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Clostridium celerecrescens, often misidentified as “Clostridium clostridioforme group,” is involved in rare human infection cases
Diagnostic Microbiology and Infectious Disease 74 (2012) 299–302
Contents lists available at SciVerse ScienceDirect
Diagnostic Microbiology and Infectious Disease j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / d i a g m i c r o b i o
Case Reports
Clostridium celerecrescens, often misidentified as “Clostridium clostridioforme group,” is involved in rare human infection cases☆ Philippe Bouvet a,⁎, Guylène K'Ouas a, Alain Le Coustumier b, Michel R. Popoff a a b
Institut Pasteur, Centre National de Référence des Bactéries Anaérobies et du Botulisme, Unité Bactéries anaérobies et Toxines, 25-28 rue du Docteur Roux, F-75724 Paris cedex 15, France Centre Hospitalier de Cahors, Laboratoire de Biologie, Cahors, France
a r t i c l e
i n f o
Article history: Received 18 January 2012 Accepted 25 June 2012 Available online 15 August 2012 Keywords: Clostridium celerecrescens Misidentification Rare Clostridium species “Clostridium clostridioforme group”
a b s t r a c t Misidentification of rare Clostridium species often originated from the environment as clinically relevant species is problematic. A strain isolated from a traumatic leg wound first identified as C. clostridioforme was finally identified as the rare Clostridium celerecrescens. Two similar misidentifications are reported in the literature. In order to help the phenotypic differentiation of C. celerecrescens from the close species of the “C. clostridioforme group”, an identification table and differential susceptibilities to 4 selected antibiotics are proposed. Once a clinical isolate is referred to this group, identification should be definitively confirmed by unambiguous methods such as 16s rDNA sequencing. © 2012 Published by Elsevier Inc.
1. Introduction Strictly anaerobic Gram-positive spore-forming bacilli of the genus Clostridium are normal inhabitants of the mammal gut and of the environment. The genus comprises a great number of described species some of which are of major clinical importance. Rare or difficult-to-identify Clostridium species may be found responsible for infection in humans or animals, and because of the large number of species in the genus and the lack or misunderstanding of differential phenotypic markers, they may be misidentified with clinically relevant species. A Clostridium strain isolated from a traumatic leg wound that was firstly identified as C. clostridioforme or the so-called "Clostridium clostridioforme group” was finally identified by 16S rDNA sequencing as the rare C. celerecrescens. Gram-positive or Gram-variable Clostridium with tapered ends may be difficult to identify to the species level and sometimes to the genus level. The species C. clostridioforme previously comprised several groups of organisms that have been reclassified into separate species, including C. clostridioforme, C. hathewayi, C. bolteae, and, more recently, C. aldenense and C. citroniae (Finegold et al., 2005; Song et al., 2003; Steer et al., 2001; Warren et al., 2006). These close species are frequently referred to as the “C. clostridioforme group” (hereafter abbreviated as the “Cc group”). A new close glycopeptide-resistant species, C. lavalense, has been identified in 2010 (Domingo et al., ☆ The GenBank accession number for the 16S rRNA gene sequence of strain AIP148.09 is JQ246092. ⁎ Corresponding author. Tel.: +33-1-40-61-35-09; fax: +33-1-40-61-31-23. E-mail address: [email protected] (P. Bouvet). 0732-8893/$ – see front matter © 2012 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.diagmicrobio.2012.06.024
2009). The aim of this work was to propose practical means that could help in the differentiation of these close species.
2. Materials and methods 2.1. Patient and bacterial strains studied The strain was isolated from a traumatic wound in a febrile 20year-old male who consulted the emergency unit 2 weeks after being injured by a piece of iron coming from a coin used for cutting wood. After removing it and local sampling, the patient was discharged with an antibacterial treatment (1 g amoxicillin + clavulanic acid 3 times a day) for 7 days. The recovery was complete. The sample yielded a pure and moderately abundant culture of a strictly anaerobic, Gram-positive but becoming rapidly Gram-negative, thin spore-forming diplobacillus with tapered ends. Routine identification using Rapid ID 32A strips (BioMérieux, Marcy-l'Etoile, France) gave a profile corresponding to a very good identification of Clostridium clostridioforme except for a positive reaction for indole production. Regarding the resistance to cefotaxim, the strain was suspected to be Clostridium hathewayi, a Clostridium species close to C. clostridioforme. Then the strain was sent to the National Reference Center (NRC) of Anaerobic Bacteria and Botulism (Institut Pasteur, Paris, France) for confirmation of identification (strain AIP148.09). The type strain of C. lavalense strain CIP109511 T (=CCUG 54291 T) was also included in the study since some data (Rapid ID 32A profile and susceptibility to antibiotics) were absent in the description of the species (Domingo et al., 2009).
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2.2. Identification done at the NRC
3. Results
Strains were characterized using reference methods (JousimiesSomer et al., 2002). Rapid ID 32A galleries (BioMérieux) were used according to the manufacturer's instructions.
3.1. Phenotypic identification of strain AIP148.09
2.3. Molecular identification Amplification of the 16S rDNA gene, sequencing, and analysis of sequence were done as described previously (Carlier et al., 2004). 2.4. Antibiotic susceptibility From published studies (Domingo et al., 2009; Song et al., 2003; Warren et al., 2006), 4 antibiotics whose data are available for all species of the “Cc group” were selected: amoxicillin + clavulanic acid, a third-generation cephalosporin (ceftriaxone), a fluoroquinolone (moxifloxacin), and vancomycin. Estimations of the MICs of strains AIP148.09 and CIP109511 T were determined using the Etest method (bioMérieux). Interpretations of MICs in terms of susceptibility were done according to the criteria of the Comité de l'Antibiogramme of the French Society of Microbiology for susceptibility testing of anaerobes (CASFM, 2012) (see Table 2). β-Lactamase enzymes were searched for using nitrocephin disks (Fluka, Buchs, Switzerland).
