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Journal of Clinical Microbiology, July 2006, p. 2416-2422, Vol. 44, No. 7
0095-1137/06/$08.00+0     doi:10.1128/JCM.00116-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Clostridium aldenense sp. nov. and Clostridium citroniae sp. nov. Isolated from Human Clinical Infections

Yumi A. Warren,1 Kerin L. Tyrrell,1 Diane M. Citron,1* and Ellie J. C. Goldstein1,2

R. M. Alden Research Laboratory, Santa Monica, California 90404,1 UCLA School of Medicine, Los Angeles, California 900732

Received 18 January 2006/ Returned for modification 5 April 2006/ Accepted 26 April 2006


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ABSTRACT
 
One hundred eight isolates were previously identified in our laboratory as Clostridium clostridioforme by colonial and cellular morphology, as well as biochemical tests. Recent studies have indicated that there are actually three different species in this C. clostridioforme group: C. hathewayi, C. bolteae, and C. clostridioforme. Our isolates were reexamined using biochemical and enzymatic tests and molecular methods. Forty-six isolates were reidentified as C. hathewayi, 34 as C. bolteae, five as C. clostridioforme, and one as C. symbiosum. Twenty-two strains were identified only to the genus level by 16S rRNA gene sequencing, and although they are microscopically and morphologically indistinguishable from the above-mentioned three species, they are phenotypically different and only 96 to 98% similar by gene sequencing. Twenty of these 22 strains were indole positive and formed two novel species. We propose Clostridium aldenense sp. nov. and Clostridium citroniae sp. nov. as names for these new species. They are differentiated from each other by results for raffinose, rhamnose, {alpha}-galactosidase, and ß-galactosidase: positive, negative, positive, and positive, respectively, for the former species and negative, positive, negative, and negative, respectively, for the latter species. The type strain of C. aldenense is RMA 9741 (ATCC BAA-1318; CCUG 52204), and the type strain of C. citroniae is RMA 16102 (ATCC BAA-1317; CCUG 52203).


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INTRODUCTION
 
Clostridium species are endospore-forming, obligately anaerobic, gram-positive bacilli. By this simple definition, the genus includes species with diverse morphological, phenotypic, and biochemical properties and is the largest anaerobic genus (7, 8, 21, 31, 33, 37). These species are found widely in the environment, form a part of the normal enteric microflora of humans and animals, and are capable of causing endogenous and exogenous infections (1, 6, 8, 12, 17, 32). In fact, Clostridium species are one of the predominant anaerobes in the human intestinal tract (34, 35) and are a major cause of endogenous infection.

Until recently, identification of Clostridium organisms has been accomplished by biochemical tests and gas-liquid chromatography. While the conventional macrotube biochemical tests have been the most accurate methods of identification (20), they are expensive and time-consuming (5, 17, 35). Although the rapid methods that measure preformed enzymes are convenient, their accuracy has not been adequate for identifying clostridia (1, 5, 9, 20, 27). Therefore, basic characteristics, such as Gram stain and colony morphology, continue to play an important role in their identification (1, 2, 20). However, this approach also has limitations because some Clostridium strains stain gram negative and others do not consistently exhibit spores. For this reason, some clostridia, such as Clostridium symbiosum and C. clostridioforme, were previously included within gram-negative genera (9, 15). More-advanced techniques, such as 16S rRNA gene sequencing, have made identification more accurate and rearrangement by phylogenetic relatedness possible within the genus, and consequently new genera have been defined (7, 35).

C. clostridioforme is one such example, and two new species have already been described, C. hathewayi (32) and C. bolteae (28). The three species are morphologically and microscopically indistinguishable from each other, appearing as cigar-shaped, gram-negative bacilli, often connected end to end, and sometimes mistaken for fusobacteria; however, susceptibility to the 5-µg vancomycin special potency disc distinguishes them from fusobacteria (1, 15). Spores are rarely seen, but when present, they are oval and subterminal.

Identification of C. clostridioforme is important for several reasons. Susceptibility testing is generally not done in clinical laboratories, although resistance to antibiotics by C. clostridioforme has been noted, and some strains produce ß-lactamase (2, 6, 13, 24, 25). In addition, C. clostridioforme strains produce a higher metabolic enzyme activity among the intestinal microflora, which leads to the generation of toxic or carcinogenic metabolites (22, 28). Although most Clostridium spp. are not generally considered to be invasive, C. clostridioforme organisms have been isolated from osteomyelitis (30), blood (8, 19), liver abscess (8), subgingival area (38), and diabetic foot infections (D. M. Citron and E. J. C. Goldstein, Abstr. 43rd Annu. Meet. Infect. Dis. Soc. Am., abstr. 316, 2005).

