CA1290271C - Material and method for promoting growth of anaerobic bacteria - Google Patents

Abstract

MATERIAL AND METHOD FOR
PROMOTING GROWTH OF ANAEROBIC BACTERIA
ABSTRACT

A material end method for promoting the growth of anaerobic bacteria which includes a nutrient media containing a hydrogen donor and sterile membrane fragments of mitochondria having an electron transfer system which reduces oxygen to water. Dissolved oxygen in the medium is removed by adding the sterile membrane fragments to the nutrient medium and holding the medium at a temperature of about 10°C to about 60°C until the dissolved oxygen is removed.

Description

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6~ 09 Fleld_o~ the Inv~ntion The pre~ent invention rel~tes ~ænerelly to promotln~
the ~rowth of ~n~erobiG bacterl~ and ln particul~r relate~
to thæ u~e of ~terillzed ~itochondri~l derivative for the production o~ ~n~erobic conditions in ~edia to promote the ~roYth of eneerobes.
hany o~ the bacterla present in natural environments ure ~ensitive to oxygen and vlll not grow in it8 presence.
When the e orgeni~ms ~known es anaerobes) ~re brought into the laboratory, lt 18 often neces~ary to employ cumbersome phy~lcal and chemicel techniques in order to get them tn 3row. Some of these bacteria produce disea~e o~ man and related species. Other produce importsnt indu~trial end products ~uch a8 meth~ne, hydrogen and various alcohols The manipulati~n of the~e or~enlsms i~, to BOme deyree, limlted by the ease vlth ~hlch they can be gro~n.
In order to grow anaerobe~, it i8 neces~ary for the medium in ~hich the ~naerobe i~ to ~e gro~n to be ~ub~tunti~lly free of oxygen. Oxygen can be removed fairly efficiently fram such media by sparglng Yith hlyh purity nltrogen or ather inert gas. Ho~ever, the~e ll~u~d media are sub~ect to foaming 80 that this process pre~ent~ a number of mechanical di~iculties. Further, ~fter spar~ing 18 ~topped, the medium i~ ea~ily recontaminated with oxygen.
Oxy~en c~n al~o ~e removed ~rom liquid ~edis by the additian of reducin~ egent~. However, mo~t of the~e agent~
are strong reducing agents snd any re~idual agent or it~
hy-products in the media tends to inhibit the ~ubsequent grow~h of anaerobe~ in the medla. Also, the reducin~ a~ent which is aonsumed durin~ the initial removal of oxy~en is not avallable to act upon oxy~en which mlght later find it~
~ay into thæ system.
In the aase of a solid medium, ~uch a~ ugarf oxy~en '' ~ : . ' . -.
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`, ' ~, ~29~7~l ~20-109 remov~l ~ith ~n inert gæs i~ difficult to occompli~
b~a~u~e thæ oxy~en in the ~edium come~ to ~quilibrium ~itb the ine~-t stmo~phere ~lo~ly end never reeche~ ~ zero cnncentration. Reduoina ~ent~ aan be ~daled to ~ ~olid ~edi~ but ~0aln there is stlll the proble~ of the inhibitlon of unaeroblc grovth in the medl~ due to any residual reducing agent or $ts by-products a8 vell ~ the problem of provlding the sy~tem with the ebility to con~ume 4ny oxygen ~hich might l~ter flnd it~ ~ay into the sy~tem.
United St~tes Petent 4,476,2Z4, i~sued October 9, lg84, to Ho~ard I. Adler, di~close~ that sterilized membr~ne ~r~gments from cert~ln oxygen-con~uming b~cteria may be inaorpor~ted into medie to remove oxygen rapidly ~nd completely from the medi~. Thi~ petent te~che~ that us~e of these membrene fregments o~ercome~ m~ny of the problems recited ~bove.
The principle object o~ this invention is to provide materi~l Yith similar oxygen-consuming propertie~, but derived from a distinctly di~ferent, non-bacteri~l ~ource.
~ore 0peci~ically, this invention provide~ oxygen consu~ing enzyme ~ystems from mitochondri~ obta$ned ~rom a v~riety of higher non-bacteri~l org~nlsms, ~a well as e method for uslng ~uch systems to grou eneerobic becteri~. The oxygen-con~uming en~yme system~ of this invention, obtelned from mitochondrie exhibit properties whlch make them useful for inarea~ing the r~n~e ~f ~naerobic becterla th~t can be 0rown u~ing the membr~næ fregment~ obt~ined from bacteria di~clo~ed in U.S. 4,476,~24. Other ob~ects end edv~nt~e~
of the lnvention will become ~pperent ~rom the follo~ing description~ of verioua embodiments end examplea of the invention.
In ~ccordence uith thi~ invention, ~hen membr~ne fr~gments from oxygen-consumlng mltochondriA are ~290Z~l 620-1~9 incorporated into medle, elther liquid or solid, o~y~en 1~
r~pldly ~nd completely removed from the ~edi~. ~ ~ill ~e pointed out, in ~ome in~tsnces it 18 neces~ary to lncorporate in the ~edis a ~all smount of a sultable hydro~en donor ~o th~t reduotlon o~ the oxy~en to ~ter 18 ~acilitsted. The ~terlle me~brane fr~gments of this inventlon cQn be employed ~ithout fo~ming problemE. They are non-toxic and the pre~ence of these membranes haE no adver~e effect upon the 0ro~th of the an~erobic becteria in the medi~, even when used st high levels, ~uch a~ ~
ten-fold exce~. Further the presence of the membrene ragments ln the medla provldes the medl~ ~lth the ~spacity to reduce additlsnal oxygen ~hich later may enter the ~y~tem 80 thAt extreme methods of ~ealing the ~yEtem are not required.
A greet number of ~ungi, yeasts, and plent~ end ~ni~m31 h~ve mitochondria thet reduceE oxy~en to uater, if a aultable hydrogæn donor i8 present in the ~ediu~. Some o~
the ~ourceE of oxygen reducln~ mem~r~nes ~rom these mitochondrie are: beef hesrt muscle, potsto tuber~, spinech, SacchQro~Yces, NeuroE~ora, A~PerqilLus. Eualena ond Chla~Ydomonss. The process of produclng the useful me~brane ~ragmentE involveE the follo~ing ~teps:

1. Yea~t, fung~l cellEr algae snd protozo~, having mitochondria membraneE containing ~n electron trans~er ~y~tem whlch reduaeE oxygen to water can be isolated, are ~ro~n under suit~ble condition~ o~ aative aerat:Lon ~nd a temperature ~hich i~ conducive to the growth of the cell~, u~ually about 200C to 450C in Q ~roth media. Alternately, mltochondria may be obtained from cell~ of animal or plsnt origLn.
2. The cellE are collected by centri~ugation or fil~retion, and are va~hed ~Lth diEtilled vster.

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3. For the prepar~tlon of crude ~itochondrial ~embr~ne ~rRgment~ ~ concentr~ted ~uspen~ion of the oell~ i~
treeted to bre~k up the cell ~ell~ and ~ltoahondr~a.
This 1~ ~ccompllshed by kno~n mean~, for ~xample by ultresonlc treatment or by pas01n~ the s~pension several tlme~ through a French pre~sure cell æt 20~000 p8i .

4. The cellular debrl~ i~ removed by low speed centrifu~ation or by microfiltration tcros~-flow filtretion).

5. The ~upernatant or f$1trate i~ subJeated to high speed centrifugation ~175,000Xg at 5~C) or ultrafiltretion.

6. For the preperation of materiel of higher purity, the cellE Df ~tep 2 ere Ru~pended in e buffer conteining l.OM sucro~e end are treated by meen~ which ~reak up the cell ~ells or membranes but lee~e the mltochondria intect. This is accompli~hed by kno~n means, ~or example, by ~ltrasonic treat~ent, pa~ge t~rough a French pressure cell at lo~ pre~sure, enzym~tic di~estion or high Rpeed blendin~ with ~la~
beed~.