The bacterium was a Gram-positive bacillus but becoming rapidly Gram negative with tapered ends. Central to subterminal spores were rarely observed. Cells were found motile. Culture supernatant fluids were nontoxic for mice. With the use of special-potency antibiotic disks, the strain was susceptible to vancomycin (5 μg) (which is predictive of a Gram-positive bacterium), kanamycin (1000 μg), and metronidazole (4 μg), but was resistant to colistin (10 μg), which is the profile mostly encountered with Clostridium. Lecithinase and lipase were negative on egg yolk agar plates. Gelatin and milk were not modified. Production of catalase was negative. Indole was produced. Nitrates and nitrites were not reduced. Abundant gas was produced in trypticase-glucose-yeast extract broth. Acid was produced from arabinose, cellobiose, fructose, galactose, glucose, maltose, mannose, mannitol, melibiose, raffinose, rhamnose, ribose, salicine, trehalose, xylose, and esculin, but not produced from glycerol, inositol, lactose, melezitose, sucrose, and sorbitol. Esculin was hydrolyzed. Strain AIP148.09 was finally referred to the group of saccharolytic, nonproteolytic group of Clostridium and particularly toward species belonging to the so-called “Cc group” but did not fit any acceptable profile. Using Rapid ID 32A gallery strain AIP gave the profile 45312000 corresponding to a very good identification
Table 1 Enzymatic activity tests and profile numbers (Rapid ID 32A gallery) and production of acid from some sugars for differentiation of species of the “C. clostridioforme group”, C. lavalense, and C. celerecrescens.a Rrapid ID 32A Characters (digit no., value)b: α-Galactosidase (1, 4) β-Galactosidase (2, 1) α-Glucosidase (2, 4) β-Glucosidase (3, 1) α-Arabinosidase (3, 2) β-Glucuronidase (3, 4) β-N-Acetyl-glucosaminidase (4, 1) Mannose (4, 2) Raffinose (4, 4) Indole production (5, 2) Alkaline phosphatase (5, 4) Arginine arylamidase (6, 1) Proline arylamidase (6, 2) Leucyl glycine arylamidase (6, 4) Leucine arylamidase (7, 2) Pyroglutamic acid arylamidase (7, 4) Rapid ID 32A profile numbers from Warren et al. (2006)—additional reference in parentheses
C. celerecrescens AIP148.09
C. aldenense
C. bolteae
C. citroniae
C. clostridioforme C. hathewayi
C. lavalense CIP 109511T
+ + + + + − + − − + − − − − − − −
+ (−) + − − +/− − − − (+) − (+) +/− +/− − − (+) − (+) − − 41267000 41000400 41000600 41202000
+ + + − − − − − − − − − − +/− − − 45000400 45000000
− − − − − − − +/− − + +/− +/− − − +/− − 00026000 00006000
+ (−) + (−) − (+) − − (+) − (+) − − − (+) − − (+) − − − − − 00000000 41000000 41000400
+ + + + + + + (−) +/− +/− − − − − +/− − +/− 45710000 45710040 45710400 45770440 (Woo et al. 2008)
− + − − − − − − − + − − − − − − −
00002120 (1)
45160000 (1)
45760440 (1)
0100200 (1)
41444000 (1)
45710400 (1)
+ − + +
+ + +/− +
Rapid ID 32A profile numbers 45312000 (1) from our laboratory (no. of strains) Production of acid from Lactose Melezitose Rhamnose Salicine
w
+ /− − + +
41206000 41266000 01206000 (1)
+/− − − − (+)
−(+) + +/− − (+)
− − + −
+ − − +
+ = Positive; +w = weakly positive; − = negative; +(−) = positive (rarely negative); −(+) = negative (rarely positive); +/− = positive or negative. a Data were obtained from published studies (Domingo et al., 2009; Palop et al., 1989; Song et al., 2003; Warren et al., 2006; Williams et al., 2010; Woo et al., 2008), from some strains studied in our laboratory with 16S rDNA gene sequenced (2 Clostridium hathewayi, 2 C. clostridioforme, 1 C. aldenense, 1 C. citroniae), and from C. lavalense type strain CIP 109511T (=CCUG 54291T) studied in our laboratory. b Coding of results in Rapid ID 32A strips is based on the following principle: 8 digits; 3 characters per digit; in a digit, a value is determined according to the character found positive (value 1 for character no. 1, 2 for character no. 2, and 4 for character no. 3) and 0 if negative. Values obtained for each character in a digit are then added up. The following tests were always found negative: urease, arginine dihydrolase, β-galactosidase-6-phosphate, reduction of nitrates, phenylalanine arylamidase, tyrosine arylamidase, alanine arylamidase, and glycine arylamidase.
P. Bouvet et al. / Diagnostic Microbiology and Infectious Disease 74 (2012) 299–302
(%id = 99.4%; T = 0.72) of C. clostridioforme except for a positive reaction for indole production (6% of strains positive in the API database) (Table 1). 3.2. Molecular identification The 16S rDNA gene sequence that was determined (1435 nucleotides; GenBank accession number JQ246092) was found to be 99.2% related to Clostridium celerecrescens (GenBank NR_026100) which was first isolated from a methanogenic cellulose–enriched culture (Palop et al., 1989) but also from the environment or from animals. Its sequence was only 92.6% related to that of the type strain of C. clostridioforme (GenBank M59089). Both species belong to the Clostridium coccoides rRNA group of organisms (rRNA cluster XIVa of Clostridium (Collins et al., 1994) which are the usual inhabitants of human fecal microbiota unlike other mammals.