Since 1984, we have isolated and identified 149 strains of C. clostridioforme by using standard methods (14, 16). Finegold et al. (9) reported the biochemical reactions that distinguished C. hathewayi, C. bolteae, and C. clostridioforme. Based on their results, we reexamined 108 isolates of our C. clostridioforme group strains recovered since 1988 to reidentify them according to the newly described criteria. As a result, 46 isolates were reidentified as C. hathewayi, 34 as C. bolteae, five as C. clostridioforme, and one as C. symbiosum. The remaining 22 isolates did not match the above-mentioned species biochemically, and identification to the genus level was accomplished by 16S rRNA gene sequencing. Among those 22 Clostridium species, 20 strains were different from all of the other species by being indole positive and by other key biochemical reactions, suggesting that they are novel species. We propose these new strains as Clostridium aldenense sp. nov. and Clostridium citroniae sp. nov.


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MATERIALS AND METHODS
 
Clinical isolates and phenotypic identification. All 108 isolates were previously frozen in 20% skim milk maintained at –70°C; 95 isolates were from intra-abdominal specimens, four from pelvic specimens, five from skin, three from blood, and one from an unknown source. They were subcultured at least twice on Brucella blood agar plates with hemin and vitamin K1 (Hardy Diagnostics, Santa Maria, CA). The type strains of C. clostridioforme (ATCC 25537), C. bolteae (ATCC BAA-613), and C. hathewayi (CCUG 43506) were also included.

Prereduced anaerobically sterilized (PRAS) biochemicals (Anaerobe System, Morgan Hill, CA) included in this study were arabinose, esculin, glucose, lactose, maltose, mannitol, mannose, melezitose, raffinose, rhamnose, salicin, sorbitol, starch, sucrose, trehalose, xylose, gelatin, and indole-nitrate. The tubes were inoculated with 0.2 ml of a 0.5 McFarland standard bacterial suspension in Brucella broth by use of a syringe and incubated at 37°C for 72 h. The pH of each tube was measured using a pH meter (Orion Research Incorporated, Boston, MA). A pH of 5.59 or lower was positive, 5.90 or greater was negative, and 5.60 to 5.89 was considered a weak reaction. Indole was tested in PRAS indole-nitrate tubes (Anaerobe System) and by a spot indole test (Anaerobe System). Preformed enzymes were tested using Rapid ID 32A (bioMerieux, Durham, NC) and RapID ANA II (Remel, Lenexa, KS) systems, according to the manufacturers' package inserts.

Genotypic identification. Isolates that were of questionable identification by phenotypic testing were sequenced to confirm their identifications. Cellular DNA was extracted using a DNeasy tissue kit (QIAGEN, Inc., Valencia, CA). Amplification of 16S rRNA genes used two universal primers, 8UA and 907B (positions 8 and 907, Escherichia coli numbering) (4, 29). PCR was performed with an initial 3 min at 95°C; followed by 30 s at 95°C, 30 s at 55°C, and 60 s at 72°C, repeated 34 times; and a final extension cycle at 72°C for 5 min. PCR products were electrophoresed in a 1% agarose gel and purified using a QIAquick gel extraction kit (QIAGEN, Inc., Valencia, CA). Purified DNA was sequenced directly (Laguna Scientific Laboratory, Laguna Beach, CA) with a Biotech Diagnostic Big Dye (Biotech Diagnostics, CA) sequencing kit on an ABI 377 sequencer (Applied Biosystems, Foster City, CA). The resulting sequences were compared with sequences in the GenBank database by using BLAST software (3, 18), and the closest relatives of the unknown isolates were determined.

For the type strains of C. aldenense sp. nov. and C. citroniae sp. nov., an additional 16S subregion was amplified using universal primers 774A and 1485B (positions 774 and 1485, E. coli numbering) (4, 29). Sequences for each of the overlapping subregion pairs were aligned together by using MEGA version 3.1 (18), creating ~1,400-bp sequences. A multiple alignment of these almost-full-length sequences was created using MEGA3 and aligned using the native implementation of CLUSTAL W in the alignment explorer tool of MEGA3, followed by further manual correction using MEGA3 and FinchTV (Geospiza, Inc., Seattle, WA).

