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).     

Isolation and characterization of a black-pigmented Corynebacterium sp. from a woman with spontaneous abortion

An unusual black-pigmented coryneform bacterium was isolated from the urogenital tract of a woman who experienced a spontaneous abortion during month 6 of pregnancy. Biochemical and chemotaxonomic analyses demonstrated that the unknown bacterium belonged to the genus Corynebacterium. Phylogenetic analysis based on 16S rRNA sequences (GenBank accession no. AF220220) revealed that the organism was a member of a distinct subline which includes uncultured Corynebacterium MTcory 1P (GenBank accession no. AF115934), derived from prostatic fluid, and Corynebacterium CDC B8037 (GenBank accession no. AF033314), an uncharacterized black-pigmented coryneform bacterium. On the basis of chemotaxonomic and phylogenetic evidence, this organism probably represents a new species and is most closely related to the uncharacterized Centers for Disease Control and Prevention group 4 coryneforms. Our strain is designated CN-1 (ATCC 700975).     

Corynebacterium riegelii sp. nov., an Unusual Species Isolated from Female Patients with Urinary Tract Infections

Four strains of an unknown coryneform bacterium were isolated in pure culture from females with urinary tract infections. Strong urease activity and the ability to slowly ferment maltose but not glucose were the most significant phenotypic features of this catalase-positive, nonmotile, nonlipophilic, rod-shaped bacterium which served to distinguish it from all other presently defined coryneform bacteria. Chemotaxonomic investigations demonstrated that the unknown bacterium belonged to the genus Corynebacterium. Comparative 16S rRNA gene sequence analysis revealed that the isolates were genealogically identical and represented a new subline within the genus Corynebacterium, for which the designation Corynebacterium riegelii sp. nov. is proposed. The type strain of Corynebacterium riegelii is CCUG 38180 (DSM 44326, CIP 105310).     

Corynebacterium imitans sp. nov. isolated from patients with suspected diphtheria.

A 5-month-old boy of a Romanian family traveling via Ukraine to Poland developed a respiratory disease that resembled and that was initially diagnosed as pharyngeal diphtheria. The child recovered after treatment with antidiphtheria antitoxin. A coryneform bacterium had been isolated from a nasopharyngeal specimen from the child and was initially identified as an atypical Corynebacterium diphtheriae strain. Seven adults who had contact with either the child or an adult contact person also developed symptoms of pharyngeal diphtheria, were also treated with antitoxin, and recovered uneventfully. Coryneform bacteria similar to that originating from the index patient were also isolated from the throat swabs of three adults. Detailed biochemical and chemotaxonomic investigations revealed that the coryneform bacteria belonged to the genus Corynebacterium and could be differentiated from all other defined species of this genus. Ribotyping and pulsed-field gel electrophoresis demonstrated that all four patients' isolates were of clonal origin. The diphtheria toxin gene and its product were not detected either by PCR assays or by the Elek test, making a possible disease association of the Corynebacterium more unlikely. Comparative 16S rRNA gene sequence analysis revealed that the coryneform bacterium represented a new subline within the genus Corynebacterium, for which the name Corynebacterium imitans sp. nov. is proposed. The type strain is NCTC 13015 (DSM 44264; CCUG 36877).     

Two similar but atypical strains of coryneform group A-4 isolated from patients with endophthalmitis.

Corynebacterium species and other coryneform organisms isolated from clinical specimens are frequently considered contaminants. We isolated two strains of a gram-positive organism from the vitreous fluid of two patients with endophthalmitis who had previously received intraocular lens transplants. The biochemical characteristics and gas chromatographic patterns of both isolates were similar to those of coryneform group A-4 strains. Major differences included esculin hydrolysis, nitrate reduction, growth pigment, and lactic acid production. These two strains along with a limited number of strains collected at the Special Bacterial Pathogens Laboratory (Division of Bacterial Diseases, Centers for Disease Control, Atlanta, Ga.) may represent a subgroup of coryneform group A-4. Results of in vitro susceptibility testing performed with antimicrobial agents commonly used to treat patients with bacterial endophthalmitis underscore the importance of determining MBCs for slow-growing organisms. This report cautions microbiologists not to discard organisms frequently considered contaminants when isolated from body fluids that are normally sterile and from patients receiving local steroids.     