7. The cell~lar debri~ from step 6 i~ removed by dlfferentlal centrifuyatlon or filtretion.

8. The supernatant or retentate from step 7 i8 pas~ed through e French Pres~ at 20,000 p~i to break the mitochandrie into small pieces.
g. Mitochondrial debris from ~tep 7 i~ removed by centrifugation ~t l~,OOOXg ~or eppro~imately 15 minute~
or by microfiltration.
10. The æupern~t~nt or filtr~te from ~tep 9 18 ~ub~ected to high speed centrifugation ~175,000%g et 50C) or ultrefiltr~tion.
11. The pellet or ~etentate from step 5 tcrude mitochondrisl fr~ment~) or the pellet or retentete .

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620-~09 rom ~tep 10 ~purl~ied ~itochondri~l ~e~br~ne ~r~gments~ ~re re~u~pended in ~ bu~fe~ sol~tion ~t ~ pH
o~ ~bout 7.0 to ~bout 7.5. A preferred buffer ~olutlon i~ ~.02~ ~slution o~ N-2-hydroxyethylpipera21ne-N'-2-ethane ~ul~onlc acid ~ HEPES ) .
12. The mem~rsne fragments in the bu~:Eer 801ution are then passed under pressure throu~h B filter hsvi~g openin~ of a s~ze whiah ~111 retain ~ny intact microor~anisms to efect Qterilization. Openings o about 0.2 micron~ ere satiæ~actory.
13. The sterilized ~u~pension i~ then prefer~bly used promptly or stored or use st about -20C or lt may be freeze dried.

In use, a ~msll amount o~ t~e ~terlle ~embrsne ~rsgment ~u~pen~ion i8 added to a liquid ~edium which is to be u~ed ~or the ~rowth o the snaerobic bacteri~ (a~out 25 to ~000 m~ of ragment~ per llt~r o medium). The ~edium iB
permitted to stsnd for a short period o~ ti~e at ~
temperature o~ ~rom about 10C to about 60-C until the oxygen 18 consumed. Thls action take~ up to sbout 20 to 30 minutes, depending upon the concentration o the sterile membrane ~ragment~ and the tempersture. At concentrations o~ about 500 mg~l ~nd temperatures of ebout ~50C, removsl i e~ected in rom about 2-8 minutes. After the oxygen i~
removed, an inoculum o anaerobic bacteria i~ introduced into the medium. The inoculated medium is then incubated ~or the ~rovth period et the proper temperature or the bacteria which are to be ~ro~n. Pre~erably, the air 0pace aho~e the liquld medium ln itQ aontainer i~ flooded ~lith en inert 0es such as nitro0en. This minimi~e~ the amount o~
oxygen thnt mu~t be removed by the membrane system and prolon~ the li~e o the oxy~en-con~uming syste~. This al80 ~ives assurance th~t, i there 18 an accidental leak o air lnto the ~y~tem, the ~ystem ~ill con~ume that ~ir .

