Patent Abstract:
there is provided a method of isolating novel lactic acid utilizing bacteria from human faeces , as well as novel strains so obtained . the use of the novel lactic acid utilising bacteria in therapy , including prophylactic therapy , is described and is of particular relevance for lactic - acidosis , short bowel syndrome and inflammatory bowel disorders such as crohn &# 39 ; s disease and ulcerative colitis . a probiotic comprising the live lactic acid utilising bacteria is also described .

Detailed Description:
1 . certain human colonic anaerobic bacteria , including a . caccae strains , are strong and efficient utilisers of lactic acid . 2 . certain human colonic anaerobic bacteria , including a . caccae strains , are strong and efficient producers of butyric acid . 3 . certain human colonic anaerobic bacteria , including a . caccae strains , convert lactic acid to butyric acid . a faecal sample was obtained from a healthy adult female volunteer that had not received antibiotics in the previous 6 months . whole stools were collected , and 1 g was mixed in 9 ml anaerobic m2 diluent . decimal serial anaerobic dilutions were prepared and 0 . 5ml inoculated into roll tubes by the hungate technique , under 100 % co 2 ( byrant , 1972 ). bacterial strains were isolated by selection as single colonies from the nutritionally rich medium in anaerobic roll tubes as described by barcenilla et al . ( 2000 ). the isolates were grown in m2gsc broth and the fermentation end products determined . butyrate producing bacteria were re - purified using roll tubes as described above . strains l1 - 92 , s d8 / 3 , s d7 / 11 , a2 - 165 , a2 - 181 , a2 - 183 , l2 - 50 and l2 - 7 were all isolated using this medium . omitting rumen fluid and / or replacing the sugars with one additional carbon source such as dl lactate increased the selectivity of the roll tube medium and this medium was used to isolate strain s d6 1l / 1 . strains g 2m / 1 and sm 6 / 1 were isolated from medium where dl - lactate was replaced with mannitol ( 0 . 5 %). separately , non - rumen fluid based media routinely used for isolating selenomonas sp ., namely ss and sr medium ( atlas , 1997 ) was used to isolate other strains . inoculating sr medium roll tubes with dilutions of faecal samples resulted in the isolation of strain sr1 / 1 while the ss medium resulted in the isolation of strains ss2 / 1 , ss3 / 4 and ssc / 2 . a . caccae and other human colonic bacterial isolates consumes lactic acid and acetic acid and produces butyric acid when grown in rumen fluid table 1 summarises the fermentation products formed by twelve strains of anaerobic bacteria when grown under 100 % co 2 in a rumen fluid - containing medium containing 0 . 5 % lactate ( m2l ) or 0 . 5 % lactate , 0 . 2 % starch , 0 . 2 % cellobiose and 0 . 2 % glucose ( m2gscl ) as the energy sources . ten of these strains were isolated from human faeces as described above in example 1 . strains 2221 and ncimb8052 are stock collection isolates not from the human gut and are included for comparison . table 1 demonstrates that three strains , l1 - 92 ( a . caccae ), sd6 1l / 1 and sd 6m / 1 ( both e . hallii - related ) all consumed large amounts of lactate (& gt ; 20 mm ) on both media examined , m2l and m2gscl , and produced large quantities of butyric acid . a . caccae l1 - 92 in particular consumed large amounts of lactate and produced large amounts of butyrate . acetate is also consumed by all three strains . the other 9 butyrate producing bacteria tested either consumed relatively small amounts of lactate , or consumed no lactate , on this medium . l - lactate concentrations were determined enzymatically and glucose concentrations were determined by the glucose oxidase method ( trinder , 1969 ). analyses were conducted in a robotic clinical analyser ( kone analyser , konelab corporation , finland ). a . caccae and other human colonic bacterial isolates consumes lactic acid and acetic acid and produces butyric acid when grown in rumen fluid free medium table 2a shows the utilisation and production of formate , acetate , butyrate , succinate and lactate , on this occasion performed using the rumen fluid - free medium ycfa ( duncan et al . 