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2009; Palop et al., 1989; Song et al., 2003; Warren et al., 2006; Williams et al., 2010; Woo et al., 2008); from some strains studied in our laboratory with 16S rDNA gene sequenced (Clostridium hathewayi [2 strains], C. clostridioforme [2 strains]], C. aldenense [1 strain], and C. citroniae [1 strain]); and from the C. lavalense type strain CIP109511 T studied at the NRC (Table 1). Seven enzymatic tests (α/β-galactosidase, α/β-glucosidase, α-arabinosidase, β-glucuronidase, β-N-acetylglucosaminidase) and indole production seemed the most useful characters for identification (characters in italics, Table 1). Except for C. hathewayi, which was positive for all these 7 enzymatic tests and does not produce indole (a few negative strains for β-N-acetylglucosaminidase may exist), other species have most often between 1 and 3 positive tests. C. celerecrescens is close to C. hathewayi with 6 positive enzymatic tests (β-glucuronidase test result was found negative) and a positive production of indole. Fermentation of melezitose also differentiates these 2 species (positive for C. hathewayi and negative for C. celerecrescens) (Table 1).
3.3. Antibiotic susceptibility β-Lactamase enzymes were not detected. Susceptibility to antibiotics has been found of help in the identification of species of the “Cc group”. Susceptibilities of these species to the selected antibiotics (amoxicillin + clavulanic acid, ceftriaxone, moxifloxacin, and vancomycin) retrieved from the literature are presented in Table 2. Susceptibilities of strains AIP148.09 and CIP109511T are also presented in Table 2. Among these species, C. hathewayi is the only species to be resistant to third-generation cephalosporins (cefotaxim or ceftriaxone), but our C. celerecrescens isolate is resistant too (MIC 256 μg/mL). C. celerecrescens AIP148.09 is susceptible to moxifloxacin with a MIC of 0.38 μg/mL, whereas species of the “Cc group” and C. lavalense have an intermediate susceptibility or a resistance to this antibiotic (Table 2). Resistance to vancomycin (MIC N256 μg/mL) was found in C. lavalense (Domingo et al., 2009) as well as in C. bolteae, C. clostridioforme, and C. hathewayi in Marvaud et al. (2011). This resistance to vancomycin is due to a VanB gene, part of conjugative transposon Tn 5382 (closely related to Tn 1549) (Marvaud et al., 2011). As this resistance determinant is located on a mobile element, one could not exclude the discovery of vancomycin-susceptible C. lavalense isolates. 3.4. Identification table for species of the “Cc group”, C. lavalense, and C. celerecrescens Phenotypic identification of this rare species, C. celerecrescens, is rather difficult and leads frequently to the “Cc group”. In order to help in the differentiation of these related species, we attempted to rebuild an identification table using dispersed available Rapid ID 32A data and sugar fermentation data from published studies (Domingo et al.,
4. Discussion Phenotypic identification of the rare species C. celerecrescens in clinical microbiology by using reference methods as well as by identification galleries is rather difficult and leads frequently to the “Cc group”. It is clear that species of the “Cc group” are involved in a variety of severe infections (Finegold et al., 2005; Linscott et al., 2005; Ogah et al., 2012; Williams et al., 2010). Most cases of bacteraemia are mostly due to mixed flora, but the species C. clostridioforme was considered as particularly virulent. Our strain of C. celerecrescens was isolated from a traumatic leg wound. Two other human cases of infections with C. celerecrescens (an abscess of an open bone fracture and a relapse of posttraumatic osteomyelitis) were recently published (Glazunova et al., 2005; Mischnik et al., 2011). In these 3 cases, phenotypic identifications lead to the “Cc group” or unlikely identifications, and, finally, definitive identifications were only obtained after 16S rDNA sequence analysis. Whereas the virulence of C. celerecrescens is not well known and probably low, its misidentification as a virulent Clostridium species is problematic. Our strain was identified as C. clostridioforme by Rapid ID 32A gallery with a very good identification score and as belonging to the “Cc group” at the NRC. Among species of the “Cc group”, the more recent Rapid ID 32A database (APIweb v. 3.2) contains only C. clostridioforme. With the use of this database, Rapid ID 32A profile numbers of strains belonging to the different species of the group (data from Warren et al., 2006), as well as data obtained from field strains with 16S rDNA sequence available (Table 1) gave frequently C. clostridioforme as identification, with more or fewer tests in opposition but also unlikely identifications. In order to help in
Table 2 In vitro susceptibility of C. celerecrescens, C. lavalense, and species belonging to the “C. clostridioforme group” to 4 selected antibiotics. Antibiotic
C. celerecrescens AIP148.09a
C. aldenense
C. bolteae
C. citroniae
C. clostridioforme
C. hathewayi
C. lavalense CIP109511Ta
Amoxicillin + clavulanic acid Ceftriaxone Moxifloxacin Vancomycin References
S
S (n = 13)b
R (n = 34)
S (n = 7)
S (n = 5)
S (n = 46)
S
R S S This study
S (n = 13) R (n = 13) S (n = 13) Warren et al., 2006
S (n = 34) I/R (n = 53) S (n = 5)c Finegold et al., 2005; Song et al., 2003; Warren et al., 2006
S (n = 7) R (n = 7) S (n = 7) Warren et al., 2006
S (n = 5) I/R (n = 26) S (n = 1)c Finegold et al., 2005; Song et al., 2003; Warren et al., 2006
R (n = 46) I/R (n = 92) S (n = 46)c Finegold et al., 2005; Warren et al., 2006
S I R Domingo et al., 2009 and this study
R = Resistance; I = intermediate susceptibility; S = susceptibility. a MIC estimations (μg/ml) for amoxicillin + clavulanic acid, ceftriaxone, moxifloxacin, and vancomycin were 0.75, 256.0, 0.38, and 1.5, respectively, for C. celerecrescens AIP 148.09 and 0.038, 3.0, 2.0, and N256.0, respectively, for C. lavalense CIP 109511T. According to the recommendations of the Comité de l'antibiogramme of the French Society of Microbiology (CASFM, 2012), interpretative criteria for amoxicillin + clavulanic acid, ceftriaxone, moxifloxacin, and vancomycin are as follows: MICs (μg/ml) of susceptible strains are ≤4, ≤4, ≤1, and ≤4, respectively; MICs (μg/ml) for resistant strains are N8, N32, N2, and N8, respectively. b Numbers of strains studied are noted in parentheses. c Vancomycin-resistant strains have been described (Marvaud et al., 2011).