and inaure that the ~ro~th of the ~naRrobic bact~ria ~111 not be retarded.
In the case of ~ ~olid medium, ~uch ~ a~sr, the membrane preparation i~ preferebly ~dded to the medlum ln a molten ~tate et ~pproximately 450C at a level of about 25 to 3000 mg of ~ragment~ per litær of mediulm. The medium i8 then poured into Petri di~he~, or the llke, in whlch it iR
held at a temperature of i'rom about 10C to about 600C
until the oxy~en ic consurned, u8ually in e perlod of time less than 20 to 30 minuteR. The time of removal depend~
upon the tempersture and concentration of the ~terile membrane fragment~, a~ pointed out above. The medium i~
then inoculated ~ith the anaerobe to be gro~n and inaubeted at the proper tempereture for gro~th. Again, the Petri di~hes Rhould pre~erably be maintained in an atmo~phere o inert gas, ~uch a~ nitrogen, but good re~ult~ can be obtein~d on rapidly 0rowing ~naerobe~ ~ithout auch e precaution ~ince the membrane sy~tem 18 capable o~
con~uming rea~onable amounts Df oxygen from any air ~hich may leak into the dish.
In the event that a ~ynthetic media i~ employed, it may be neceR~ary to add e ~mall amount of a hydrogen donor ~hiah doe~ not interere ~ith the growth of the selected ~naerobia bacteria. Suitable hydrogen donors ~re sodium lactate, ~odium ~uacinute, alpha-glycerol pho~phate, alphe-keto gluterate or ~odium formate. Mo~t natural medle da not require the additiDn o~ a hydrogen donor, but ~ith some media, perticularly synthetiG media, the addition oi' the hydro~en donor l~ neaes~ary or the ~terile membr~ne ~ra~ments to perorm their oxygen removin~ function.
Some l8 species of anaerobic bacteria representin~ 8 diferent ~enera ~ill flouri~h in media which has had lt~
oxy~en removed by the ~terile membrene ay~tem of this lnventlon. ~ecau~e o the fa~t that the ~terile membrane fraament~ are in the form of particle~ ~hlcb do not ~L29[3~7~L

620-1~9 _,9 _ penetr~te the cell ~11~ the ~yatem doe~ ~ot ~dversely ~ffect the ~naerobe~ ~ein~ ~ro~n. This 1B ln contr~t to ~y~tem in ~hlch ~ chemicel reducln~ ~gent 18 used to ~cco~pll~h the re~ov~l o~ dissolved oxy~en ~hen the residunl reducing a~ent or lt8 by-product Dey pene~r~te the cell ~ell~ Thu~, becnu~e the ~neerobic bacteri0 ere bein~
~rown under more natursl condition~, they flouriRh at a greeter rate than in medie which he~ been treated ~ith ~uch reducin~ agents.

EXAMPLE I

A nutrient broth i~ ~noculeted with SaccheromYces cervisiae ~ATCC i8790). The nutrlent broth employed i nalt Extract Hroth ~old by Dico Laboratories, Detroit, Michlgan. The ino~ulated broth i melnteined at 240C end i~ ~ctively eerated. The groYth 1B continued until it i~
ln the late logartthm~c phane.
The broth conteining the SaccharomYce~ cervisiag cells i~ centrifuged at 4,000Xg to harvest the yeast cell~ The cells are ~eshed wlth diRtilled ~eter end centriuged.
This ~shing end centrifuging are repeated.
The hervested cell~ ~re cooled to n-2 C ~nd suspended in two volumes of a bufi'er containing l.OM
~ucro~e, O. 02n tris-~hydroxymethyl)amino methsne ~Tri~) and 0.0001 sodium ethylene diamine tetre anetete ~ EDTA ) at pH
7.4. To thi~ ~u~pension i8 edded an equal volume of gles~
beads ~averege diemeter 0.15mm) and the mixture is homo~enized et 15,000 RPM in a steel blender for ~-4 minutes. The ~less beeds are allowed to æettle and the ~upernatent is decanted.
The supernetant i~ centrifu~ed at 2400Xg ~or ~0 minutes to remove unbroken cells ~nd debris. The ~itochondria are ~arve~ted from the supernatent by centri~ugin~ at 20,000X~
for 15 minutes.

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62~-lOg The ~itochondri~ ~re then resu~pended ln 0.02M HEPES
buf~er ~t pH 7.5 ~CalBiochem-Behring ~orporatlon, L~Jolla~
C~liornl~) 3nd p~s~ed three time~ through e French pressure cell ~t 20,000 p8i.
The ~uspenslon o~ the membrane frayments in the buffer solutian i~ then pes~ed throuyh R 0. 22 micron fllter under pres~ure to produce the sterile membrane ~ragment~ to be used to pr~duce anaerobio~is in the media to be used for gro~inH anaerobe~ The su~pen~ion of membrane fragments is etored at a~out -200C. The dry ~eight o~ the 601ids in the ~u~penelon i~ about 30 ma/ml which i8 determined by desiccating æamples over phosphorou~ pentoxide in a vacuum.

