Intestina] hacterial fiora in normal adulte in the U.K.

S. PF.ACH

Interest in anaerobes as norma! flora began early in the present century fl-3] and during the last 15 years it has been established that non-sporing anaerobes are prt'sf'nt as norma! flora at various sites in the body [4, 5J andare hy far the most numerous of the indigenous flora of man. The human mouth and intestina! tract are of particular interest to bacteriologists stu· clying anaerobic hactt•ria hecause they provide very rich sources of isolates.

The mrmth

Because of easy access to atmospheric oxygen the mouth is not generally eonsidered to be an anaerobic environment; however. deep in the gingiYal nevices facoltative hacteria lower the oxygen potential and produce local anaf'xobic conditions.

The indigenous flora of the oral cavity is compie x and anaerohic bacteria are numerous (Tahle 1). Many origina] studies concentrated on the micro· hial flora of saliva, since samples were easily ohtainahle. Streptococcus sali­l'arius was found to be the predominant organism (Table 2). For severa! ?''f'ars now, however, it has heen recognised that certain haderial types preferentially colonize clifferent sites in the mouth and the sampling nf thest' ~ites has hrought to ligbt microhial flora which is present. T n addition to 8 .. ~alivarius, the soft tissues of the mouth such as the surface of the gums, tnngue and cheeks are preferentially colonized by S. sanguis, while S. mutans colonill.:ed the hard surfaces of the teeth. Possible differences in inherent ahilities to attach to different sources may account for these ohservations [6, 7J. Bifidohacteria, fusobacteria and spirocbaetes may be isolated from salivary samples [8 9,J and hoth Bacteroides melaninogenicus and B. fJralis bave heen isolated from gingival cavities. lt should be noted that strains of B. malaninogenirus isolated from the mouth bave heen shown to he bio-

'" I'Ff:ZIU"I 111 HA'I'TERI _4,A.I·.HOI!I 1\0'\ -,f'UHH:··I\1

clwmicall~- Jiffen•nt fwm strains isolated from other an·a~< nfthe g-astroint1·­

stinal tract [lOJ. l>t·ntal plaqut• contaim a relativdy high prOf>Ortion ofCraul

positin· rod~, e~pecially Actinom_yce.~ SfWt:ie,.. and thi>< is often uccompaui,·d h,- high leni~ of nillonellae (11 J and anat•robir Graru nq~atin.:' rod~.

Tooth &urfarr•

Ginf(ival scrapin,.:•

Oral flora

l"fyp;·-•1 ••••. ,,.,., ''"""" , ... , "'' 1--------

Au.,.,,,,. ..

1 O' -l o·

lU' IO"

IO' IO' ' i '---·--·-- --------'

Oral flora

1-,_ l '

S. sangui'

S. mulans

Bifidohaeto·ria, Fu•obactrria Spirochaetr~

BorJeroides melanirW,I(enicus

Hurteroidn oralis

Veilloiwllar

Actiuomy"c~

Bacteroidc;

S T F

Saliva. soft tissu<''

Soft ti~sut·,.

Saliva

~ Gingival ~a,·itir> ~

l

l t """"' "'"""" ------'------··--·

-

I'K<I.GH Il

The ,tomach pmvit!es an impnrtant barril'r tu ruicrohial 0\l:'fgro\\th

11 f the small bml-'elp2]. ,\_rid stomach t'ontents are normally "tt>rile [9] as

tht~ pH nf thc stmnadl eontPnt falls the number of bactPria per millilitrt~

01 f contcnts falls eurre~pmuliugly. Tlw haett·ricidal a<'tion is thnught tn lw

due to fret• llplnwhloric acid. Ora:<ar and Cnll. [9] havc shown that immeflia· tl'!y after a mt>al a count of I0·'-Hl7 bacteria:'ml of gastric juice can be recor·

dt•tl allfl indudes Stn•proet)Cei, F.ntrrnhacteria, Bactrruide~ allfl Bifidobaete­ria, tlt·riHJ from the rnnuth antl from the meal. As the pH falls the hacterial