2002 ) containing no added energy source , or with 32 mm lactate ( ycfal ) or lactate plus 23 mm glucose ( ycfalg ) as added energy sources . separately table 2b reveals the levels of the two isomers of lactate ( d and l ) remaining at the end of the incubations and the concentration of glucose metabolised during the incubations . five additional new lactate - utilising isolates were discovered using the semi - selective medium as described earlier and are included in tables 2a and 2b , although one of these ( ss 3 / 4 ) proved to - consume a relatively small amount of lactate only on the ycfal medium ( table 2a ). analysis of the consumption of the d and l isomers reveals that three strains ( ss2 / 1 , ssc / 2 and sr1 / 1 ) preferentially consumed d lactate . partial repression of lactate consumption by glucose was observed on this medium with a . caccae l1 - 92 , and almost complete repression for sl 6 / 1 / 1 and ss 3 / 4 . the previously isolated e . hallii strain l2 - 7 ( barcenilla et al ., 2000 ) behaved in a similar manner to sl 6 / 1 / 1 . the higher glucose concentration in this medium compared with m2gscl is likely to explain the difference in behaviour of a . caccae compared with table 1 . the remaining five strains showed no evidence of repression of lactate utilisation in the presence of glucose although it is possible they use the glucose before switching to lactate . butyrate levels exceeding 30 mm were obtained for four strains on ycfalg medium . results : the three e . hallii - related strains ( l - 27 , sl 6 / 1 / 1 , sm 6 / 1 ) and the two a . caccae strains ( l1 - 92 and p2 ) were able to use both the d and l isomers of lactate during growth either on dl lactate or dl lactate plus glucose ( fig3 ). the four remaining new isolates sr1 / 1 , ssc / 2 , ss2 / 1 and ss3 / 4 however showed a strong preference for using d - lactate . in most strains , except ss3 / 4 and l2 - 7 , some utilisation of lactate was detectable following 24 hours incubation even when glucose was initially present in the medium ( fig3 ). table 2a table 2a . fermentation products formed or utilised ( u as indicated by minus values ) by human gut isolates incubated on yeast extract - casitone - fatty acids medium ( ycfa ); ycfa supplemented with lactate ( ycfal ); and ycfa supplemented with glucose and lactate ( ycfalg ). the initial concentration of glucose added to the medium was 23 mm and 32 mm lactate was added that contained 15 . 5 mm l - lactate . a strain identity is based on 16s rrna sequence information (% identical residues with closest relative is shown ). see figure 1 for sequence information . all strains except 2221 and 8052 ( table 1 ) were isolated as described in example 1 . strain id closest relative a isolation medium medium formate acetate p / u butyrate succin lactate p / u ss2 / 1 cl . indolis ( 95 %) selenomonas selective ycfa 0 . 02 ± 0 . 04 − 4 . 25 ± 4 . 68 2 . 24 ± 0 . 26 0 . 39 ± 0 . 03 ycfal 0 . 18 ± 0 . 02 − 12 . 51 ± 1 . 27 12 . 98 ± 0 . 19 − 15 . 27 ± 2 . 53 ycfalg 10 . 10 ± 1 . 05 − 24 . 32 ± 1 . 03 35 . 69 ± 1 . 13 − 13 . 95 ± 2 . 70 sr 1 / 1 ruminococcus obeum selenomonas ycfa − 5 . 42 ± 1 . 77 2 . 33 ± 0 . 03 0 . 36 ± 0 . 12 hucb 12 * ruminantium ycfal 0 . 76 ± 0 . 19 − 13 . 35 ± 2 . 27 14 . 15 ± 0 . 17 − 15 . 04 ± 0 . 