Phylogenetic trees were constructed with MEGA3 by using the neighbor-joining method (26). Reliability of the inferred trees was estimated using bootstrap analysis (500 repetitions). The resulting trees were visualized using the tree explorer tool of MEGA3.

Antimicrobial susceptibility testing. Susceptibilities of seven C. citroniae, 13 C. aldenense, 34 C. bolteae, five C. clostridioforme, and 46 C. hathewayi strains were determined against 11 antimicrobial agents, including ampicillin-sulbactam (Pfizer, Roerig Division, Groton, CT), piperacillin-tazobactam (Wyeth Pharmaceuticals, Pearl River, NY), ertapenem (Merck & Co., Rathway, NJ), cefoxitin (Merck & Co.), ceftriaxone (Roche Laboratories, Inc., Nutley, NJ), moxifloxacin (Bayer Corporation, Mt. Prospect, IL), levofloxacin (Johnson & Johnson, Springhouse, PA), chloramphenicol (Sigma, St. Louis, MO), penicillin (Sigma), clindamycin (Voigt Global Distributing, Kansas City, MO), and metronidazole (Searle, Inc., Skokie, IL). Antimicrobial powders were reconstituted according to the manufacturers' instructions, and serial twofold dilutions were made. Susceptibility testing was performed by the agar dilution method according to the procedure described in CLSI (formerly NCCLS) document M11-A6 (23). When the penicillin MIC result was ≥1 µg/ml, ß-lactamase was tested using Nitrocef disks (Hardy Diagnostics, Santa Maria, CA).

Nucleotide sequence accession numbers. The almost-complete sequences of the type strains of C. aldenense and C. citroniae were deposited in GenBank under accession numbers DQ279736 and DQ279737, respectively.


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RESULTS AND DISCUSSION
 
Phenotypic differentiation of five species in the C. clostridioforme group and three reference strains is shown in Table 1. The table is a summary of PRAS biochemicals and rapid systems.


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  		TABLE 1. Phenotypic differentiation for five species in the C. clostridioforme group and three reference strains

Out of 108 isolates, 46 were reidentified as C. hathewayi by positive results for lactose, melezitose, and N-acetyl-ß-glucosaminidase (NAG), except for one isolate that was melezitose negative, which made the overall agreement only slightly less than 100%. This isolate was confirmed as C. hathewayi by 16S rRNA gene sequence analysis. Five C. clostridioforme strains were positive for lactose and negative for melezitose and NAG as expected, and their identifications were confirmed by PCR. There were 34 C. bolteae strains, all solely identified by PCR. Unlike results from a previous report (9), our strains showed variable reactions in lactose, which resulted in difficulty in differentiating between C. bolteae and C. clostridioforme, although further testing determined that o-nitrophenyl-ß-D-galactopyranoside could distinguish the species: C. clostridioforme showed positive results and C. bolteae negative.

Preformed-enzyme tests evaluated by the RapID ANA II system by Marler et al. (20) showed that the reaction patterns of C. clostridioforme were varied and that the reactions could change depending on the medium type and the manufacturer from which the colonies were taken. We also noticed that the RapID ANA II system produced two distinct patterns; however, our pattern differences were due to species differences only. As Table 2 shows, C. hathewayi produced significantly more positive reactions with the RapID ANA II system and somewhat more with Rapid ID 32A than C. bolteae and C. clostridioforme and there was more strain variability for many of the tests. Because C. clostridioforme and C. bolteae exist in the same genetic line but are separate from C. hathewayi (Fig. 1 and 2), it is understandable that they showed variations and that the preformed-enzyme systems produced two distinct profile patterns, one for C. hathewayi and the other for C. bolteae/C. clostridioforme, which are not in the database (9, 20).


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  		TABLE 2. Profile numbers for each species, obtained by use of Rapid ID 32A and RapID ANA II systemsa


Figure 1
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  		FIG. 1. Unrooted tree showing the phylogenetic positions of Clostridium aldenense sp. nov. and C. citroniae sp. nov. within the C. clostridioforme group rRNA cluster of organisms. The tree was constructed using the neighbor-joining method based on a comparison of approximately 700 nucleotides. Bootstrap values, expressed as percentages of 500 replications, are given at the branching points. GenBank accession numbers are given in parentheses.