t'ount declim•s and n•lativdy few hacteria art: f(mnd when the stomttch

conh•nts ha\ c fallcn helow ahout p H 3. Data nbtained using intubation tcchniqut·~ l9. 13-15] havc shown that

tht· upper rt•gions of the small bowel (JuodemJm, jejunum and upper

ileum) bave a ~parse microhial flora eonsisting predominantly nf Gram-po­~iti\'e facultativt• organisms. fn the luwt>r small intestine ana(•rnbes, inclu­.ling Bactt•roidPs, occur and there is a rich(~r more permanent flora. The

luwt>r ih•um, huwP.ver, stili maintains a rdatively low total conct>ntration ,f urganisms (HP-10a'ml) and rl'prt•sents a transitional zone bctween thc

~pttr~t~ flora of tht~ uppt·r gastrnintrstinal tract aud tlll' rit~h fiura of tht~ largo~ lmw••l [16),

Ba(·terial f/om of the /arp,e hrnt'l'l

Distai to the ilt•ocaecal valve a striking l'hangt• in lhc numilcr aliti

typt•s of huc.teria r.an lm Hl'l"ll [14, 15J. Thl're is au incrPase in anaerohic pupulations, Bacteroides and Bifidobacteria hecome the tlominat mil'ro­

organisms. outnumhering the at>rohic ami facultativl" fiora hy 1,000 to 10,000: l. The intraahdominal colon is ohviously tliffìcult to .~ampie, llllt

ÌntHhation .~tut!ies [14. lSJ together uith stndies of the eontents of healthy

.tppendicrs ohtainml at op(•ration [17] su~g~">lt that within hroad limits. thP faet~al fiora is rqtresl'nlative nf thl' ltadt•rial fiora of the largt· howel.

'l'lw rulalt fal'ml jltlfa

"dH'llll' l is a dit~gnunmatic rt·pn•st•utalion uf tht• fat~cal Hora of Euro·

]""an mlults. Thl' numt•rit·al tlominancc uf tht• nou-~poring: anat•robf'~ in

l'aP•·•·~ is w•·ll t•sublishl't! j2, 18, 19J . Hat:tt•ria rtnmnonly rt•g:anh·d as t~weal

l~olatt·~. "-!~· tlw Entt•rohaetl'ria, thf" Entt•roetH'('i and the Clostritlia. in faet

··••nsti tut t· <mly a .~n1all propurtion of t h t' total haett•rial flora. V aritlll~ aut httrs ••~ing- tlifft'ring: haet;•riologi•~al to·ehnif]ttes gt•nt·rally agrt't' that lll 111 - 10 11

1 ia!.lo• l.at·tt•ria ('\l li lu~ rt'C'O\'t'ft'tl from l g (\q•t-"eight) of fan'f'S. In thf'

.K. onlv a/mut !OH nf tht•st• art· facultatiYe baetl'ria and ~poring a!Hif'rolw~.

---------

IO l"iFF..ZIU~I ilA IIATTERI ANAEROBI NU~ ~POI<.Ua:"ll

A study iu 19ìl by H ili and Coli. [20] a t St. .Mary's H ospitai w Londuu showed that tht- facce;; of pen;om: living in tht' {T.l\.. and America f!Cneral!~­

contained higher numhers of Bacteroides and ff.v.-er Enterococci than di•l

the facce~ of groups of peopl•~ living on high carhobydrate diet~ in Uganda. India and Japan, Thus, thl" ratio ofthe numher uf an<wrobf'~ to tlw numlwr

of aNoht's difl'ered in thcsc two populatiom. In addition Sareinat· (Gra!ll

positiH sporing cocci) were isolah·d only from 1!-"roups of JWOple e-unsumìn~·

diet8 rich in vcp;l'tahlf' matkr. In Britaìn, such peoplt• \\'t•n• Btri('t '''!!t'· tarians [21].