89 ycfalg 9 . 53 ± 2 . 03 − 22 . 47 ± 1 . 40 35 . 77 ± 1 . 50 − 13 . 71 ± 0 . 40 sl 6 / 1 / 1 e . hallii m2 + 0 . 5 % lactate ycfa − 4 . 96 ± 3 . 26 1 . 42 ± 0 . 23 huca 15 * ycfal − 18 . 51 ± 0 . 96 21 . 06 ± 1 . 06 − 29 . 93 ± 0 . 60 ycfalg − 9 . 22 ± 2 . 52 20 . 78 ± 1 . 52 − 2 . 43 ± 0 . 70 sm 6 / 1 e . hallii ( 98 %) m2 + 0 . 5 % mannitol ycfa 0 . 09 ± 0 . 03 − 2 . 61 ± 2 . 36 1 . 42 ± 0 . 05 ycfal 0 . 21 ± 0 . 1 − 7 . 20 ± 2 . 08 6 . 54 ± 0 . 43 − 6 . 27 ± 1 . 27 ycfalg 20 . 68 ± − 10 . 95 ± 29 . 2 ± − 25 . 82 ± ss 3 / 4 ruminococcus gnavus selenomonas selective ycfa 4 . 75 ± 2 . 20 6 . 10 ± 0 . 27 1 . 09 ± 0 . 47 huca19 * ss 3 / 4 ruminococcus gnavus selenomonas selective ycfa 4 . 75 ± 2 . 20 6 . 10 ± 0 . 27 1 . 09 ± 0 . 47 huca19 * ycfal 6 . 68 ± 2 . 09 6 . 19 ± 0 . 34 − 9 . 78 ± 2 . 56 ycfalg 0 . 54 ± 0 . 13 5 . 06 ± 4 . 28 8 . 66 ± 0 . 53 3 . 86 ± 1 . 09 ssc / 2 cl . indolis ( 95 %) selenomonas selective ycfa 0 . 25 ± 0 . 04 − 0 . 16 ± 1 . 32 2 . 37 ± 0 . 09 0 . 48 ± 0 . 03 ycfal 0 . 36 − 12 . 12 13 . 49 − 13 . 78 ycfalg 10 . 98 ± 1 . 27 − 25 . 35 ± 2 . 87 36 . 10 ± 0 . 49 − 13 . 34 ± 1 . 28 l1 - 92 a . caccae m2gsc ycfa 0 . 00 ± 0 . 08 − 2 . 35 ± 2 . 03 1 . 99 ± 0 . 09 ( type strain ) ycfal − 0 . 05 ± 0 . 10 − 21 . 98 ± 2 . 45 23 . 35 ± 1 . 16 − 28 . 92 ± 0 . 54 ycfalg 1 . 49 ± 0 . 13 − 26 . 83 ± 0 . 58 36 . 81 ± 3 . 61 − 12 . 01 ± 1 . 32 l2 - 7 e . hallii m2gsc ycfa 0 . 02 ± 0 . 01 − 1 . 58 ± 1 . 73 0 . 63 ± 0 . 03 0 . 00 ± 0 . 00 ycfal 1 . 09 ± 1 . 55 − 14 . 77 ± 0 . 93 22 . 58 ± 0 . 76 − 30 . 47 ± 0 . 00 ycfalg 3 . 93 ± 3 . 38 12 . 78 ± 0 . 94 5 . 80 ± 0 . 97 1 . 67 ± 0 . 47 * clone library sequences , uncultured ( hold et al ., 2002 ) total lactate ( mm ) remaining in the tubes at the end of the 24 h incubation period and separately the concentration of the two forms d and l . total cell pellets from 1 ml cultures grown on m2gsc medium ( 24 h ) that were resuspended in 50 μl of sterile d . h 2 o served as templates for pcr reactions ( 0 . 5 μl per 50 μl of pcr reaction ). 16s rrna sequences were amplified with a universal primer set , corresponding to positions 8 - 27 ( 27f , forward primer , agagtttgatcmtggctcag ) and 1491 - 1511 ( rp2 , reverse primer acggctaccttgttacgactt ) of the escherichia coli numbering system ( brosius , 1978 ; weisberg , 1991 ) with a mgcl 2 concentration of 1 . 5 mm . pcr amplifications were performed using the following conditions : initial denaturation ( 5 min at 94 ° c . ), then 30 cycles of denaturation ( 30 s at 94 ° c . ), annealing ( 30 s at 51 ° c . ), and elongation ( 2 min at 72 ° c . ), and a final extension ( 10 min at 72 ° c .). the amplified pcr products were purified using qia quick columns ( qiagen gmbh , germany ) according to manufacturer &# 39 ; s instructions and directly sequenced using a capillary sequencer ( beckman ) with primers 27f , rp2 , 519f ( cagcmgccgcggtaatwc ) and 519r ( gwattaccgcggckgctg ) ( corresponding to positions 518 - 535 of the e . coli numbering system ) and 926f ( aaactcaaakgaattgacgg ) and 926r ( ccgtcaattcmtttragttt ) corresponding to positions 906 - 925 ). two independent pcr products were sequenced per strain . similarity of the 16s rrna sequences ( minimum 1444 bases ) of the isolates with other organisms was compared with all sequence data in genbank using the blast algorithm ( altschul , 1990 ). three lactate utilising strains , anaerostipes caccae l1 - 92 and two strains of eubacterium hallii ( sm 6 / 1 and l2 - 7 ) were incubated alone and in co - culture with b . adolescentis l2 - 32 on ycfa medium modified to contain reduced casitone ( 0 . 1 %) and 0 . 2 % soluble starch as an added energy source . the inoculated tubes were incubated for 24 h at 37 ° c . b . adolescentis l2 - 32 was enumerated on mann ragosa sharpe ( mrs ) medium containing 2 . 