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the differentiation of these related species, we propose an identification table (Table 1) and differential susceptibilities of these species to 4 selected antibiotics (Table 2). Based on these data, our C. celerecrescens isolate was found to be close to C. hatheway with a similar enzymatic activity profile (Rapid ID 32A gallery) except for a negative β-glucuronidase activity, a positive reaction for indole, and a negative result for melezitose fermentation (Table 1). Both species had a common resistance to third-generation cephalosporin, but our C. celerecrescens isolate was susceptible to moxifloxacin, whereas other species of the “Cc group” and C. lavalense presented an intermediate susceptibility or a resistance (Table 2). It is obvious that these data need to be refined with the study of additional wellcharacterized strains. Published articles relating misidentified microorganisms by conventional methods are regularly increasing (Petti et al., 2005). It is obvious that the number of new bacterial species is growing exponentially, and, consequently, the performance of commercially available biochemical galleries that give an exact identification is decreasing partly due to the limited number of characters available. Diagnostics of rare bacteria by conventional methods as well as of bacteria with unusual phenotypic profiles needs to be confirmed by a more objective tool, e.g., 16S rDNA gene sequencing (Woo et al., 2008). Since rare bacterial species such as C. celerecrescens may be responsible for infections in humans, recently developed identification systems used in clinical microbiology like matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS) should include in their databases corresponding entries. This point has been recently highlighted by Mischnik et al. (2011) who failed to identify their C. celerecrescens isolate by MALDI-TOF MS. Databases elaborated by identification gallery suppliers or other identification systems suppliers should be refined by integrating strains originated from diverse locations (environment, different microbiota, etc.) and unambiguously identified by 16S rDNA sequencing. Unambiguous identification of unusual strains in clinical microbiology is more and more needed to improve our knowledge of reservoirs, routes of transmission, antibiotic susceptibility, and improvement of treatments, and to allow identification of novel bacterial species. Dedication We dedicate this communication to the memory of our colleague Jean-Philippe Carlier deceased in 2008 who has made a major contribution to the identification and taxonomy of anaerobes.
Acknowledgment The authors warmly thank the CRBIP (Biological Resource Centre of Institut Pasteur, Paris) for the gift of the type strain of Clostridium lavalense CIP 109511 T. References Carlier JP, K'Ouas G, Bonne I, Lozniewski A, Mory F. Oribacterium sinus gen. nov., sp. nov., within the family ‘Lachnospiraceae’ (phylum Firmicutes). Int J Syst Evol Microbiol 2004;54:1611–5. CASFM. Recommandations 2012. Paris, France: Comité de l'antibiogramme de la Société Française de Microbiologie; 2012. Collins MD, Lawson PA, Willems A, Cordoba JJ, Fernandez-Garayzabal J, Garcia P, et al. The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species combinations. Int J Syst Bacteriol 1994;44:812–26. Domingo MC, Huletsky A, Boissinot M, Helie MC, Bernal A, Bernard KA, et al. Clostridium lavalense sp. nov., a glycopeptide-resistant species isolated from human faeces. Int J Syst Evol Microbiol 2009;59:498–503. Finegold SM, Song Y, Liu C, Hecht DW, Summanen P, Kononen E, et al. Clostridium clostridioforme: a mixture of three clinically important species. Eur J Clin Microbiol Infect Dis 2005;24:319–24. Glazunova O, Raoult D, Roux V. First identification of Clostridium celerecrescens in liquid drained from an abscess. J Clin Microbiol 2005;43:3007–8. Jousimies-Somer HR, Summanen P, Citron DM, Baron EJ, Wexler HM, Finegold SM. Anaerobic bacteriology manual. 6th ed. Belmont, California: Star Publishing Company; 2002. Linscott AJ, Flamholtz RB, Shukla D, Song Y, Liu C, Finegold SM. Fatal septicemia due to Clostridium hathewayi and Campylobacter hominis. Anaerobe 2005;11: 97–8. Marvaud JC, Mory F, Lambert T. Clostridium clostridioforme and Atopobium minutum clinical isolates with vanB-type resistance in France. J Clin Microbiol 2011;49: 3436–8. Mischnik A, Zimmermann S, Bekeredjian-Ding I, Egermann M. Relapse of posttraumatic osteomyelitis due to Clostridium celerecrescens. Infection 2011;39:491–4. Ogah K, Sethi K, Karthik V. Clostridium clostridioforme liver abscess complicated by portal vein thrombosis in childhood. J Med Microbiol 2012;61:297–9. Palop ML, Valles S, Pinaga F, Flors A. Isolation and characterization of an anaerobic, cellulolytic bacterium, Clostridium celerecrescens sp. nov. Int J Syst Bacteriol 1989;39:68–71. Petti CA, Polage CR, Schreckenberger P. The role of 16S rRNA gene sequencing in identification of microorganisms misidentified by conventional methods. J Clin Microbiol 2005;43:6123–5. Song Y, Liu C, Molitoris DR, Tomzynski TJ, Lawson PA, Collins MD, et al. Clostridium bolteae sp. nov., isolated from human sources. Syst Appl Microbiol 2003;26:84–9. Steer T, Collins MD, Gibson GR, Hippe H, Lawson PA. Clostridium hathewayi sp. nov., from human faeces. Syst Appl Microbiol 2001;24:353–7. Warren YA, Tyrrell KL, Citron DM, Goldstein EJ. Clostridium aldenense sp. nov. and Clostridium citroniae sp. nov. isolated from human clinical infections. J Clin Microbiol 2006;44:2416–22. Williams OM, Brazier J, Peraino V, Goldstein EJ. A review of three cases of Clostridium aldenense bacteremia. Anaerobe 2010;16:475–7. Woo PC, Lau SK, Teng JL, Tse H, Yuen KY. Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories. Clin Microbiol Infect 2008;14:908–34.