In a continuation nf thf' study at St. Mary's Hospital. t~ompurÌnf!: tllt' faf'cal flora of pt>rsons iu tlw P.K. ,\-ith y1ersonf; in otlH'r l'ountrie;;, 196 strains of non-sporing anat•roht>~ ""-Ne isolatf'd front the faece:, of 34 pt•opl•· living on a mixed 'Western dif't in the U.:k.. ~in America. Tbesl' isolati:'.~

were compared with 215 straim; isolated from the fa1•ce;;; of 45 people eomm·

ming carhohydrate rich dit>t~ in Uganda. India and Japan [22J. Ali tht· isolates could be assigned to genera. The predominant groups of non---8ptl·

ring strict anaerobes in faPces were found to be similar forali thc populatiou" studied: thf'y fell into three main genera: Bacteroides, Bifidohacteria and

Euhacterium. Minor component~ of the anaerohic flora consisted of Fu~o­

bacteria and Propionihacteria. Although similar gronps of organism;; wcr1·

isolated from aH the faccal samples, with Bacteroides the most common isolates, ther{' were difl'erences in the distrihution and relative proportirm,.

of these organism8. Faeccs from Britain and America appeared to bave a

Main flt'nera of bacteria in facce,.

FAECAL FLORA

t -1

90 ~" Non sporing strict anaerobes

______ l_ ___ ~

y

90 "o Bacteroides

Bifidobacteria Eubacteria

l

l j.

10 "., Fusobacteria

Propionibacteria Lactobacilli

Anaerobic cocci

10 ", Enterobacteria

Enterococci Spore forming bacili•

Clostridia.

I'F.ACH 13

larger proportion of Gram negati veorganisms (Bacteroides spp. and Fuso­hacteria) than did those from people from India, Uganda and Japan. The consequently largf'r proportion of Gram positive organisms found in faeces from these countries seemed to be due mainly to the contribution of the Eubacteria (Table 3). These differences were shown to exist when the popu­lations were taken as a whole. On close examination of individuals within a population four types of faecal Rora appeared to exist, i.e. persons with (l) mainly Bacteroides, (2) mainly Bifidobacteria, (3) mainly Eubacteria, (4) mixed Rora. As a whole, the populations reftected the most common type of individuai Rora. Almost ali the Bacteroides isolates could be shown to bt"long to the B. fragilis group of organisms (Table 4). Two subspe· eies of B. fragilis are common in faeces - vulgates and thetaiotamicron. These subspecies of B. frasilis, amongst others, bave now been a:fforded full

TABLE 3

Percentage frequency of genera of non~ sporillfJ anaerobes among ieolates from faeees ol populations consuming differenl dieta

l Por<·rnU~o f .... qu•n~y, an>un~ i•ulat<• f<OID th~ _.,&tPd Il :>~,.mher -~- pupulMtion•. "f ~<nu• of i.olat•• ! ' l 1 l i·l~nti6•d BMct•rni- i Fu• .. Lao·l HHi~n- "uhac- l l'rnpioni· Loetn- l

<l<> l terium 1t.act<rium < _,_,;.,.., l boctorium bo•·iUu~-~

l'Ol'CL-I.TWN ,\l'H_> O!lèT

------c---T j

\lix<'d Western (U.S.A., England, Scotland)

lligb Carbohydrate (l'g;anda, India, Japan)

22-t. 61.6

37.7

0.9 25.0

27 .o

6. 7 3.1

23.2 5.1

TABLE. ~

Major >~pecies of non- !òporing anaerohes found in faeces -----

(~ram-ue!l.'ative rod~:

Uacwroid~s themiolamirron

Htlt'teroides rulg<lliU

(;rum-positive rod•;

Hijidabacl~rillm fldalescenli~

H•"jidubttrWillm bijÌ<lum

Hifidahar/erirlm rriksoni

Bifidabaclerillm flprofo.rims

' ' «fragili~ ~roup»

-----·

o

LI

Il

SJWCÌI"'~ ~tatus ancl an• slwwn a~ ~uch iu T<1bk 1 auwng thc BifidoharlPria.

