View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by SHAREOK repository APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 2003, p. 6321–6326 Vol. 69, No. 10 0099-2240/03/$08.00ϩ0 DOI: 10.1128/AEM.69.10.6321–6326.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved. Formate-Dependent Growth and Homoacetogenic Fermentation by a Bacterium from Human Feces: Description of Bryantella formatexigens gen. nov., sp. nov. Meyer J. Wolin,1* Terry L. Miller,1 Matthew D. Collins,2 and Paul A. Lawson2 Wadsworth Center, New York State Department of Health, Albany, New York 12201-0509,1 and School of Food Sciences, University of Reading, Reading RG6 6AP, United Kingdom2 Downloaded from Received 14 March 2003/Accepted 17 July 2003 Formate stimulates growth of a new bacterium from human feces. With high formate, it ferments glucose to acetate via the Wood-Ljungdahl pathway. The original isolate fermented vegetable cellulose and carboxym- ethylcellulose, but it lost this ability after storage at ؊76°C. 16S rRNA gene sequencing identifies it as a distinct line within the Clostridium coccoides supra-generic rRNA grouping. We propose naming it Bryantella forma- texigens gen. nov., sp. nov. http://aem.asm.org/ ⅐ ␮ ␮ Plant cell wall polysaccharides in human diets are not di- Na2MoO4 2H2O, 100 g; H3BO3, 100 g; Na2SeO4, 1.9 mg; ⅐ ␮ ⅐ gested by host enzymes (3). The cellulose and hemicellulose in NiCl2 6H2O, 92 g; nitrilotriacetic acid, 15 mg; thiamine vegetables and fruits are digested in the colon (2, 11, 27) and HCl, 2.0 mg; D-pantothenic acid, 2.0 mg; nicotinamide, 2.0 mg; are fermented by the colonic microbial community to molar riboflavin, 2.0 mg; pyridoxine ⅐ HCl, 2.0 mg; biotin, 10.0 mg; ratios of ca. 56 acetate: 22 propionate: 22 butyrate (4, 32) and cyanocobalamin, 20 ␮g; p-aminobenzoic acid, 100 ␮g; folic ␮ ⅐ ⅐ H2,CO2, and CH4 (32). Bacteria that digest filter paper (FP) acid, 50 g; cysteine HCl H2O, 0.5 g; rumen fluid, 100 ml; or Avicel are relatively unimportant in the human colon. Be- sodium acetate, 2.5 g; sodium formate, 2.5 g; trypticase, 2.0 g. tian et al. (1) and Wedekind et al. (31) showed that the fre- Resazurin (1 mg/liter) was added as an oxidation reduction on May 27, 2015 by UNIV OF OKLA LIBRARIES quency of individuals that harbor them is low and, when potential indicator. The pH was adjusted to 7 with NaOH prior present, their concentrations are ca. 0.001 times the concen- to gassing with 100% CO2 and the addition of NaHCO3. After tration of all anaerobic bacteria. We hypothesized that bacteria dispensing into serum bottles and autoclaving under CO2,a that use amorphous cellulose found in vegetables, but not the sterile solution containing 0.125 g each of cysteine and sodium crystalline cellulose in FP, are present in the colon. A goal of sulfide/ml (30 ␮l per ml of medium) was added prior to inoc- this study was to enumerate and isolate human colonic bacteria ulation. Incubation was at 37°C. that use vegetable cellulose. Table 1 shows the results of MPN analyses of enema samples MPN study. We used a purified cellulose preparation from of patients presenting for flexible sigmoidoscopy. Five tubes of cabbage for most-probable-number (MPN) comparisons of the B1C plus the indicated substrate were inoculated with each concentrations of bacteria that use FP cellulose, vegetable dilution of the enema samples. Disappearance of substrate in cellulose, and starch in human fecal suspensions. A modifica- inoculated tubes indicated the presence of hydrolytic bacteria. tion of the method described by Ehle et al. (8) was used to FP hydrolysis was found in only 3 out of 15 subjects. The prepare a cellulose-enriched fiber preparation (VCP) from concentrations of FP-digesting bacteria were appreciably lower fresh white cabbage (29). The hydrolysis of1gofVCPwith 2 than those for bacteria that used CS or VCP. Our results on N HCl solubilized 287 mg of reducing sugar (glucose equiva- the frequency and concentrations of FP-digesting bacteria con- lent) (22). The insoluble residue contained 138 mg of glucose firm those of Betian et al. (1) and Wedekind et al. (31). VCP- equivalents when measured by the anthrone procedure (26). digesting bacteria were found in 11 of 15 subjects at ca. 16 Distilled water suspensions of the powder were ball milled for times higher concentrations than those of FP-digesting bacte- 18 h at 25°C prior to addition to media. ria. CS-digesting bacteria were present in all subjects exam- MPN analyses were run concurrently with 0.8% VCP, 0.5% ined, and their concentrations, similar to