Contents lists available at SciVerse ScienceDirect
Diagnostic Microbiology and Infectious Disease j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / d i a g m i c r o b i o
Case Reports
Clostridium celerecrescens, often misidentified as “Clostridium clostridioforme group,” is involved in rare human infection cases☆ Philippe Bouvet a,⁎, Guylène K'Ouas a, Alain Le Coustumier b, Michel R. Popoff a a b
Institut Pasteur, Centre National de Référence des Bactéries Anaérobies et du Botulisme, Unité Bactéries anaérobies et Toxines, 25-28 rue du Docteur Roux, F-75724 Paris cedex 15, France Centre Hospitalier de Cahors, Laboratoire de Biologie, Cahors, France
a r t i c l e
i n f o
Article history: Received 18 January 2012 Accepted 25 June 2012 Available online 15 August 2012 Keywords: Clostridium celerecrescens Misidentification Rare Clostridium species “Clostridium clostridioforme group”
a b s t r a c t Misidentification of rare Clostridium species often originated from the environment as clinically relevant species is problematic. A strain isolated from a traumatic leg wound first identified as C. clostridioforme was finally identified as the rare Clostridium celerecrescens. Two similar misidentifications are reported in the literature. In order to help the phenotypic differentiation of C. celerecrescens from the close species of the “C. clostridioforme group”, an identification table and differential susceptibilities to 4 selected antibiotics are proposed. Once a clinical isolate is referred to this group, identification should be definitively confirmed by unambiguous methods such as 16s rDNA sequencing. © 2012 Published by Elsevier Inc.
1. Introduction Strictly anaerobic Gram-positive spore-forming bacilli of the genus Clostridium are normal inhabitants of the mammal gut and of the environment. The genus comprises a great number of described species some of which are of major clinical importance. Rare or difficult-to-identify Clostridium species may be found responsible for infection in humans or animals, and because of the large number of species in the genus and the lack or misunderstanding of differential phenotypic markers, they may be misidentified with clinically relevant species. A Clostridium strain isolated from a traumatic leg wound that was firstly identified as C. clostridioforme or the so-called "Clostridium clostridioforme group” was finally identified by 16S rDNA sequencing as the rare C. celerecrescens. Gram-positive or Gram-variable Clostridium with tapered ends may be difficult to identify to the species level and sometimes to the genus level. The species C. clostridioforme previously comprised several groups of organisms that have been reclassified into separate species, including C. clostridioforme, C. hathewayi, C. bolteae, and, more recently, C. aldenense and C. citroniae (Finegold et al., 2005; Song et al., 2003; Steer et al., 2001; Warren et al., 2006). These close species are frequently referred to as the “C. clostridioforme group” (hereafter abbreviated as the “Cc group”). A new close glycopeptide-resistant species, C. lavalense, has been identified in 2010 (Domingo et al., ☆ The GenBank accession number for the 16S rRNA gene sequence of strain AIP148.09 is JQ246092. ⁎ Corresponding author. Tel.: +33-1-40-61-35-09; fax: +33-1-40-61-31-23. E-mail address: [email protected] (P. Bouvet). 0732-8893/$ – see front matter © 2012 Published by Elsevier Inc. http://dx.doi.org/10.1016/j.diagmicrobio.2012.06.024
2009). The aim of this work was to propose practical means that could help in the differentiation of these close species.
2. Materials and methods 2.1. Patient and bacterial strains studied The strain was isolated from a traumatic wound in a febrile 20year-old male who consulted the emergency unit 2 weeks after being injured by a piece of iron coming from a coin used for cutting wood. After removing it and local sampling, the patient was discharged with an antibacterial treatment (1 g amoxicillin + clavulanic acid 3 times a day) for 7 days. The recovery was complete. The sample yielded a pure and moderately abundant culture of a strictly anaerobic, Gram-positive but becoming rapidly Gram-negative, thin spore-forming diplobacillus with tapered ends. Routine identification using Rapid ID 32A strips (BioMérieux, Marcy-l'Etoile, France) gave a profile corresponding to a very good identification of Clostridium clostridioforme except for a positive reaction for indole production. Regarding the resistance to cefotaxim, the strain was suspected to be Clostridium hathewayi, a Clostridium species close to C. clostridioforme. Then the strain was sent to the National Reference Center (NRC) of Anaerobic Bacteria and Botulism (Institut Pasteur, Paris, France) for confirmation of identification (strain AIP148.09). The type strain of C. lavalense strain CIP109511 T (=CCUG 54291 T) was also included in the study since some data (Rapid ID 32A profile and susceptibility to antibiotics) were absent in the description of the species (Domingo et al., 2009).
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2.2. Identification done at the NRC
3. Results
Strains were characterized using reference methods (JousimiesSomer et al., 2002). Rapid ID 32A galleries (BioMérieux) were used according to the manufacturer's instructions.
3.1. Phenotypic identification of strain AIP148.09
2.3. Molecular identification Amplification of the 16S rDNA gene, sequencing, and analysis of sequence were done as described previously (Carlier et al., 2004). 2.4. Antibiotic susceptibility From published studies (Domingo et al., 2009; Song et al., 2003; Warren et al., 2006), 4 antibiotics whose data are available for all species of the “Cc group” were selected: amoxicillin + clavulanic acid, a third-generation cephalosporin (ceftriaxone), a fluoroquinolone (moxifloxacin), and vancomycin. Estimations of the MICs of strains AIP148.09 and CIP109511 T were determined using the Etest method (bioMérieux). Interpretations of MICs in terms of susceptibility were done according to the criteria of the Comité de l'Antibiogramme of the French Society of Microbiology for susceptibility testing of anaerobes (CASFM, 2012) (see Table 2). β-Lactamase enzymes were searched for using nitrocephin disks (Fluka, Buchs, Switzerland).