B. adtJk.~cenlis was tlw comnHmt•$t Ì1-olate; B. b~fìdum and B. eriksfllli w..r•·

also common. 1\fo><t of the !:'uhactt>ria appeared tn lll' E. aerf!facie11.~-

Jn this papcr discu;,~ion has lwc•n confiut>d to tbc hactl'.riul flora of tlw

lumt>n nf tho: ~astwinte;~tinal trarl. However. iu addition tht·n· appcoar.~

to exi~t a mierohial flora which i~ r.lo.sl'ly associatl"ll with th•· int.c.~tinal

mucosa, prohahly colonizing th•· layer of mucu~ which oycrlie~ the celi~

[23. 24]. lu a study at St. Bartholomew's Hospital in London [25J a mirrH­bial flora was found to lH' assoòated with tissue t'rum variou~ part.s of tlll'

human gastrointt>stiual tract. Large howel tissu•• yidded grcaler numhN~ of hacteria (IO';g of tis~tw) thau did tissue from th~ small howel (10~ 1 g of tissuc). From ali sitP~. about half of tllf' isolatcs were aerohic or facultati,-,.

bacteria and half wcn- strictly anaerohie. Mucosa] flora. therefort>, unliko·

the fcacal flora, doo•s not appcar to lw duminated hy anaerohic hactcriu. Within hroad limit~ thr hacterial flnra of the intestine appcars to }w stahlo·

an d self regulating. Sc•veral factor" may he involveò in its regulation ano!

tht'sc are slwwn in Tahlc• 5. Thc inflm·nct> of òict un tlu- faccal flora of various J'opulatiom ha~

already hcc~n mentionetl. Difl"erent dictary groups may han•. difl"t'rl'!nt pro­

portions of various bactcria in their faec~s. In addition it has hecn knov. n for

many years [26], that infants fed wholly on hreast milk haw a particularly characteristk faecal flora dominatcd by large numhers of Bifidohactt>ria.

In 1974, Willis and C:oll.l2iJ showt>d that thc composition of hrl'ast milk is important in d••termining thi.~ flura ami Bullen. Hogers and Leigh [28J showed that certain factors in human hreasl milk may prot~d tht' infant

against coloni.zation hy enteropathogenic strains of E. coli. As pre\ iomly discussed. f!:HStric acid provido•s an important barricr

to bacterial ovcrgrowth in th•· small bowd. This action may he morlifìt'tl t o !';ome extent hy tlw ncutralizing effeet of food wht>n tht' stumach is full.

As in any mi,.;ed flora, hat:terial interactions play a largt• part in df'termining the flora of tht• gatrointestinal tract. Thest' interactions may Jw either

~ynergistie (e.!-(. tl1t' produrtion of anaerohic eondition~; through the m1•ta-

Factor~> inftuencing the intestinal Rora

I

ii ))if't

Hactrrial iutt>raf"t;ou'

Cll•trointf"•till<ll muhility

l ____ _ L~~ozym•·

Ga8tric af"id

Bi l,.

!;pecifir \llltibod~

PEACH 15

holism of oxygen by aerobic bacteria) or competitive (e.g. the production of colicines by coliform bacteria). Direct compet1twn for nutrient sources may also play a role in determining the dora of the colon and faeces.

Tolerance to bile is used by bacteriologists in culture media to select for intestina} organisms and will ohviously exert a selective effect in vivo, particularly in the small bowel. Similarly, the gastrointestinal mobility associated with the small bowel will limit bacterial populations in this area.

Finally, lysozyme and ·specific antibody - particularly lgA, may play a role in confining gut bacteria to the gastrointestinal tract.

In conclusion, the gastrointestinal tract support a rich and varied bacte­.rial dora, particularly as far as the non-sporing anaerobic hacteria are con­cerned. Under normal circumstances the host lives happily in association with these organisms, however if this dora is disturhed or displaced, serious consequences result and often become the problema of the clinical hacte­riologist.

Summary (lntestinal bacterial flora in normal adults in U.K.). --- The A. reports on the composition of the bacterial flora heing present ali through the intestine of norma! adults in the United Kingdom, listing the numher and families of the aerobes and anaerohes occupying the various portions of the alimentary tract.

Emphasis is then laid on a comparison hetween the norma!« British» flora in the colon and rectum, where the ratio of anaerohes to aerobes is 100 to l, and the fiora found in norma) adults in Africa, India and Japan, stressing the major differences in the numbers of clostridia and non-sporing anaerobes.

