ABSTRACT
Nineteen new Corynebacterium species or taxa described since 1995 have been associated with human disease. We report the characteristics of 72 strains identified as or most closely resembling 14 of these newer, medically relevant Corynebacterium species or taxa, as well as describe in brief an isolate of Corynebacterium bovis, a rare pathogen for humans. The bacteria studied in this report were nearly all derived from human clinical specimens and were identified by a polyphasic approach. Most were characterized by nearly full 16S rRNA gene sequence analysis. Some isolates were recovered from previously unreported sources and exhibited unusual phenotypes or represented the first isolates found outside Europe. Products of fermentation, with emphasis on the presence or absence of propionic acid, were also studied in order to provide an additional characteristic with which to differentiate among phenotypically similar species.
Nineteen species or taxa consistent with the genus Corynebacterium and recovered from human clinical material have been described or validated since 1995 (10, 11). Many of these species were first described from European laboratories and have not as yet been reported from human clinical specimens in other geographic areas. We report here the characteristics observed for 73 strains of Corynebacterium which had been studied with a polyphasic approach and could be assigned to one of 15 newer or rarely recovered species or a recently described taxon group.
Many strains exhibited phenotypic features which differed from those presented in the original descriptions of the new species, which were occasionally based on a single strain or small number of strains. Each of the newer or rare species found here, as well as most other medically relevant Corynebacterium species, was studied for the detection of propionic acid as a fermentation product. This had been found previously to provide additional chemotaxonomic data with which to discriminate among phenotypically close Corynebacterium species (7, 8).
Strains and methods.
The strains studied are described in Table 1. Nearly all were referred from Canadian provincial reference centers as isolates in pure culture and were recovered from human clinical material. Identifier numbers are those of the Special Bacteriology Laboratory of the National Microbiology Laboratory (NML), Health Canada, except where otherwise described. Some of the older cultures were originally unidentifiable or poorly identifiable until retrospectively characterized by molecular identification methods. Detailed clinical information on underlying diseases of the patients was generally not available.
Newer or rare Corynebacterium species in Canadaa
Conventional biochemical tests, salt tolerance, Christie-Atkins-Munch-Peterson (CAMP) reaction test, tyrosine hydrolysis, esculin or bile esculin hydrolysis, starch hydrolysis, DNase production, and type and degree of hemolysis on 5% sheep blood agar were done as described previously (2) or by standard methods (19, 20). The Corynebacterium bovis strain was the only isolate studied for O-nitrophenyl-β-d-galactopyranoside (Sigma-Aldrich Canada Ltd.) production; C. bovis has been described as being positive (19). Reduction of both nitrate and nitrite was added to the panel of tests after the description of C. simulans (31). Lipophilia was determined by comparing growth after 72 h at 37°C in brain heart infusion broth (Becton Dickinson, Sparks, Md.) with that in brain heart infusion broth with 1% (vol/vol) Tween 80 (Becton Dickinson) (22).
Semiquantitative estimates of growth on plate agar were evaluated as no growth or from 1+ (poor) to 4+ (excellent). Extended (48 to 72 h) incubations were required for some taxa. For each strain, temperature preference was determined by estimating semiquantitatively the amount of growth on 5% sheep blood agar after incubation in air at 25°C, 37°C, or 42°C; its atmospheric preference after incubation in air, 5% CO2, or anaerobically (Anaerocult anaerobic jar; EM Science, Gibbstown, N.J.) at 37°C; and ability to grow on media lacking blood, such as tryptic soy agar (Becton Dickinson). Products of glucose fermentation for the taxa shown in Table 2 were analyzed here as described previously (1) or as described in other publications. Vibriostatic disk O129 was applied to most of these taxa for at least one strain, and a zone of 11 to 36 mm was considered to show sensitivity while one of 0 to 10 mm was considered to show resistance (34).