Figure 2
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  		FIG. 2. Unrooted tree showing the phylogenetic positions of Clostridium aldenense sp. nov. and C. citroniae sp. nov. (in boldface type) within the C. coccoides rRNA cluster of organisms. The tree was constructed using the neighbor-joining method based on a comparison of approximately 1,390 nucleotides. Bootstrap values, expressed as percentages of 500 replications, are given at the branching points. GenBank accession numbers are given in parentheses.

The remaining 22 strains produced biochemical and enzymatic results that differed from those of the other three species. As the bootstrap tree shows (Fig. 1), there appeared to be two distinct groups of organisms, 13 strains in one and seven strains in the other. The 16S rRNA gene sequences of these strains did not match at the 99% level to any of the known bacterial species, while the closest known relatives were C. bolteae and C. clostridioforme. The first group showed 96 to 97% identity to C. bolteae and 95 to 96% identity to C. clostridioforme. The second group had 97 to 98% identity to both C. bolteae and C. clostridioforme. The phylogenetic tree (Fig. 2) indicated that they fit in Clostridium cluster XIV A as defined by Collins et al. (7). In addition, unlike the other three species, these strains were indole positive, indicating two new species in the C. clostridioforme group.

The MIC results are presented in Table 3. Only the ranges and the MIC50s are listed for one of the two new groups and for C. clostridioforme due to the low numbers of isolates tested. A total of 67 strains were tested for penicillin, of which 41 strains had an MIC of ≥1 µg/ml and were tested for ß-lactamase. Thirteen (62%) out of 21 isolates that were resistant to penicillin (MIC of >1 µg/ml) and 2 (10%) out of 20 isolates that were intermediate (MIC of 1 µg/ml) produced ß-lactamase. Therefore, penicillin nonsusceptibility and ß-lactamase production did not always correlate. In addition, the number of C. clostridioforme group organisms that produce ß-lactamase was much higher in our study than in the study by Alexander et al. of isolates recovered between 1984 and 1993 (1). By species, 11 (65%) C. bolteae isolates, three (12%) C. hathewayi isolates, and one (20%) C. clostridioforme isolate produced ß-lactamase in our study, while none of the new species was positive.


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  		TABLE 3. In vitro susceptibilities to 11 antimicrobial agents

Many clinical laboratories do not identify anaerobes, especially to the species level (12). However, identifying some of the clinically common clostridial isolates is necessary because susceptibility is often species specific, and the importance of performing susceptibility testing on clostridial isolates other than C. perfringens has been shown in several studies (10, 11, 36). Our susceptibility results were different from those of the study by Finegold et al. (9) in that most strains in all five species appeared resistant to moxifloxacin by use of FDA-defined breakpoints. In general, C. bolteae seems more resistant than other species in this group, presenting higher MICs for penicillin, ampicillin-sulbactam, and piperacillin-tazobactam and more strains producing ß-lactamase.

In this study, we identified two new species in the C. clostridioforme group. We propose C. aldenense sp. nov. and C. citroniae sp. nov. as names for these novel species. C. aldenense and C. citroniae are genetically closely related to C. bolteae and C. clostridioforme but are more distant from C. hathewayi (Fig. 2). C. aldenense and C. citroniae could be differentiated by their profile numbers obtained by Rapid ID 32A but not by RapID ANA II (Table 2). By individual testing, they could be differentiated by results for raffinose, rhamnose, {alpha}-galactosidase, and ß-galactosidase, as shown in Table 1.

Prior to the identification of these newly described species, C. clostridioforme was one of the most frequently isolated Clostridium species in clinical specimens (21, 27). In a recent intra-abdominal study, we isolated 1,190 anaerobes from 401 patient specimens (unpublished data). There were 158 Clostridium strains, comprising 13% of the total. Thirty-seven of these (23%) were in the C. clostridioforme group: 19 (51%) C. hathewayi strains, 11 (30%) C. bolteae strains, two (5%) C. clostridioforme strains, four (11%) C. aldenense strains, and one (3%) C. citroniae strain. C. clostridioforme was in reality one of the least frequently encountered species among this group.