The A. finally highlights the importance of the factors governing the intestina! bacterial ecology, as well as the serious consequences likely to arise from the alteration of such flora.

RiaMunto, (Flora batttJrica intestinale in adulti sani in U. K.). - L'A. riferisce la composizione della fiora batterica presente lungo tutto l'intestino rli adulti normali nel Regno V nito, elencando il numero e le famiglie dei germi anaerobi ed aerobi che occupano i vari tratti dell'apparato digerente.

Successivamente si sofferma a confrontare la flora normale presente nel rolon e nel retto, dove gli anaerobi sono in una proporzione di 100 a l ri-8petto agli aerobi, con quella presente in adulti normali in Africa, India e Giappone, mettendo in risalto la differenza che si rileva tra numero di cio­stridi e di anaerobi asporigeni.

Conclude infine sottolineando l'importanza dei fattori che rt"golano la i~cologia batterica intestinale e le serie conseguenze che possono derivare da una modificazione ;if•lla Rora stessa.

"' lNFt:ZlUNI HA BATTERI A"'AEKOBI NO.> SPURH;J:NI

l. l.EWJt.OWICZ, X. l'iO!. R<"cht·rclit'h ~urla flon• microhit•nut' de la buut'h<" dc,. nourris,on.-.

Arrh. Med, Exp. Anut. Parhol. 13: 6:1:!.

'' SA:>;HIIJI"'E. c\. G. 1\1.11. The fal."cal flora of adult~ with particular attcntion to indi~·j. dual differenct·~ and their relationhhip to thl' cffects of ~-arÌOII' di<'h. l. Individuai diflt'rencf's un normlll dit•t. ./. Infert, Di.•. 48: 541-.~I><J.

3. 1'liUHAS. C. G. A. & HARt:, H. 1954. Th,. clas,.ification of anaer<lhic cut•d and their iso!>~·

tion iu norma! human hein!l'» and J>athological proc<'~Sf'> . ./. Clin. Pathol. 7: 30(1.

4. Rol>EBI'fiY, T. 1962. ~firroor#tmisms Indip;nwus to .1\-[un. :\e\\ Yurk, l_i.S.A .

. ). SMITH, L. D. & lifli,UEMA,, L\', 1968. The Pat/wf;f'nir AnaProbic Hucteri'l. Blacksl.erf!:., \"irj!:inìa. ll.S . ..\.

6. \-A~ HotTTE. J .. GioBoNs, H. J. & BAlliGHOST. S. B. 1970. Adherence a~ a determìnanl of th1· preseuce nf Slreplocorru~ .sali1•arius ami SITeploe<.>CTu.< sun,rzuis on tlw tooth sur· facf', Archs. Ora/ Bio/. 15~ 102~>.

GiBBONS, R. ,J. & VAN Houn; .. T. 1971. Selective bacterial adherence to ora! tl!lithelinl

surfa<"<' and it~ rol.- a~ an t"cologi~al determinant. lr(ff'l. }mmun. 3: 56j-57l.

8. RICHAHDSOK. U. L. & JOKES, M. 1958. A bacterioloJ;:ical censo. of human saliu. ,/. Denr. R~s. 37: 697-700.

!J. DHASAH, 8, 5 .. 5HINER, H. & McLEOD, J:<: . .M, 1969. Studie,; Oli thf' iotestina] fior<~

of thc jla~troim.-~tinal tract in h<"althy and achlorhydric rwrsnn~. Gastroenterolog,. 56: 71·-79.

10, MACFARLANI;, T, \'L 1974. Dtnlibtry :md Bacteroidl'"~. Symposium an Bu.:-reroidrs In· ferlions. The W est of Scotlaud Clinica! Microhiolojl~ Group, Glasgow.

II. HAHDIE, J. M. & Bown:~;r;-, G. H. 1973. The uormal microbial fiora of the mouth. In: Tlw Tliarma/ Mirrobial Flora of Man, F. A. Skinnl:'r aud J. G. Carr (Eds,) Acad<mlir· Pre&b, London.