Corynebacterium strains classified by production of propionic acida
API Coryne and API Zym strips were used as described by the manufacturer (Biomérieux, Montréal, Canada). Cellular fatty acid composition extraction and analysis were done after growth on 5% sheep blood agar at 35°C to 37°C in 5% CO2 for 24 to 72 h, depending on the species' requirements (2). Version 3.9 or version 4.0 of the MIDI method Aerobe (MIDI, Newark, Del.) and library generation system software (MIDI) were used to make entries for an in-house library, LCDC1. Each strain analyzed was compared to LCDC1 and CLIN (MIDI version 3.9 or 4.0) libraries. The methods to extract, amplify, and sequence the 16S rRNA gene, yielding about 1,400 to 1,500 bp, have been described (1). Establishing the closest known relatives was done by performing database searches of GenBank with the Blast program or of in-house data.
Assignment to specific Corynebacterium species was made based on demonstrating phenotypic characteristics consistent with literature values for that taxon group, with differences otherwise being noted in Table 1. Identifications were made based on a strain’s having a cellular fatty acid composition and products of fermentation consistent with species of the genus Corynebacterium (2, 7, 8, 10) and based on a significantly high degree of identity (>98.0% to 100%) with respect to 1,300 to 1,500 bp of 16S rRNA gene sequence when aligned with sequences derived from validated Corynebacterium species (derived from GenBank), with the Corynebacterium species assigned being first choice upon sequence comparison. Some strains from the years 1981 to 1998 did not have 16S rRNA gene sequence analysis done but were otherwise consistent with the relevant species description. Therefore, some C. glucuronolyticum bacteria were identified based on production of β-glucuronidase and other features (14), some CAMP-positive isolates which fermented glucose only slowly plus other features were identified as C. coyleae (17), and some C. mucifaciens bacteria were identified based on the observance of characteristic yellow, mucoid colonies and other relevant traits (15).
The 16S rRNA gene sequences for 20 of 73 strains reported here have been deposited in GenBank and are further described in Table 1. Identification was based on characteristics described in standard texts (10, 11) as well as for C. argentoratense (26), C. bovis (13, 19), C. confusum (9), C. coyleae (17), C. durum (21, 24), C. falsenii (29), C. glucuronolyticum (14), C. imitans (12), C. kroppenstedtii (3), C. mucifaciens (15), C. riegelii (16), C. simulans (31), C. sundsvallense (4), C. thomssenii (33), and a black-pigmented Corynebacterium taxon group (28).
Of the 15 Corynebacterium species or taxa described here, 11 (C. argentoratense, C. confusum, C. coyleae, C. falsenii, C. glucuronolyticum, C. imitans, C. kroppenstedtii, C. mucifaciens, C. riegelii, C. simulans, and C. thomssenii) have not been previously reported as being recovered outside Europe. The C. argentoratense strain (NML 98-0075, GenBank accession no. AF537589) was recovered from a blood culture, not from a throat specimen, lower respiratory tract, or ear (25, 26), and lacked significant chymotrypsinase activity. C. bovis identified by contemporary methods appears to be a very rare human pathogen (5, 13). This strain of C. bovis, a blood culture isolate (NML 99-0033, GenBank accession no. AF537590), was oxidase positive and did not produce β-galactosidase by two methods (API Coryne and API Zym) but was otherwise consistent with that species (13).
The abscess-derived strain of C. confusum (NML 99-0145, GenBank accession no. AF537591) found here contained tuberculostearic acid as a cellular fatty acid and produced propionic acid as a fermentation product (Table 2), but unlike the isolate described previously (9), this isolate was CAMP positive. Glucose was utilized if conventional methods were used, but this isolate was glucose negative with the API Coryne strip, as found previously (9). Such isolates would have to be further discerned from C. propinquum or C. coyleae by the CAMP reaction, production of propionic acid, nitrate reduction, and tyrosine hydrolysis.
The originally described strains of C. coyleae were derived from sterile body fluids, including blood cultures and pleural fluid (17). We report here the first recoveries of this species from urine and from an abscess. 16S rRNA gene sequence data for one of the urine isolates (NML 97-0171) has been deposited as GenBank accession no. AF537592. Some strains tested with the O129 disk were found to be resistant. Acid was produced from ribose, fructose, and mannose in addition to glucose, as found by Funke et al. (17), which aids in differentiation from other taxa.