0 % agar with a final concentration of 0 . 5 % propionate and the three butyrate producing strains , were enumerated on m2 medium containing 0 . 5 % dl lactate . results : in most human diets , resistant starch is considered to be the most important energy source for microbial growth in the large intestine ( topping , 2001 ). the major amylolytic species in the human colon are generally considered to be bacteroides and bifidobacterium spp . ( macfarlane , 1986 ; salyers , 1977 ). bifidobacteria produce acetate and lactate from carbohydrate substrates , typically in the molar ratio of 3 : 2 . since the lactate utilisers isolated here either do not utilise starch or utilised it weakly , as a growth substrate in pure culture , it was of interest to co - culture them with a starch - degrading bifidobacterium strain in order to establish whether they could remove the lactate formed . the recently isolated , actively amylolytic b . adolescentis strain l2 - 32 was used for these experiments . as shown in tables 3a , 3b and fig2 , co - culture with any one of three lactate utilisers tested , with starch as the growth substrate , resulted in complete conversion of the l - lactate , and some of the acetate , formed by b . adolescentis l2 - 32 into butyric acid . this corresponded with greatly increased growth of the lactate utilisers in the presence of the b . adoliscentis l2 - 32 , as determined by selective plating . viable counts ( cfu ml − 1 ) after 24 hours growth for l1 - 92 , sm 6 / 1 and l2 - 7 were , respectively , 2 . 4 × 10 8 , 1 . 0 × 10 7 and 8 . 0 × 10 6 , in the absence of b . adolescentis , and 1 . 7 × 10 9 , 6 . 8 × 10 8 and 5 . 4 × 10 9 , in the presence of b . adolescentis l2 - 32 . growth of b . adolescentis l2 - 32 was unaffected by co - culture ( mean 4 . 3 × 10 8 cfu ml − 1 ). there may have been some contribution of starch hydrolysis products that escape uptake by the b . adolescentis l2 - 32 , in addition to lactate and acetate , to the growth of the lactate utilisers . this might account for the apparent effectiveness of e . hallii sm 6 / 1 in co - culture , even though this strain used rather little in pure culture when supplied with lactate alone . table 3a fermentation profiles for bifidobacterium adolescentis l2 - 32 and three lactate utilisers when incubated alone or in co - culture for 24 hours at 37 ° c . on modified ycfa medium ( modified to contain 0 . 1 % casitone ) containing 0 . 2 % soluble starch . culture / total co - culture formate acetate butyrate lactate l - lactate l2 - 32 4 . 29 ± 0 . 92 51 . 04 ± 5 . 44 0 5 . 00 ± 0 . 09 5 . 16 ± 0 . 45 l1 - 92 0 . 01 ± 0 . 01 34 . 99 ± 0 . 93 1 . 57 ± 0 . 26 0 . 40 ± 0 . 69 0 sm 6 / 1 0 35 . 25 ± 2 . 15 0 . 75 ± 0 . 06 0 . 27 ± 0 . 27 0 l2 - 7 0 . 04 ± 0 . 06 35 . 70 ± 0 . 44 0 . 83 ± 0 . 02 0 0 l2 - 32 + l1 - 92 4 . 29 ± 0 . 04 44 . 82 ± 1 . 13 7 . 62 ± 0 . 66 0 . 61 ± 0 . 53 0 l2 - 32 + sm 6 / 1 4 . 81 ± 1 . 08 48 . 17 ± 6 . 47 6 . 23 ± 1 . 15 0 0 l2 - 32 + l2 - 7 5 . 16 ± 1 . 37 43 . 88 ± 3 . 74 7 . 35 ± 0 . 27 0 . 36 ± 0 . 01 0 total viable counts ( cfu per ml ) of bifidobacterium adolescentis l2 - 32 and three lactate utilisers following 24 hours at 37 ° c . in monoculture utilisers were selected for on m2 + 0 . 5 % lactate roll tubes following time courses were followed in batch culture for growth on glucose , lactate or glucose and lactate ( fig4 ). e . hallii l2 - 7 when grown with dl - lactate used all of the added lactate together with some acetate , producing more than 20 mm butyrate ( fig4 ). less butyrate , but significant formate , was produced during growth on glucose , or on glucose plus lactate , and lactate utilisation was almost abolished by the presence of glucose . hydrogen production in 24 hours was 12 μm ml − 1 for growth on glucose , 15 . 