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.     

Microbiological and clinical features of Corynebacterium urealyticum: urinary tract stones and genomics as the Rosetta Stone

Corynebacterium urealyticum , formerly known as coryneform CDC group D2, was first recognized to be involved in human infections 30 years ago. It is a slow-growing, lipophilic, asaccharolytic and usually multidrug-resistant organism with potent urease activity. Its cell wall peptidoglycan, menaquinone, mycolic and cellular fatty acid composition is consistent with that of the genus Corynebacterium . DNA–DNA hybridization studies and 16S rDNA sequencing analysis have been used to determine the degree of relatedness of C. urealyticum to other corynebacterial species. The genome of the type strain consists of a circular chromosome with a size of 2 369 219 bp and a mean G + C content of 64.2%, and analysis of its genome explains the bacterium's lifestyle. C. urealyticum is a common skin colonizer of hospitalized elderly individuals who are receiving broad-spectrum antibiotics. It is an opportunistic pathogen causing mainly acute cystitis, pyelonephritis, encrusted cystitis, and encrusted pyelitis. More infrequently, it causes other infections, but mainly in patients with urological diseases. Infections are more common in males than in females, and treatment requires administration of antibiotics active against the organism in vitro , mainly glycopeptides, as well as surgical intervention, the latter mostly in cases of chronic infection. Mortality directly associated with infection by this organism is not frequent, but encrusted pyelitis in kidney-recipient patients may cause graft loss. The outcome of infection by this organism is reasonably good if the microbiological diagnosis is made and patients are treated appropriately.     

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).     

Bacteremia due to a novel Microbacterium species in a patient with leukemia and description of Microbacterium paraoxydans sp. nov

A yellow-pigmented coryneform rod was isolated from the blood of a child with acute lymphoblastic leukemia who was perfused with a central venous catheter. The culture bottles were positive twice, at a 2-month interval. The isolate was identified as a Microbacterium sp. and studied along with five other similar strains. Phenotypic, chemotaxonomic, and genetic characteristics indicated that they are closely related to Microbacterium oxydans but that they belong to a distinct species, for which the name Microbacterium paraoxydans sp. nov. is proposed. The type strain of M. paraoxydans is CF36(T) = DSM 15019(T). The G+C content of its DNA is 69.9 mol%.     

Identification of Turicella otitidis isolated from a patient with otorrhea associated with surgery: differentiation from Corynebacterium afermentans and Corynebacterium auris.

Turicella otitidis is a newly described coryneform bacterium isolated from middle ear fluids. We report here on the diagnosis of a strain isolated from otorrhea. The API Coryne system (bioMérieux, Marcy I'Etoile, France) used alone failed to differentiate T. otitidis, Corynebacterium afermentans, and Corynebacterium auris (ANF group). Biochemical tests such as DNase, enzymatic reactions (API ZYM; bioMérieux), and carbon substrate assimilation tests (Biotype 100; bioMérieux) allow presumptive identification. However, only chemotaxonomy and molecular biology can achieve unequivocal differentiation among these three species.     

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.     

Infections caused by antibiotic-resistantCorynebacterium group D2

A coryneform bacteria known as Corynebacterium group D2 pending definitive taxonomic classification, has emerged as a human pathogen. Although first described 16 years ago, the organism has only recently been implicated in clinical infections. It differs from all previously recognized species of Corynebacterium, and in common with Corynebacterium jeikeium exhibits a broad spectrum of antimicrobial resistance, being sensitive to only vancomycin, teicoplanin and some quinolones. The microorganisms is mainly involved in urinary tract infections, and its strong urease activity seems to play an important role in its pathogenicity. Corynebacterium jeikeium and group D2 are very different from the clinical and microbiological point of view. Corynebacterium group D2, like other coryneform bacteria, may be isolated from healthy skin in hospitalized patients, mainly females, while Corynebacterium jeikeium is more prevalent in males. Microbiologists should be aware that such a slow-growing microorganism may be responsible for clinical infections and should not be overlooked in routine cultures of clinical specimens.     

Evaluation of API Coryne system for identifying coryneform bacteria.