Three of the C. durum isolates described here were also reported in brief by Rassoulian Barrett et al. (21). C. durum is one of the few Corynebacterium species which can utilize mannitol and hydrolyze esculin, and these two tests were variable among our isolates. Members of this species have been recovered from respiratory tract specimens (24) or throat cultures of healthy volunteers (30) but not from blood cultures or an abscess, as found here. C. durum blood culture isolate NML 99-0047 (GenBank accession no. AF537593) did not reduce nitrate and fermented lactose. Several Canadian strains produced β-galactosidase, the detection of which had been observed previously (30). The C. falsenii strain reported here (NML 97-0205, GenBank accession no. AF537594) did not exhibit a yellow pigment which intensified with time but otherwise was highly consistent with the species description (29).
Table 1 test results for 11 strains of C. glucuronolyticum were as reported by Funke et al. (14). However, recovery of C. glucuronolyticum or its junior synonym C. seminale from blood cultures, disk fluid (NML 98-0013, GenBank accession no. AF537595), or peritoneal fluid (NML 98-0098, GenBank accession no. AF537596) had not been reported previously (6, 14, 23). Isolates of C. imitans found here were primarily recovered from blood cultures, including NML 01-0063 (GenBank accession no. AF537597), and all isolates were resistant to the O129 disk. Four out of six strains reduced nitrate by either the conventional or rapid strip method, and two of six did not reduce nitrate by either method, as described previously by Funke et al. (12).
C. kroppenstedtii was originally described based on characteristics of one strain from the sputum of a woman with pulmonary disease (3). Strains here were also recovered from respiratory specimens as well as from pus and blood cultures. One strain reduced nitrate (NML 87-0354, from pus), and one blood culture isolate (NML 00-0244, GenBank accession no. AF537598) was weakly reactive by conventional methods for mannitol and lactose but did not hydrolyze esculin. The remaining three strains hydrolyzed esculin, as found by Collins et al. (3).
Twenty-three strains of C. mucifaciens are described in Table 1, with 21 of 23 isolates being found to be highly consistent with the description by Funke et al. (15). One blood culture isolate, observed to have typical microbiological and morphological characteristics for this species, was selected for deposit in GenBank (NML 87-0436, GenBank accession no. AF357599). Many of the clinical sources shown in Table 1 had been reported previously by Funke et al. (15), with the exception of recovery from dialysate (NML 95-0019) and peritoneal fluid (NML 95-0189 and 95-0369). Blood culture isolate NML 01-0118 (GenBank accession no. AF537600, with 99.1% identity to C. mucifaciens [Gen Bank accession no. Y11200]) was found to have some aberrant characteristics in that it was observed to have an only slightly mucoid colony, exhibited weak beta-hemolysis, and was reactive in both sucrose and maltose with both the rapid strip and conventional methods.
It is interesting that, by phylogenetic analysis of the 16S rRNA gene sequences, C. coyleae, C. afermentans, and C. mucifaciens were most closely related (97.7% to 98.5% identity) with each other (15) but at times are difficult to differentiate even after using a polyphasic approach, for phenotypically aberrant strains. From Table 1, blood culture isolate NML 97-0160 (GenBank accession no. AF357601), by 16S rRNA sequence analysis, was closest to (98.3% identity with) C. mucifaciens (GenBank accession number Y11200) (Table 1) but differed from typical strains of that species by lacking pigment, having a smooth, not mucoid colony, hydrolyzing urea, and being resistant to the O129 disk. This and other aberrant strains may actually represent additional taxonomic diversity, requiring further study.
C. riegelii was originally recovered from urine specimens from women with urinary tract infections (16). Here, we report recovery from blood cultures, including NML 99-0185 (GenBank accession no. AF537602), as well as from urine (NML 97-0139).