5 μmol ml − 1 for growth on lactate and 10 . 9 μmol ml − 1 for growth on glucose plus lactate . a . caccae l1 - 92 similarly produce larger quantities of butyrate when grown on lactate compared with growth on glucose , when formate was also a product . this strain was able to use lactate once glucose had been exhausted , following inoculation into glucose plus lactate medium . strain ss2 / 1 is likely to represent a new species , since its closest relative ( 95 % identity in 16s rrna sequence ) is the non - butyrate producing clostridium indolis . this strain was able to use d -, but not l -, lactate following glucose exhaustion in lactate plus glucose medium ( fig5 ). again formate was not a significant product when lactate was the sole energy source but 4 . 7 μmol ml − 1 hydrogen was formed . a . caccae strain l1 - 92 was able to consume up to 30 mm dl lactate , along with 20 - 30 mm acetate during batch culture incubation for 24 hours at 37 ° c . with the production of & gt ; 20 mm , and up to 45 mm butyrate ; this occurred also when glucose was added as an alternative energy source ( table 1 ). lactate or lactate plus glucose thus resulted in very much higher production of butyrate than observed with 23 mm glucose alone , when only & lt ; 15 mm butyrate was formed . furthermore none of the 74 strains screened previously by barcenilla et al . ( 2000 ) produced more than 25 mm butyrate when tested in m2gsc medium . lactate consumption is not a general characteristic of butyrate - producers , and six of the strains screened in table 1 failed to consume lactate in m2gscl medium . six further strains that are highly active lactate utilisers ( defined for example as net consumption of at least 10 mm of lactate during growth to stationary phase or for 24 hours in ycfalg or ycfal medium at 37 ° c .— see table 2a ) were obtained following deliberate screening of new human faecal isolates for lactate utilisation . at least two of these ( sl 6 / 1 / 1 and sm 6 / 1 — tables 1 , 2 ) are related to eubacterium hallii . ( table 2a ), based on determination of their 16s rdna sequences . these isolates again consume large quantities of lactate and produce high levels of butyrate in vitro . with one exception where considerable glucose repression occurred ( strain sl 6 / 1 / 1 ), significant lactate utilization occurred in the presence of glucose ( table 2 ). three strains ( ss 2 / 1 , sr 1 / 1 and ssc / 2 ) showed preferential utilization of d - lactate , whereas the two e . hallii - related strains sm 6 / 1 , sl 6 / 1 / 1 and a . caccae l1 - 92 utilise both isomers ( table 2b ). the two stereoisomers differ in their toxicity in the human body , with the d - isomer being regarded as the more toxic ( chan et al ., 1994 , hove et al ., 1995 ). the present invention thus provides a means of utilising both d and l lactate isomers or preferentially utilising d - lactate in preference to l - lactate . a . caccae and newly isolated bacteria related to e . hallii and cl . indolis were shown to consume up to 30 mm dl , d or l lactate , along with 20 - 30 mm acetate during batch culture incubation and convert this energy in to production of at least 20 mm , and up to 45 mm butyrate . furthermore , these strains were shown to convert all of the l - lactate produced by a starch - degrading strain of bifidobacterium adolescentis into butyrate when grown in culture . this is the first documentation demonstrating the conversion of lactate to butyrate by human colonic bacteria , some of which are likely to be new species . altschul , s . f ., gish , w ., miller , w ., myers , e . w . and lipman , d . j . 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