The taxonomy within the genus Clostridium has been in flux during the last 10 years. The C. hathewayi and C. bolteae strains in our study were recovered as early as 1998, confirming the clinical importance of C. hathewayi first reported by Elsayed and Zhang in 2004 (8). Interestingly, the two novel species described in this study were isolated from our clinical specimens even earlier: C. aldenense in 1994 and C. citroniae in 1988.

Description of C. aldenense sp. nov. Clostridium aldenense (Al.de.nen'se. N.L. neut. adj. aldenense, pertaining to R. M. Alden Research Laboratory and its first patron, Rose M. Alden Goldstein) produces colonies that are 1 to 2 mm in diameter, flat, opaque to white, and nonhemolytic on Brucella blood agar plates after 48 h at 37°C. The cells are 0.8 to 1.1 µm by 2 to 5 µm and stain gram negative. Spores are rarely seen, as with other species in the C. clostridioforme group.

Biochemically, the organisms are differentiated from existing species in the C. clostridioforme group by exhibiting a positive indole reaction. They produce acid from glucose, maltose, mannose, raffinose, sucrose, and xylose but not from cellobiose, esculin, mannitol, melezitose, rhamnose, sorbitol, or starch. They do not hydrolyze urea, starch, or gelatin or reduce nitrate. Fermentations of arabinose, lactose, salicin, and trehalose and esculin hydrolysis are variable. Enzymatically, {alpha}-galactosidase and ß-galactosidase are positive, but arginine dihydrolase, ß-galactosidase-6-phosphate, {alpha}-glucosidase, ß-glucosidase, ß-glucuronidase, N-acetyl-ß-glucosaminidase, arginine arylamidase, proline arylamidase, leucyl glycine arylamidase, phenylalanine arylamidase, leucine arylamidase, pyroglutamic acid arylamidase, tyrosine arylamidase, alanine arylamidase, and glycine arylamidase are negative. Variable reactions are produced by {alpha}-arabinosidase and alkaline phosphatase.

The type strain of C. aldenense is RMA 9741 (ATCC BAA-1318; CCUG 52204).

Description of C. citroniae sp. nov. Clostridium citroniae (Ci.tro'ni.i. N.L. gen. n. citronii, named after Diane M. Citron for numerous contributions to clinical anaerobic bacteriology as a clinical microbiologist and educator) produces colonies that are 1 to 2 mm in diameter, flat, opaque to white, and nonhemolytic on Brucella blood agar plates in 48 h at 37°C. The cells are 0.8 to 1.1 µm by 2 to 5 µm and stain gram negative. Spores are rarely seen, as with other species in this group, including C. aldenense sp. nov.

Similarly to C. aldenense, the organisms are indole positive and produce acid from glucose, maltose, mannose, rhamnose, sucrose, trehalose, and xylose but not from cellobiose, esculin, lactose, mannitol, melezitose, raffinose, salicin, sorbitol, or starch. They do not hydrolyze urea, esculin, starch, or gelatin or reduce nitrate. Fermentations of arabinose are variable. Alkaline phosphatase is positive, but arginine dihydrolase, {alpha}-galactosidase, ß-galactosidase, ß-galactosidase-6-phosphate, {alpha}-glucosidase, ß-glucosidase, {alpha}-arabinosidase, ß-glucuronidase, N-acetyl-ß-glucosaminidase, arginine arylamidase, proline arylamidase, leucyl glycine arylamidase, phenylalanine arylamidase, leucine arylamidase, pyroglutamic acid arylamidase, tyrosine arylamidase, alanine arylamidase, and glycine arylamidase are negative.

The type strain of C. citroniae is RMA 16102 (ATCC BAA-1317; CCUG 52203).


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ACKNOWLEDGMENTS
 
This study was supported in part by Merck & Co., Inc.

We thank Vreni C. Merriam for critical review of the manuscript and Helen T. Fernandez for excellent technical assistance. We thank the Wadsworth Anaerobe Laboratory, especially Yuli Song and Denise R. Molitoris, for some of the type strains and help with PCR methods.


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FOOTNOTES
 
* Corresponding author. Mailing address: R. M. Alden Research Laboratory, 2001 Santa Monica Blvd., Suite 685W, Santa Monica, CA 90404. Phone: (310) 453-7820. Fax: (310) 453-7670. E-mail: rmarl{at}verizon.net. Back


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Journal of Clinical Microbiology, July 2006, p. 2416-2422, Vol. 44, No. 7
0095-1137/06/$08.00+0     doi:10.1128/JCM.00116-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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