Two strains of C. simulans were found in this review. One of these isolates, NML 81-0613 (GenBank accession no. AF537603), had been recovered from bile and was initially identified as resembling Centers for Disease Control (CDC) group G-1. This strain had been studied along with other lipophilic coryneforms, including CDC group G strains, by Riegel et al. and found to be like members of the Corynebacterium genus but could not be definitively assigned to any existing species at that time (22, 27). After molecular studies and demonstration of nitrite reduction, this isolate could be definitively assigned to C. simulans but differed from the description by Wattiau et al. by utilizing maltose (31). The second strain of C. simulans derived from a blood culture (NML 00-0186, GenBank accession no. AF537604) was aberrant by repeatedly being catalase negative but otherwise consistent with the species description by Wattiau et al.
C. sundsvallense isolates found here include one from a draining sinus, described in brief previously (4) (NML 93-0639, GenBank accession no. AF537605), and a blood culture (NML 99-0121, GenBank accession no. AF537606). C. sundsvallense and C. thomssenii were both described within a short time period in 1999, based on the description of three strains and one strain, respectively, and share a number of commonalities biochemically (4, 33). C. sundsvallense and C. thomssenii (GenBank accession no. Y09655 and AF010474, respectively) are closely related by comparative 16S rRNA gene sequence analysis (98.9% identity; data not shown). The C. thomssenii isolate found here (NML isolate 97-0130, GenBank accession no. AF537607) was recovered from an air sample of an operating theater being studied due to contamination by a coryneform. The sequence derived from this strain had 99.9% identity with C. thomssenii (GenBank accession no. AF010474) and was consistent with that species except that oxidase was weakly positive and that N-acetyl-β-glucosaminidase could not be demonstrated, in spite of several attempts.
Two black-pigmented Corynebacterium-like strains similar to the isolate described by Shukla et al. (28) have been characterized here. That isolate is referred to in GenBank as “Corynebacterium nigricans,” currently a nonvalidated species name. These bacteria were recovered from a vulval ulcer (NML 91-0032) and vaginal specimen (NML 92-0360, GenBank accession no. AF537608), sources similar to that of the Shukla isolate. The strains here were pyrazimamidase and alkaline phosphatase negative with the API Coryne strip. Ribose and glycogen were negative with both the API Coryne strip and conventional methods, differing from the strain reported by Shukla et al. (28).
Products of fermentation were reviewed for each taxon group in Table 2. All taxa produced small or moderate volumes of acetic, lactic, and/or succinic acid. The presence or absence of propionic acid appears to be species dependent, as originally postulated (7), and so observation of that product may be useful for discerning between phenotypically close taxa.
In summary, we demonstrated with a polyphasic approach that rare or new Corynebacterium species could be recovered in geographic areas outside Europe. Some isolates were observed to have characteristics or were recovered from clinical sources which had not been described previously, which may prove useful for clinical microbiology laboratories attempting to identify Corynebacterium species.
Nucleotide sequence accession number.
The following accession numbers (NML isolate designations in parentheses) have been reported to GenBank: AF537589 (98-0075), AF537590 (99-0033), AF537591 (99-0145), AF537592 (97-0171), AF537593 (99-0047), AF537594 (97-0205), AF537595 (98-0013), AF537596 (98-0098), AF537597 (01-0063), AF537598 (00-0244), AF537599 (87-0436), AF537600 (01-0118), AF537601 (97-0160), AF537602 (99-0185), AF537603 (81-0613), AF537604 (00-0186), AF537605 (93-0639), AF537606 (99-0121), AF537607 (97-0130), and AF537608 (92-0360).
ADDENDUM
Two additional Corynebacterium species have been described since submission of our manuscript, Corynebacterium aurimucosum sp. nov. (32a) and Corynebacterium appendicis (32b). It is not known if the Canadian reference center has encountered any strains of these species.
ACKNOWLEDGMENTS
The assistance of Shaun Tyler and the staff of the DNA core facility of the NML is gratefully acknowledged.
FOOTNOTES
- Received 9 May 2002.
- Returned for modification 22 July 2002.
- Accepted 19 August 2002.
- American Society for Microbiology
