Note Bioscience Microflora Vol. 20 (2), 53-57, 2001 Evaluation of Methanethiol and Hydrogen Sulfide Production by Standard Strains of Intestinal Bacteria and Isolates from Pig Feces KazunariUSHIDA,1* Naoko OSHIMA,1 Atsushi TANIMURA,1 KohjiMIYAZAKI,1 KOJIMA1 Yoichi and SusumuTAKAKUWA2 1Laboratory ofAnimal Science , KyotoPrefectural University, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan 2LaboratoryofEnvironmental Microbiology , DepartmentofEducation, Kyoto Women's College, Higashiyama-ku, Kyoto605-8501, Japan ReceivedDecember 11,2000; Accepted forpublication, March 19, 2001 Methanethiolandhydrogen sulfide generated from feces present an environmental issue in the intensive field of animal agriculture.We have identified four strains of clostridia and five others as methanethiol producers from a rangeof laboratorystrains, which may be present in the large intestines of livestock.We have also isolated one Clostridium perfringensfrom pig feces as a potentproducer ofmethanethiol. Unlike methanethiol from L-methionine, manylaboratory strainsproduced hydrogen sulfide from L-cystine. Keywords: intestine; feces; methanethiol; hydrogen sulfide Volatile sulfur compounds, such as hydrogen sulfide, Forty-three bacterial species related to intestinal (fe- methanethiol, and dimethyl sulfide, are generated in the cal) bacteria were used (Table 1). We have selected large intestine of mammals because of the bacterial me- most of these bacteria because they are very likely to tabolism of free sulfate via dissimilatory sulfate reduc- be present in pig feces. Most species selected have been tion and desulfhydration of sulfur amino acids. Vola- detected in pig or human intestines (10, 14, 17, 20). tile sulfur compounds are potentially deleterious to the One dairy strain of Lactobacillus, L. delbrueckii, was host, for example in damaging colonocytes (19), and also involved. Most strains were obtained from the Ja- are potentially related to ulcerative colitis and colon pan Collection of Microorganisms (JCM) (Wako, Ja- cancer (8, 18) as well as to hepatic encephalopathy (4, pan). Some strains were from the Deutsche Sammulung 13). Volatile sulfur compounds also produce malodor von Mikroorganismen and Zellkulturen (DSM) originating from animals in intensive farming systems (Braunschweig, Germany), the American Type Culture (26), which has become a serious problem in animal Collection (ATCC) (Manassas, VA, USA). Prevotella agriculture in Japan (9). Sulfate-reducing bacteria are ruminicola NIAH 7-31 and Streptococcus bovis 30 were one of the major agents responsible for cecal and co- supplied by Dr. Minato (National Institute of Animal lonic hydrogen sulfide (3), probably in feces. However, Health, Tsukuba, Japan). Prevotella bryantii B14 was the bacteria responsible for desulfhydration of sulfur supplied by Dr. Flint (Rowett Research Institute, Ab- amino acids in the large intestine and feces have not erdeen, UK). The remaining strains were from our cul- yet been defined; however, some industrial and oral- ture collection. These strains, except for Lactobacil- methanethiol and hydrogen-sulfide producers have been laceae, were cultured twice in GAM broth (Nissui, identified (5, 12, 15, 22, 23, 25). Tokyo, Japan). A portion (0.2 ml) of the preculture was In this experiment, we have examined a range of labo- transferred in the late exponential phase (OD660= 0.4- ratory strains for their activity of desulfhydration of L- 0.6, overnight culture in most cases) to test media (5 methionine and L-cystine. The former reaction produces ml) with and without L-methionine or L-cystine (0.5%, methanethiol, which is the most strictly controlled vola- w/v). The bacteria belonging to Lactobacillaceae were tile sulfur compound because its stench is 10 times cultured in MRS medium (Difco Laboratories, Sparks, stronger than other volatile sulfurs (11). We have also MD, USA) instead of GAM broth. The culture was isolated and identified bacteria that desulfhydrated maintained under anaerobiosis with O2-free CO2 in a methionine or L-cystine from pig feces. 10-ml serum vial closed with a butyl rubber stopper *Corresponding author . Mailing address: Laboratory of Animal Science, (Bellco Laboratories, Vineland, NJ, USA) at 37°C. The Kyoto Prefectural University, Shimogamo, Sakyo-ku, Kyoto 606-8522, Ja- test cultures were continued for 48 hr and 24 hr, re- pan. Phone/Fax: +81-75-703-5620. E-mail: [email protected] spectively, for L-methionine and L-cystine. 