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 produced hydrogen nine to produce methanethiol, ammonia, and a-ketobu- sulfide 100-700 ,ƒÊmol. L-Cystine could be converted to tyrate. The enzyme activity has been detected or purified L-cysteine and further desulfhydrated to hydrogen

in a range of aerobic and anaerobic bacteria, and the sulfide. The reaction is catalyzed by cysteine archaebacteria include those belonging to the genera desulfhydrase. The enzyme, known as cystathionine y- Pseudomonas, Aeromonas, Lactococcus, Lactobacil- lyase (4.4.1.1), produced a free thiol group, a keto acid

lus, Brevibacterium, Fusobacterium, Eubacterium, component, and ammonia from sulfur amino acids L- Bacteroides, Clostridium, and Halobacterium (5 7, 12, cysteine and methionine, and amino acid derivatives.

15, 16, 22, 23, 25). However, the number of species Because the enzyme has an essential role in amino acid related to the intestinal bacteria were limited to the fol- and sulfur metabolism, it must be distributed for a wide lowing: C. sporogenes; two oral fusobacteria, F. range of organisms (1). In fact, 36 protein sequences

nucleatum and F. periodonticum; and an unidentified for this enzyme from a variety of animals (three spe- Bacteroides and Eubacterium. The detection of high cies), higher plants (two species), fungi (two species),

methanethiol production in C. sporogenes in this ex- archaea (three species), and bacteria (nine species) are

periment is in line with the detection of the enzyme now available in the protein database through the Entrez activity of this bacterium. According to the present re- search (NCBI, Bethesda, MD, USA). Therefore it is

sult, C. bifermentans should have the same enzyme. probable that many laboratory strains have this enzyme Our fresh isolate Clostridium sp. Sl, most probably C. activity.

perfringens, should also have the same enzyme. The In conclusion, many fecal and/or intestinal bacteria

importance of clostridia in producing methanethiol in generated hydrogen sulfide, but those that generated the large intestine and in feces is clear because all tested methanethiol were limited. In the present experiment, clostridia produced methanethiol with some high-po- methanethiol producers could also be recognized as

tential species. In the present experiment, F. necro- potent hydrogen sulfide producers. Therefore most

phorum was the only strain tested as fusobacteria that probable contributors to methanethiol and hydrogen produced methanethiol. Fusobacteria are often consid- sulfide production by desulfhydration of sulfur amino ered to be sulfide producers in oral cavities (5, 16). As acids in the large intestine and feces are those belong- a major colonizer of the large intestine, fusobacteria ing to the genus Clostridium. These are the targets of were suggested as contributors to intestinal and fecal the control for fecal malodor.

methanethiol production. Most bacteroides tested here VOLATILE SULFUR PRODUCTION BY ANAEROBIC BACTERIA 57

Acknowledgments. This work was supported by a methioninase) from Clostridium sporogenes . Cancer Res

Grant-in-Aid for Scientific Research (B) (2) No . 33: 1862-1865.

10460119 from the Japan Society for Promoting Sci- (13) Merino GE, Jetzer T, Doizaki WM, Najarian JS. 1975. Methionine-induced hepatic coma in dogs. Am J Surg 130: ence. 41-46.

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