AIM--To identify rapidly and accurately coryneform bacteria, using a commercial strip system. METHODS--Ninety eight strains of Corynebacterium species and 62 additional strains belonging to genera Erysipelothrix, Oerskovia, Rhodococcus, Actinomyces, Archanobacterium, Gardnerella and Listeria were studied. Bacteria were identified using conventional biochemical tests and a commercial system (API-Coryne, BioMèrieux, France). Fresh rabbit serum was added to fermentation tubes for Gardnerella vaginalis isolates. RESULTS--One hundred and five out of the 160 (65.7%) organisms studied were correctly and completely identified by the API Coryne system. Thirty five (21.8%) more were correctly identified with additional tests. Seventeen (10.6%) organisms were not identified by the system and three (1.9%) were misidentified. CONCLUSIONS--The system was a good alternative for identification of coryneform organisms. When occasionally performed with some additional tests, this method permits reliable and rapid identification of coryneform organisms compared with conventional methods.     

Human infections caused by Brevibacterium casei, formerly CDC groups B-1 and B-3.

Forty-one clinical strains of CDC coryneform groups B-1 and B-3 were compared biochemically, by analysis of cell wall sugars, amino acids, and cellular fatty acids, and by DNA relatedness to the type strains of Brevibacterium casei, Brevibacterium epidermidis, and Brevibacterium linens. Twenty-two strains were shown to be B. casei, while five other strains formed a phenotypically inseparable genomospecies in the same genus. The remaining isolates were genetically heterogeneous, and most are probably members of the genus Brevibacterium. They were not further identified, but they were biochemically distinguishable from B. casei. Eleven of the clinical strains of B. casei were isolated from blood, and two each were isolated from cerebrospinal fluid and from pleural fluid. At least five isolates were from multiple blood or cerebrospinal fluid cultures. To our knowledge, these strains are the first described clinical isolates identified as B. casei, which was previously considered to be a nonpathogenic species.     

Recognition of Dermabacter hominis, formerly CDC fermentative coryneform group 3 and group 5, as a potential human pathogen.

Thirty strains of fermentative coryneform-like bacteria designated CDC fermentative coryneform group 3 and coryneform group 5 were compared biochemically by cellular fatty acid analysis and by DNA relatedness with the type strain of Dermabacter hominis, ATCC 49369. DNA from 22 strains of both CDC groups showed 69 to 96% relatedness (hydroxyapatite method) to labeled DNA from ATCC 49369 and to DNA from CDC group 3 strain G4964, and the strains are considered to belong to D. hominis. The remaining eight strains were genetically but not phenotypically differentiable from D. hominis. They were genetically heterogeneous, but hybridization results indicated that they probably belong to the genus Dermabacter. Thirteen of the 22 D. hominis strains and all 8 of the other Dermabacter strains had been isolated from blood, which indicates the pathogenic potential of this species and genus.     

Characterization of Brevibacterium spp. from clinical specimens.

Nonfermenting coryneform bacteria identified as Brevibacterium spp. were isolated from routine clinical specimens. Four strains were derived from peritoneal fluid and has presumably been involved in the pathogenesis of continuous ambulatory peritoneal dialysis peritonitis. Another five isolates most probably represented skin contaminants. Cell wall and lipid analyses confirmed the genus identification. Strains in this taxon are difficult to distinguish from other biochemically inactive and nonmotile coryneform species but show characteristics cellular fatty acid profiles. In vitro susceptibilities to commonly used antibiotics were determined.     

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.     

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.     

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.     

Differentiation of Corynebacterium spp., Listeria spp., and related organisms by using fluorogenic substrates.

A total of 228 strains of Arcanobacterium haemolyticum, Corynebacterium spp., Erysipelothix rhusiopathiae, and Listeria spp. were investigated for their abilities to hydrolyze 60 different fluorogenic 4-methylumbelliferyl-linked and beta-naphthylamide-linked substrates within 6 and 24 h of incubation. The hydrolysis of a group of 16 fluorogenic substrates, and in particular, the glycosidase tests, in most cases showed high separation values at the genus level. When used in combination with other biochemical tests, these tests improved the differentiation of coryneform bacteria and phenotypically similar organisms.