12. GHA'. J. D. A. & SlJI:"iEH. M. 1967. lnfiuenPc of gastric pH on gastric and jejunal fiora. Gut. 8: 574-581.

13. Sm~EH. M .. WA1"ERS. T. E. & GoAY, J. D. A. 1963. Cnlture .tudie~ of thE- gastroint•·· stinal lract with a nrwly devi8ed capsule. Ga&ITaenlerology. fS: 625-632.

14. GoHBACII. S. L., :\AHAS. L LEHNER, P. L & WFIST.!>I!'-0, L. 1967. Studies of Ìlll!:'stinal mìcrofiora. I. Effrcts of diet, age and p<"riodìc samplìnp; on numh.-rs offllecal microor·

ganisms in nian. Gmtroenterotogy. 53: 845-355.

l.). GoHBACH, S. L, Pt,ANT, A. G., 1\"AUA, L.&: \\'EINSTEI::>ò. L, 1967. Studi<"s of intestìual

microfiora. IL Microorganisms of thf' small ìnte~tim· :md thdr relatiomhip to ora] and farenl fiora. Ga.stromrerolo/{Y· 53; 856-867,

16. GoHBACH, S. I.. 1971. Int<"stinal mkrofiora, Prof!"r"~" in ga~troenterolog). Gastro."'·

terolagy. 60: lll0-1122.

17. SEEJ.IGEH, H.&. \\.-ER:--oER, H. 1963. Il.!:'l'"her<"hPs qualitati\"('& et qnantitatin·s sur la

fiore intestìnaiP d!:' l'homme. -~nn. lmt, Past~ur. 105: 9ll-6936.

1!1. EGGEHTII, A. H.&. GAGr>;O"i, G, H. 1933. The ba.-teroid.-~ of hnmau fat-cef<. ]. Bal."terio/.

25: 389-413.

l 17

l'l. f.ElllS. K. H. & RF.TTI;F.R, L. F. l'IlO. :'\on ~poring anaetobic haeteria nf thl' inte­

,tiual trac·t. (. Oceurr<.>nce and ta.-.;onomic r~lation~hip- . .f. Rocl~riol, .W: 287-:107.

~H. HU.L. M.]., CRnWTIIF.H, .L 5., DR.II.SAR. 8. S., HAWKSW(IRTII. GAIIRIEI.LE •. \.RIES.YIH•

Pi'ljE & W!LI.IAMS, H.. E. O. 1971. Bal't<·ria and t hl' at"tiology uf canc"r of the largc

bowel. Lunu!, i: 9~-liJO.

::!1. CaowTIIER, .T. S. 1971. Suuinll<'!'lllrir•u/i in human faeces . ./.M ed. Jlirrobiol. 4: :IB-3~0.

~:?. f'EAUI, S .. FERNANDF.Z, F. JoH."o"~!IN. K. & DRASAR, B. 5.197-l. The nOII-A(IOtÌn" anat•ro­hit• bact~ria in htunan faece~. ], Jhd. Microbiol, 7: 213 22!.

:::1. "\EJ.~ON, D. P. & ~lATA. l.. J. 1970. Bartl'tial flora ussoriatf'd with t h!." hnman ~~:a•troin·

tt·~tinal muco>"l- (;R8ltoentuolo/(.f· 58: .~6-lll.

:!l. PLAUT, A. E.. GoRB.-!..CH, S. L., 'iAIIA~. L., Wu;>;~TEI~, L., 5PA~K~EBEL, G. & LEVITA"·

1{. (907. The microbialllora of hmnan ~mali intf'stinal muco~a and llnid~. Ga.,rtlPrUero­

l"I'Y· 53: 868-!173.

:!~. PKACH. 5., LUCK, R., KATZ, Jl •• Tnuo, f. & T.-I.BAQCHUI, S. 1978 .. \luco•al a~~ociatt>d

hactt>rial flora of the inte~tine in J>~lit•nts with Crohn's <\i,._·a~" and in ~ rontroll!,"roup.

1;ut. 19: 10:'14-IOU.

:!7. W11.1.1~ •. \.T., lku,f:N, J. J. & WII.LIAMS. K. !9H. Brra~t milk •uhstitut<•. a bat·t<>rio­

lo~o~icul ~tudy. Hril. _U~d . ./. 3: 67.