The bacterium was a Gram-positive bacillus but becoming rapidly Gram negative with tapered ends. Central to subterminal spores were rarely observed. Cells were found motile. Culture supernatant fluids were nontoxic for mice. With the use of special-potency antibiotic disks, the strain was susceptible to vancomycin (5 μg) (which is predictive of a Gram-positive bacterium), kanamycin (1000 μg), and metronidazole (4 μg), but was resistant to colistin (10 μg), which is the profile mostly encountered with Clostridium. Lecithinase and lipase were negative on egg yolk agar plates. Gelatin and milk were not modified. Production of catalase was negative. Indole was produced. Nitrates and nitrites were not reduced. Abundant gas was produced in trypticase-glucose-yeast extract broth. Acid was produced from arabinose, cellobiose, fructose, galactose, glucose, maltose, mannose, mannitol, melibiose, raffinose, rhamnose, ribose, salicine, trehalose, xylose, and esculin, but not produced from glycerol, inositol, lactose, melezitose, sucrose, and sorbitol. Esculin was hydrolyzed. Strain AIP148.09 was finally referred to the group of saccharolytic, nonproteolytic group of Clostridium and particularly toward species belonging to the so-called “Cc group” but did not fit any acceptable profile. Using Rapid ID 32A gallery strain AIP gave the profile 45312000 corresponding to a very good identification
Table 1 Enzymatic activity tests and profile numbers (Rapid ID 32A gallery) and production of acid from some sugars for differentiation of species of the “C. clostridioforme group”, C. lavalense, and C. celerecrescens.a Rrapid ID 32A Characters (digit no., value)b: α-Galactosidase (1, 4) β-Galactosidase (2, 1) α-Glucosidase (2, 4) β-Glucosidase (3, 1) α-Arabinosidase (3, 2) β-Glucuronidase (3, 4) β-N-Acetyl-glucosaminidase (4, 1) Mannose (4, 2) Raffinose (4, 4) Indole production (5, 2) Alkaline phosphatase (5, 4) Arginine arylamidase (6, 1) Proline arylamidase (6, 2) Leucyl glycine arylamidase (6, 4) Leucine arylamidase (7, 2) Pyroglutamic acid arylamidase (7, 4) Rapid ID 32A profile numbers from Warren et al. (2006)—additional reference in parentheses
C. celerecrescens AIP148.09
C. aldenense
C. bolteae
C. citroniae
C. clostridioforme C. hathewayi
C. lavalense CIP 109511T
+ + + + + − + − − + − − − − − − −
+ (−) + − − +/− − − − (+) − (+) +/− +/− − − (+) − (+) − − 41267000 41000400 41000600 41202000
+ + + − − − − − − − − − − +/− − − 45000400 45000000
− − − − − − − +/− − + +/− +/− − − +/− − 00026000 00006000
+ (−) + (−) − (+) − − (+) − (+) − − − (+) − − (+) − − − − − 00000000 41000000 41000400
+ + + + + + + (−) +/− +/− − − − − +/− − +/− 45710000 45710040 45710400 45770440 (Woo et al. 2008)
− + − − − − − − − + − − − − − − −
00002120 (1)
45160000 (1)
45760440 (1)
0100200 (1)
41444000 (1)
45710400 (1)
+ − + +
+ + +/− +
Rapid ID 32A profile numbers 45312000 (1) from our laboratory (no. of strains) Production of acid from Lactose Melezitose Rhamnose Salicine
w
+ /− − + +
41206000 41266000 01206000 (1)
+/− − − − (+)
−(+) + +/− − (+)
− − + −
+ − − +
+ = Positive; +w = weakly positive; − = negative; +(−) = positive (rarely negative); −(+) = negative (rarely positive); +/− = positive or negative. a Data were obtained from published studies (Domingo et al., 2009; Palop et al., 1989; Song et al., 2003; Warren et al., 2006; Williams et al., 2010; Woo et al., 2008), from some strains studied in our laboratory with 16S rDNA gene sequenced (2 Clostridium hathewayi, 2 C. clostridioforme, 1 C. aldenense, 1 C. citroniae), and from C. lavalense type strain CIP 109511T (=CCUG 54291T) studied in our laboratory. b Coding of results in Rapid ID 32A strips is based on the following principle: 8 digits; 3 characters per digit; in a digit, a value is determined according to the character found positive (value 1 for character no. 1, 2 for character no. 2, and 4 for character no. 3) and 0 if negative. Values obtained for each character in a digit are then added up. The following tests were always found negative: urease, arginine dihydrolase, β-galactosidase-6-phosphate, reduction of nitrates, phenylalanine arylamidase, tyrosine arylamidase, alanine arylamidase, and glycine arylamidase.
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(%id = 99.4%; T = 0.72) of C. clostridioforme except for a positive reaction for indole production (6% of strains positive in the API database) (Table 1). 3.2. Molecular identification The 16S rDNA gene sequence that was determined (1435 nucleotides; GenBank accession number JQ246092) was found to be 99.2% related to Clostridium celerecrescens (GenBank NR_026100) which was first isolated from a methanogenic cellulose–enriched culture (Palop et al., 1989) but also from the environment or from animals. Its sequence was only 92.6% related to that of the type strain of C. clostridioforme (GenBank M59089). Both species belong to the Clostridium coccoides rRNA group of organisms (rRNA cluster XIVa of Clostridium (Collins et al., 1994) which are the usual inhabitants of human fecal microbiota unlike other mammals.