EXAMPLE II

Ten m$croliter~ of the ~uspension o sterilized mem~rane fragment~ from Ex~mple I ie added per millil~ter of o~ygen s~turated Difco Nutrient Broth at 370C. In ~ive minutes all of the oxygen i~ removed. A portion of the nutrlent broth, tre~ted ~ith the sterilized membrene fragment~ end held until ~naero~ic ~ondition~ are obtained, i8 inoculated ~ith Clostridium di~ficile und lncubated at ~70C for 15 houre in ~ se~led container. A luxurisnt t growth ~Approximetely ~ cell~ per ml~ i8 ob~erved.
., 5 EYAMPLE III

Ten microliters of the ~terile membrane i'ragment preparetlon of Example I i~ edded per milliliter of synthetia medium ~hich is ~upplemented by the addition o~
~odium laatete to ~ final conaentration of 0.25~. The ~ynthetic medium con~i~t~ yenerally o~ inorgenic salt~, ethnnol and ~odium acetete. It i8 prepered from reagent grade chemicale. A semple o~ ~edium containing the aembraDe fr-gmente i~ neld at 37-C t~ remove oxygen ~nd ' .
: ' 1~90271 620-~09 is inoculated wlth Clostridium kluvveri. The inoc~l~tQd medlu~ i~ incubated ~t 34~C ~or 96 hours. At ~he end o~
that tlme a luxurlunt ~ro~th S ~pproximately ~ cells per mi) i8 o~served.

EXAMPLE IV

The sterile membrane fr~gment ~u~pension of Ex~mple I
18 addæd to molten agar at 45~C ~t a level o~ 10 microliter~ of ~uspension per mllliliter of agar. The agar i8 poured into a Petri di~h end held to obtnln an~erobic conditions. It ifi inoculeted ~ith C~lostridium kluYverl end incubated in the pre~ence of en lnert gas ~tmo~phere at 8 temperature o~ 340C ~or 9 period o~ 96 hour~. Colonie ~lth a diameter of approxim~tely 2 millimeter~ are observed et that ti~e.

Slmilar results are ob~erved vith sterilized membrane ~ragment~ from beef heart muscle, potato tubers, ~pinach, Sacaharomyce~. Neurospora, Asperqilius, EYg~en~ and Chl~mydomonas. In all ca~es the concentration of membr~ne fragmentfi in the sterile membr~ne suspenElon i about 2S -30 mg~ml. At 370C ell of the o~ygen i8 removed from the nutrlent media ln from about ~ to about a ~inute~ vhen the ~embrane fragment ~u~pension i~ pre~ent at from abaut 10 to about 100 milliliters per llter of broth ~250-3000 mg of fra~ments per liter of broth). At higher level~, oxygen I`emOV~l i8 more rapid th~n at lower levels.
As pointed out above, when a ~ynthetic ~edia or one not aontainlng a hydragen donor i8 employed, a hydrogen donor ~hich is competible ~lth the bacteria being cultured ~hould be employed to ~upplement the medium. The hydroger, donor, e.g. ~odlum lnctate, sodium suGcinate, alpha-glycerol pho~phate, or ~odium formate should be employed at ~ level o~ the order of 0.15 to 0.25M.