:!!\. BUJ.LEN • .1 . .1 .. ROGERS, H . .T. & LF.U:H. H. 1973. lron-bindin~ i>rotPin~ in milk ~n<l

rt>~istance to Escherichiu ~oli infet·tion in infants. Rr. Jfed . .T. 1: 69-7:!.

:!9. HOEPRICH, P. U. 1970. Etiolo~ical dia)!:nn~is nf lowt•r r<>spir~tory tract inf<><·tion. Cn/ij.

Jfed. 112: l.

l

Flora batterica intestinale in adulti normali

P. GIANFUILLI MASTRA "TONIO (11), I. LUZZI (11), M. OCCHIO NERO (t•), F. BELSITO (11). G. PANI CHI (b), E. MORERA (b),

F. ERCOLANI (b), L. PIAZZAI (b), A. L. PANTOSTI (b),

(11) Istituto .'iuperiore di S11nità, Roma (b) III Clinica .Hedica, lfnivl!nità di Roma

Negli ultimi anni l'interesse per la flora batterica intestinale è notevol­mente aumentato perché l'uso di nuove metodiche ha permesso di coltivare con più facilità i germi anaerobi così da dimostrare che questi rappresen­tano la maggior parte dei batteri presenti nell'intestino, e da permettere studi più completi sulle possibili variazioni quantitative e qualitative della flora intestinale per valutare il ruolo svolto da certi batteri nel determinismo Hia di varie forme patologiche quali ad es. il cancro del colon, sia di varia­:lioni metaboliche importanti nella economica del corpo umano [1-3].

Con il nostro studio ci siamo posti come obiettivi la rilevazione della llora predominan~ aerobica ed anaerobica nelle feci di soggetti sani, la valu­tazione del rapporto quantitativo tra germi aerobi, anaerobi non formanti spore e clostridi e l'identificazione del genere degli aerobi e degli anaerobi eosì da poter individuare nell'ambito di queste due componenti batteriche quelle maggiormente rappresentate. I risultati ottenuti con questo lavoro rappresentano dunque i dati di una Rora batterica predominante del bas!!!n intestino Ji soggetti sani non sottoposti a diete particolari o a trattamenti con chemioantibiotici o antiacidi e possono fornire una indicazione quali­tativa e quantitativa dei microorganismi che albergano nt'l colon in condi­zioni normali.

A questo fine sono ~;tati ~saminati 12 individui sani di età tra i 20 ~

i 60 anni, che presentavano regolari funzinni dell'alvo intestinale. Per ogni soggetto sono stati prelevati tre campioni di feci in giorni successivi. Ogni !:ampione veniva in parte immediatamente insemenzato in un container contenente terreno di trasporto precedentemente tarato, in modo da .ricavare dopo l'insemenzamento il peso reale dellt~ feci t>d in parte posto in un con­tainer per !>ottoporlo ad un esame microscopico diretto. Per il nostro lavoru \'t'nivano accettati solo quei campioni che rilmltavann negativi al precedentP

'" L"iFEZW"il IlA BATTF.R! A'\AHU>BI ;\O;\ :-I'ORJI,}-'\1

t~samt". 11 tf'rrf'nn di trasportn contetlt'ntl" !t• fe<'i in t:sam1· ...-t·nint introdotto

nella cappa in atmosfera di co2. H~ l' )\2· omop;t>ncizzalo l' diluito in JHU­

vette contenenti terreno di Ringer preridotto a diluizioni scalari -0.1 mi

dellt> diluizioni 10· \ 10· -~>. w-- 7 veniva pia~trato su tern·no Rf'inforcr-d Clostri­

dial Agar (RCA) preridotto. (juestf' piastn· veni' auo post1• in Gas Pack. f'stratte dalla cappa ed incuhah• a 37 °C 3 P:!!· O. l mi ddle diluizimli lO \ lO \ lo-:; Yeniva utilizzato }'t'T fH>minare piastn· di agar sangut· m·­

f'f'Rsari" per la rieerca di gf'rmi aerohi. Lt· provetlt· c1m l" diluizioni 1 O '­]O·-~ vr-nivano invece sottopposh1 a shock tt'rmico a 80 °C 20 min r poi ~~·mi­

nate su piastre dì RCA per la ricerca dei germi ~porigeni. incuhate dentru contenitori Gas Pack a 37" 3 gg.