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2009; Palop et al., 1989; Song et al., 2003; Warren et al., 2006; Williams et al., 2010; Woo et al., 2008); from some strains studied in our laboratory with 16S rDNA gene sequenced (Clostridium hathewayi [2 strains], C. clostridioforme [2 strains]], C. aldenense [1 strain], and C. citroniae [1 strain]); and from the C. lavalense type strain CIP109511 T studied at the NRC (Table 1). Seven enzymatic tests (α/β-galactosidase, α/β-glucosidase, α-arabinosidase, β-glucuronidase, β-N-acetylglucosaminidase) and indole production seemed the most useful characters for identification (characters in italics, Table 1). Except for C. hathewayi, which was positive for all these 7 enzymatic tests and does not produce indole (a few negative strains for β-N-acetylglucosaminidase may exist), other species have most often between 1 and 3 positive tests. C. celerecrescens is close to C. hathewayi with 6 positive enzymatic tests (β-glucuronidase test result was found negative) and a positive production of indole. Fermentation of melezitose also differentiates these 2 species (positive for C. hathewayi and negative for C. celerecrescens) (Table 1).
3.3. Antibiotic susceptibility β-Lactamase enzymes were not detected. Susceptibility to antibiotics has been found of help in the identification of species of the “Cc group”. Susceptibilities of these species to the selected antibiotics (amoxicillin + clavulanic acid, ceftriaxone, moxifloxacin, and vancomycin) retrieved from the literature are presented in Table 2. Susceptibilities of strains AIP148.09 and CIP109511T are also presented in Table 2. Among these species, C. hathewayi is the only species to be resistant to third-generation cephalosporins (cefotaxim or ceftriaxone), but our C. celerecrescens isolate is resistant too (MIC 256 μg/mL). C. celerecrescens AIP148.09 is susceptible to moxifloxacin with a MIC of 0.38 μg/mL, whereas species of the “Cc group” and C. lavalense have an intermediate susceptibility or a resistance to this antibiotic (Table 2). Resistance to vancomycin (MIC N256 μg/mL) was found in C. lavalense (Domingo et al., 2009) as well as in C. bolteae, C. clostridioforme, and C. hathewayi in Marvaud et al. (2011). This resistance to vancomycin is due to a VanB gene, part of conjugative transposon Tn 5382 (closely related to Tn 1549) (Marvaud et al., 2011). As this resistance determinant is located on a mobile element, one could not exclude the discovery of vancomycin-susceptible C. lavalense isolates. 3.4. Identification table for species of the “Cc group”, C. lavalense, and C. celerecrescens Phenotypic identification of this rare species, C. celerecrescens, is rather difficult and leads frequently to the “Cc group”. In order to help in the differentiation of these related species, we attempted to rebuild an identification table using dispersed available Rapid ID 32A data and sugar fermentation data from published studies (Domingo et al.,
4. Discussion Phenotypic identification of the rare species C. celerecrescens in clinical microbiology by using reference methods as well as by identification galleries is rather difficult and leads frequently to the “Cc group”. It is clear that species of the “Cc group” are involved in a variety of severe infections (Finegold et al., 2005; Linscott et al., 2005; Ogah et al., 2012; Williams et al., 2010). Most cases of bacteraemia are mostly due to mixed flora, but the species C. clostridioforme was considered as particularly virulent. Our strain of C. celerecrescens was isolated from a traumatic leg wound. Two other human cases of infections with C. celerecrescens (an abscess of an open bone fracture and a relapse of posttraumatic osteomyelitis) were recently published (Glazunova et al., 2005; Mischnik et al., 2011). In these 3 cases, phenotypic identifications lead to the “Cc group” or unlikely identifications, and, finally, definitive identifications were only obtained after 16S rDNA sequence analysis. Whereas the virulence of C. celerecrescens is not well known and probably low, its misidentification as a virulent Clostridium species is problematic. Our strain was identified as C. clostridioforme by Rapid ID 32A gallery with a very good identification score and as belonging to the “Cc group” at the NRC. Among species of the “Cc group”, the more recent Rapid ID 32A database (APIweb v. 3.2) contains only C. clostridioforme. With the use of this database, Rapid ID 32A profile numbers of strains belonging to the different species of the group (data from Warren et al., 2006), as well as data obtained from field strains with 16S rDNA sequence available (Table 1) gave frequently C. clostridioforme as identification, with more or fewer tests in opposition but also unlikely identifications. In order to help in
Table 2 In vitro susceptibility of C. celerecrescens, C. lavalense, and species belonging to the “C. clostridioforme group” to 4 selected antibiotics. Antibiotic
C. celerecrescens AIP148.09a
C. aldenense
C. bolteae
C. citroniae
C. clostridioforme
C. hathewayi
C. lavalense CIP109511Ta
Amoxicillin + clavulanic acid Ceftriaxone Moxifloxacin Vancomycin References
S
S (n = 13)b
R (n = 34)
S (n = 7)
S (n = 5)
S (n = 46)
S
R S S This study
S (n = 13) R (n = 13) S (n = 13) Warren et al., 2006
S (n = 34) I/R (n = 53) S (n = 5)c Finegold et al., 2005; Song et al., 2003; Warren et al., 2006
S (n = 7) R (n = 7) S (n = 7) Warren et al., 2006
S (n = 5) I/R (n = 26) S (n = 1)c Finegold et al., 2005; Song et al., 2003; Warren et al., 2006
R (n = 46) I/R (n = 92) S (n = 46)c Finegold et al., 2005; Warren et al., 2006
S I R Domingo et al., 2009 and this study
R = Resistance; I = intermediate susceptibility; S = susceptibility. a MIC estimations (μg/ml) for amoxicillin + clavulanic acid, ceftriaxone, moxifloxacin, and vancomycin were 0.75, 256.0, 0.38, and 1.5, respectively, for C. celerecrescens AIP 148.09 and 0.038, 3.0, 2.0, and N256.0, respectively, for C. lavalense CIP 109511T. According to the recommendations of the Comité de l'antibiogramme of the French Society of Microbiology (CASFM, 2012), interpretative criteria for amoxicillin + clavulanic acid, ceftriaxone, moxifloxacin, and vancomycin are as follows: MICs (μg/ml) of susceptible strains are ≤4, ≤4, ≤1, and ≤4, respectively; MICs (μg/ml) for resistant strains are N8, N32, N2, and N8, respectively. b Numbers of strains studied are noted in parentheses. c Vancomycin-resistant strains have been described (Marvaud et al., 2011).