53 54 K. USHIDA, et al. VOLATILE SULFUR PRODUCTION BY ANAEROBIC BACTERIA 55 Fermentation in the test media was stopped by the addition of 1 ml 6 N HCI. Gas was collected in a glass syringe, and the volume was measured. A portion (0.5 ml) of collected gas was analyzed for methanethiol and hydrogen sulfide as indicated elsewhere (24). Four mature crossbred pigs were used as donors of feces to isolate volatile sulfur-producing bacteria. The pigs were fed a diet composed mainly of a mixture of cracked corn, alfalfa meal, and meat bone meal (24). The feces from the four pigs underwent a serial 10-fold dilution with an anaerobic dilution solution as indicated by Arakaki et al. (2). A portion (0.2 ml) of each diluted sample was inoculated in triplicate into the above-men- tioned GAM broth medium containing L-methionine (0.5%, w/v) and incubated for seven days. After incu- bation, headspace gas was analyzed for methanethiol as indicated above. When methanethiol was detected, one loop of culture was transferred to the same GAM medium supplemented with L-methionine (0.5%, w/v) both on a slant and in a roll-tube in duplicate. Five to six colonies were picked from one slant or one roll- tube, and 20 to 30 colonies were isolated from each pig. All the manipulation was done under anaerobiosis with 02-free CO2. After appropriate incubation at 37•Ž, developed colonies were picked and transferred to a fresh broth medium of the same ingredients. The iso- lates were analyzed for their ability to produce meth- anethiol, and those that did were further identified. Af- ter collection and washing by centrifugation, bacterial cells were subjected to SSU (16S) rRNA gene sequenc- ing. The technique was principally the same as indi- cated elsewhere (21). Chromosomal DNA was briefly isolated and used as a template for a polymerase chain reaction to amplify 16S rDNA using 27f and 1492r primers. Amplified DNA fragments were directly se- quenced after purification by agarose gel electrophore- sis or were ligated into a pGEM-T easy vector for TA- cloning (Promega, Madison, WI, USA). Escherichia coli JM 109 (Takara Shuzo, Kyoto) was transformed by the vector, and transformants were selected by blue- white selection. Inserted fragments were sequenced after plasmid purification from at least five clones. The sequence results were analyzed by using the BLAST program in the DNA Data Bank of Japan (National In- stitute of Genetics, Center for Information Biology, Mishima). A significant production of methanethiol from L-me- thionine in the GAM medium was detected in cultures of Clostridium sporogenes, C. bifermentans, C. per- fringens, C. butyricum, Streptococcus bovis, Pepto- streptococcus hydrogenalis, Bacteroides thetaiotao- 56 K. USHIDA, et al. micron, Klebsiella pneumoniae, and Fusobacterium also produced small amounts of methanethiol. B. necrophorum (Table 1). Among them, two clostridia thetaiotaomicron was the only strain that produced a produced methanethiol at a very high concentration large amount of methanethiol. Some bacteroides con- (over 100 ƒÊmol). Little or no production was observed tribute to methanethiol production in the large intes- among other laboratory strains, especially among the tine and feces, but probably only to a limited extent lactic acid bacteria. Three anaerobic fecal bacteria were compared with clostridia and fusobacteria. Lactic acid identified as methanethiol producers from 110 isolates. bacteria were often stressed as a source of methanethiol Feces from three pigs each provided one methanethiol production, but they were limited to dairy strains such producer. One methanethiol producer was provided by as Brev. acetylicum, L., cremoris, and L. helveticus (6, the feces of each of three pigs. We failed to isolate a 7). They are detected in feces when animals ingest dairy methanethiol producer from the feces of one pig. Of products. Therefore, there is little chance for these bac- the three, two of the anaerobic rods were Gram-stained teria to be present in significant numbers in pig feces. positive, and one negative. The positive were C. Moreover, three intestinal lactobacilli, L. plantarum, perfringens by partial 16S rDNA sequences; both had L. acidophilus, and L. breve, as well as one dairy lacto- similarities to the database sequence (Genbank acces- bacillus, L. delbrueckii, produced no methanethiol in sion No. Y12669) that exceeded 99% (1319/1327 bp this experiment. Therefore the contributions of lactic and 1320/1327 bp). The negative was Fusobacterium acid bacteria to methanethiol production in pig feces varium, which had a high similarity (97%, 1296/1323 can be ignored. bp) to the database sequence (Genbank accession No. Unlike methanethiol, all laboratory strains tested pro- M58686). duced hydrogen sulfide from L-cystine. Although most A methanethiol producer must have L-methionine- of the clostridia and B. thetaiotaomicron produced siz- a-deamino-y-mercaptomethane-lyase (L-methioninase, able amounts of hydrogen sulfide from L-cystine EC 4.4.1.11) that catalyzes a-y elimination of methio- (> 1,000 ƒÊmol), the others also