those previously corn starch (CS), or 1-cm by 5-cm strips of Whatman number found by colony enumeration (30), were the highest of the 1 FP with 10 subjects and concurrently with VCP and FP polysaccharide-digesting populations (ca. 45 times higher than cellulose with 15 subjects. The medium (B1C) contained (per those for VCP-digesting bacteria). liter): NaHCO3, 5.0 g; K2HPO4, 0.3 g; KH2PO4, 0.3 g; Isolation. We isolated a novel gram-positive bacterium after ⅐ Ϫ Ϫ (NH4)2SO4, 0.3 g; NH4Cl, 1 g; NaCl, 0.61 g; MgSO4 7H2O, enrichment of 0.5 ml of 10 6 to 10 9 dilutions (five tubes per ⅐ ⅐ 0.15 g; CaCl2 2H2O, 80 mg; MnSO4 H2O, 4.5 mg; dilution) of one human fecal suspension (28) in B1C plus 0.8% ⅐ ⅐ ⅐ FeSO4 7H2O, 3.0 mg; CoSO4 7H2O, 1.8 mg; ZnSO4 7H2O, VCP. Hydrolysis was monitored by observation of the disap- ⅐ ␮ ⅐ ␮ 1.8 mg; CuSO4 5H2O, 100 g; AlK(SO4)2 12H2O, 180 g; pearance of insoluble VCP. The sample yielded an MPN of 1.1 ϫ 1010 hydrolytic bacteria per g of dry feces. Transfers were Ϫ9 * Corresponding author. Mailing address: 8 Wisconsin Ave., Del- made from a tube with 0.5 ml of the 10 dilution to the same mar, NY 12054. Phone: (518) 439-6969. Fax: (518) 474-8590. E-mail: medium with VCP. Growth was also obtained when 2.0% car- [email protected]. boxymethylcellulose type 4M6F (CMC) (Hercules Inc., Wil- 6321 6322 WOLIN ET AL. APPL.ENVIRON.MICROBIOL. TABLE 1. MPN concentrations of bacteria in human fecal No activity was detected for alkaline phosphatase, arginine a suspensions that use vegetable cellulose, FP cellulose, or starch arylamidase, arginine dihydrolase, alanine arylamidase, ␣- Total fucosidase, glutamic acid decarboxylase, glutamyl glutamic No. Bacteria concn (per Substrate no. of positive g of dry feces) Ϯ SD acid arylamidase, glycine arylamidase, histidine arylamidase, subjects leucine arylamidase, leucyl glycine arylamidase, phosphoami- Ϯ Vegetable cellulose 15 11 Log10 9.66 0.70 dase, phenyl alanine arylamidase, proline arylamidase, pyro- Ϯ FP 15 3 Log10 8.45 0.40 glutamic acid arylamidase, serine arylamidase, tyrosine aryl- Ϯ Starch 10 10 Log10 11.31 0.41 amidase, or urease. The organism was indole negative and did a Samples with more than 4 ϫ 106 bacteria per ml of fecal specimen. The not reduce nitrate to nitrite. 6 dilutions necessary to detect samples with less than 4 ϫ 10 bacteria per ml were The dependence of strain I-52T growth on the addition of not inoculated into MPN tubes. Downloaded from CMC to B1C broth was apparent from cell density measure- ments. It grew to a maximal OD660 of 1.0 (1.8-cm light path) in 24 h in a medium with 2% CMC added as the sole carbohy- mington, Del.) replaced 0.8% VCP. Transfers from the VCP drate in B1C with added formate. With VCP containing 2 mg medium were diluted and plated on WM medium with 2% of cellulose/ml (26) instead of CMC, 70% (1.4 mg) of the agar and 0.6% CMC in anaerobic roll tubes (17). WM medium, cellulose disappeared after growth for 96 h (26). No cellulose with mineral concentrations based on those used by McInerney utilization occurred when FP or Avicel was used as a growth et al. (15), contained (per liter): NaHCO3, 3.5 g; KH2PO4, substrate. Unfortunately, recent transfers from stock cultures ⅐ Ϫ http://aem.asm.org/ 0.5 g; NH4Cl, 1 g; NaCl, 0.4 g; MgCl2 6H2O, 0.33 g; maintained at 76°C for several years did not grow with CMC ⅐ ⅐ ⅐ CaCl2 2H2O, 50 mg; FeCl2 4H2O, 1.5 mg; CoCl2 6H2O, 0.2 or the VCP. Other properties of the transfers were identical to ⅐ ⅐ mg; ZnSO4 7H2O, 0.1 mg; MnCl2 4H2O, 0.03 mg; those of the original isolate. ⅐ ⅐ CuCl2 2H2O, 0.01 mg; Na2MoO4 2H2O, 0.03 mg; H3BO3, Fermentation. A brief report of the fermentation of the ⅐ 0.3 mg; Na2SeO4, 1.9 mg; NiCl2 6H2O, 0.02 mg; isolate was presented in previous publications (33, 34). Al- thiamine ⅐ HCl, 2.0 mg; D-pantothenic acid, 2.0 mg; nicotin- though 2.5 mM formate was sufficient for good growth of strain amide, 2.0 mg; riboflavin, 2.0 mg; pyridoxine ⅐ HCl, 2.0 mg; I-52T, increasing the formate concentration ca. 20-fold dramat- biotin, 10.0 mg; cyanocobalamin, 20 ␮g; p-aminobenzoic acid, ically altered the nature of the products formed from glucose ␮ ␮ ⅐ ⅐ 100 g; folic acid, 50 g; cysteine HCl H2O, 0.5 g; sodium (Table 2). Either without added formate or with 4.8 mM for- on May 27, 2015 by UNIV OF OKLA LIBRARIES acetate, 1.0 g; isobutyric acid, 0.54 ml; 2-methylbutyric, valeric, mate, succinate, lactate, and acetate are major products (Table and isovaleric acids (0.6 ml each); casein hydrolysate, 2.0 g 2). With 48 mM formate, acetate production increased consid- (Type I, No.