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the differentiation of these related species, we propose an identification table (Table 1) and differential susceptibilities of these species to 4 selected antibiotics (Table 2). Based on these data, our C. celerecrescens isolate was found to be close to C. hatheway with a similar enzymatic activity profile (Rapid ID 32A gallery) except for a negative β-glucuronidase activity, a positive reaction for indole, and a negative result for melezitose fermentation (Table 1). Both species had a common resistance to third-generation cephalosporin, but our C. celerecrescens isolate was susceptible to moxifloxacin, whereas other species of the “Cc group” and C. lavalense presented an intermediate susceptibility or a resistance (Table 2). It is obvious that these data need to be refined with the study of additional wellcharacterized strains. Published articles relating misidentified microorganisms by conventional methods are regularly increasing (Petti et al., 2005). It is obvious that the number of new bacterial species is growing exponentially, and, consequently, the performance of commercially available biochemical galleries that give an exact identification is decreasing partly due to the limited number of characters available. Diagnostics of rare bacteria by conventional methods as well as of bacteria with unusual phenotypic profiles needs to be confirmed by a more objective tool, e.g., 16S rDNA gene sequencing (Woo et al., 2008). Since rare bacterial species such as C. celerecrescens may be responsible for infections in humans, recently developed identification systems used in clinical microbiology like matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS) should include in their databases corresponding entries. This point has been recently highlighted by Mischnik et al. (2011) who failed to identify their C. celerecrescens isolate by MALDI-TOF MS. Databases elaborated by identification gallery suppliers or other identification systems suppliers should be refined by integrating strains originated from diverse locations (environment, different microbiota, etc.) and unambiguously identified by 16S rDNA sequencing. Unambiguous identification of unusual strains in clinical microbiology is more and more needed to improve our knowledge of reservoirs, routes of transmission, antibiotic susceptibility, and improvement of treatments, and to allow identification of novel bacterial species. Dedication We dedicate this communication to the memory of our colleague Jean-Philippe Carlier deceased in 2008 who has made a major contribution to the identification and taxonomy of anaerobes.
Acknowledgment The authors warmly thank the CRBIP (Biological Resource Centre of Institut Pasteur, Paris) for the gift of the type strain of Clostridium lavalense CIP 109511 T. References Carlier JP, K'Ouas G, Bonne I, Lozniewski A, Mory F. Oribacterium sinus gen. nov., sp. nov., within the family ‘Lachnospiraceae’ (phylum Firmicutes). Int J Syst Evol Microbiol 2004;54:1611–5. CASFM. Recommandations 2012. Paris, France: Comité de l'antibiogramme de la Société Française de Microbiologie; 2012. Collins MD, Lawson PA, Willems A, Cordoba JJ, Fernandez-Garayzabal J, Garcia P, et al. The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species combinations. Int J Syst Bacteriol 1994;44:812–26. Domingo MC, Huletsky A, Boissinot M, Helie MC, Bernal A, Bernard KA, et al. Clostridium lavalense sp. nov., a glycopeptide-resistant species isolated from human faeces. Int J Syst Evol Microbiol 2009;59:498–503. Finegold SM, Song Y, Liu C, Hecht DW, Summanen P, Kononen E, et al. Clostridium clostridioforme: a mixture of three clinically important species. Eur J Clin Microbiol Infect Dis 2005;24:319–24. Glazunova O, Raoult D, Roux V. First identification of Clostridium celerecrescens in liquid drained from an abscess. J Clin Microbiol 2005;43:3007–8. Jousimies-Somer HR, Summanen P, Citron DM, Baron EJ, Wexler HM, Finegold SM. Anaerobic bacteriology manual. 6th ed. Belmont, California: Star Publishing Company; 2002. Linscott AJ, Flamholtz RB, Shukla D, Song Y, Liu C, Finegold SM. Fatal septicemia due to Clostridium hathewayi and Campylobacter hominis. Anaerobe 2005;11: 97–8. Marvaud JC, Mory F, Lambert T. Clostridium clostridioforme and Atopobium minutum clinical isolates with vanB-type resistance in France. J Clin Microbiol 2011;49: 3436–8. Mischnik A, Zimmermann S, Bekeredjian-Ding I, Egermann M. Relapse of posttraumatic osteomyelitis due to Clostridium celerecrescens. Infection 2011;39:491–4. Ogah K, Sethi K, Karthik V. Clostridium clostridioforme liver abscess complicated by portal vein thrombosis in childhood. J Med Microbiol 2012;61:297–9. Palop ML, Valles S, Pinaga F, Flors A. Isolation and characterization of an anaerobic, cellulolytic bacterium, Clostridium celerecrescens sp. nov. Int J Syst Bacteriol 1989;39:68–71. Petti CA, Polage CR, Schreckenberger P. The role of 16S rRNA gene sequencing in identification of microorganisms misidentified by conventional methods. J Clin Microbiol 2005;43:6123–5. Song Y, Liu C, Molitoris DR, Tomzynski TJ, Lawson PA, Collins MD, et al. Clostridium bolteae sp. nov., isolated from human sources. Syst Appl Microbiol 2003;26:84–9. Steer T, Collins MD, Gibson GR, Hippe H, Lawson PA. Clostridium hathewayi sp. nov., from human faeces. Syst Appl Microbiol 2001;24:353–7. Warren YA, Tyrrell KL, Citron DM, Goldstein EJ. Clostridium aldenense sp. nov. and Clostridium citroniae sp. nov. isolated from human clinical infections. J Clin Microbiol 2006;44:2416–22. Williams OM, Brazier J, Peraino V, Goldstein EJ. A review of three cases of Clostridium aldenense bacteremia. Anaerobe 2010;16:475–7. Woo PC, Lau SK, Teng JL, Tse H, Yuen KY. Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories. Clin Microbiol Infect 2008;14:908–34.