still recent. In this study, conducted on 321 strains of Flavobacterium from various sources, but essentially from patients in the intensive care unit, the authors define the methods and features of identification and the clinical significance of the species of the Flavobacterium genus. One species is particularly important in medical bacteriology, Flavobacterium meningosepticum which can cause septicaemia and neonatal meningitis which are difficult to treat.     

Hardware Infection with Corynebacterium spp.: a Case Report and Review of the Literature

There are over 120 species in the genus Corynebacterium, many of which are generally non-pathogenic. Isolates are frequently recovered from human clinical specimens, but because Corynebacterium spp. are known to be colonizers of human skin and mucosal surfaces, they are often not identified to the species level due to limitations in identification methods and lack of consensus concerning their clinical relevance. In this article, we present a case report demonstrating the difficulty and importance of distinguishing different strains of Corynebacterium isolated from a patient with a post-operative spine infection and review the current literature. This case illustrates not only the role of molecular diagnostics in determining the etiology of infection, but also the diagnostic subtleties that remain for the clinical microbiologist and infectious disease practitioner regarding the genus Corynebacterium.