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~L290271 Qnaeroblc bacteria shich can be ~ucce~fully ~ro~n in nutrient medl~ treated hy the ~terlle membrane 8y8tem described herein aræ:

Clostridium difficile Clnstridium tetani Clostridium kluYveri Clostridium ~poroqenes Clo~tridium perfrlnaens S19U~IGJ¦L~ sordelli Clostridium butYricum Clostridlum biferment~n~
Clo~tridium acetobutyricum Peptofitreptococcu anaerobius Peptostreptococcu~ micro~
Pepto~treptococcus ~aqnus De~ul~ov~hrio ~ulnari~
Fu~o~acterlum nucleatu~
yiellonelia Parvula Pro~ionibacterium acne~
Eubacterium limosum Bacteriode~ fraqili~

The u~e of sterile membrane-containin~ media may be used in clinicæl labor~tories to stimulate growth of anaerobic bacteria from human patients. Sterile membr~ne cont~ining media may be used to increa~e the survival of enaerobes in medium used to transport sample~ ~rom the patient to the laboratory ~nd al~o for the determinatlon of entibiotia ~en~itivity pattern~ of anaerobic bacteria. It al~o hss use in producin~ the eneerobic conditions required in many indu~trial fermentation proce~se~. The use of Gterile me~brene fre~ments, as described above, produce little or no toxic side ef~ects ~hen usæd in amounts much greater than those re~uired to achieve oxygen-free - '. :':

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Gondltions. Small qu~ntitie~ of thæ membrene fr~gmeDts reduae a medium that i~ initially ~eturated ytth o~y~en to an en~erobic cnndition and ~aintaln that condition even t~ou0h ~mall amounts o~ eir are introduoed.
V~rious ~e~tures of the invention are ~et forth ln the appended claim~.

Claims (10)

1. A nutrient medium for growing anaerobic bacteria which includes a hydrogen donor and sterile membrane fragments derived from mitochondria having membranes containing an electron transfer system which reduces oxygen to water.

2. The nutrient medium of claim 1 in which the sterile membrane fragments are present in a concentration of from about 25 to 3000 mg/1.

3. A nutrient medium for growing anaerobic bacteria which includes a hydrogen donor and sterile mitochondrial membrane fragments containing an electron transfer system that converts oxygen to water, derived from the group consisting of beef heart muscle, potato tubers, spinach, Saccharomyces, Neurospora, Aspergillus, Euglena and Chlamydomonas.

4. The nutrient medium of claim 3 wherein the sterile membrane fragments are derived from the group consisting of beef heart muscle, potato tubers, spinach, Saccharomyces cervisiae, and Aspergillus, oryzae.

5. The nutrient medium of claim 1 wherein the hydrogen donor is selected from the group consisting of sodium lactate, sodium succinate, alpha-ketoglutarate, sodium formate and alpha-glycerol phosphate.

6. The method of removing dissolved oxygen from a nutrient medium for anaerobic bacteria comprising the steps of introducing sterile membrane fragments derived from mitochondrial membranes which contain an electron transfer system which reduces oxygen to water in the presence of a hydrogen donor to the nutrient medium and maintaining the medium containing sterile membrane fragments at a temperature of from about 10°C to about 60°C until the dissolved oxygen is converted to water.

7. The method of claim 6 further comprising the step of adding a hydrogen donor to the nutrient medium.

8. The method of claim 7 wherein the hydrogen donor is selected from the group consisting of sodium lactate, sodium succinate, alpha-ketoglutarate, sodium formate, and alpha-glycerol phosphate.

9. A method of removing dissolved oxygen from a nutrient medium for anaerobic bacteria comprising the steps of introducing sterile mitochondrial membrane fragments, containing an electron transfer system that converts oxygen to water, derived from the group consisting of beef heart muscle, potato tubers, spinach, Saccharomyces, Neurospora, Aspergillus, Euglena and Chlamydomonas, to the medium and maintaining the medium containing sterile membrane fragments at a temperature of from about 10°C to 60°C until the dissolved oxygen is converted to water.

10. The method of claim 9 wherein the sterile membrane fragments are derived from the group consisting of beef heart muscle, potato tubers, spinach, Saccharomyces cervisiae, and Aspergillus oryzae.