lO colonie ~viluppat1•si in condi:r.ioni dì aerobiosi n:nivano prclcvalt' seguendo una linea casuale segnata precetltmtemt>nlt• sul retro della capsula Petri, scegliendo le piastre con sviluppo tra 20 e 200 coloni1· e S1•minatt• su containers di Trypticase Say Agar (TSA) la cui crescita vf'niva utilizzata P"r le prove hiochimichc nect'><sarif' alla idt:>ntificazionl:' dt•lla spt'cif' hattf'rica. l.t· J'iastre all'H CA su cui erano state seminate if' dilui;-.ioui trattate con lo shock tf'rmico di 80 °C >: 20 min per la ricerca di'i clostridi vf'nivano lf'tte t• eontat,. dopo 72 h. così come le Jliastrf' di HtA chiuse i11 Ga~ Pack a 37 °C per la crt·­ilCÌta dei germi anaerobi nou sporigeni, dalle quali si prdevavann casualml'nt•· st'condo una linea tracciata nel diamPtro della pia~>tra, come pt'r gli aerobi. lO colonie asportando ancht' una parte dell'agar sangue sottostante. si st:minayano in conlainer$ di Robertson Cnoked Meat (HCM) incuhandoli a 37 °C 2 gg. Dopo 48 h i cunlainf'r,ç venivano dunque aperti e st•minati su piastre di RCA ~ di,.chi di 'ancomirina. nec~·ssaria JWT la discrimìnaziom• tra Gram -resistenti e Gr a m --'-- sensibili. su piastre di a~ar sangue da iucu· bare in condizio11i di aernhiosi per ultf'Tiore controllo ed in container.ç di glucosio brodo Ilt'cessario prr J!:li esami gas-ernmatuwafiei.

Le colonit~ cn•seiutP !'oln in condi1.ioui di anaerohiol'i su RCA veniva· 'ano dunquf' osservate pt'r il loro comportamt'nto verso la vancomieiua, esa­minate al microscopio dopo la coloraziom• con il Gram ed i relativi contaillf'r.~

di gluco~io brodo venivano quindi trattati con acido solforico 50 ~·u ed eter1•. centrifugati, il supcrnatantl' prelevato ed init>ttato nel gas-cromatografn. l profili rt-lativi a~li acidi grassi volatili, insieme al Gram f' alla morfologia rilevata al microscopio erano nella maggior parte dei casi sufficienti ad indi­care il genf'rc del germe mentre lì don· ,-j potevano essen• df'i duhhi si ew­guivano tlellt· prove hiochif!lich!·. Ad t'S. per difff'renziare i Lattohacilli dagli Eubatteri e dai Bifido nt•l caso in cui questi ultimi non fossero già chiaramente riconoscihili alresame microscopieo p1•r il loro particolan· aspetto, si eseguiva la cn•Hcita a 45 11 C positiva "'olo )l!'T i Lattohacilli, o pt•r distinguere i Peptostrt-ptncocchi dai Pt•ptococchi si eseguiva la fermenta­zione dt•l glucosio, positint per Pf'ptostreptococchi tramlt' per il solo P.

GIA:<IFRU.LI MASTII.\NTONIO F. Al.TIII 2!

micros facilmPnte però distinguibile aii'I·same microscopico pf'"r le sue esigur­dimensioni. Non mi addentro ora nei dettagli tlelle proH hiochimich.- e gas-cromatografiche perché ciò è compito di altri relatori.

Dopo questa serie di esami, si avevano completi i protocolli pt>r ngni campione e si potevano quindi ottenere le percentuali relative ai genrri ril!contrati, costruendo delle M tra i risultati ottenuti nei tre t·ampioni suc­eessivi relativi ad ogni singolo individuo esaminato. Da quf'sti dati risulta (Tab. l) rhe la concentrazione degli aerobi era di 108 -"g di feci ffl('Dtre quella degli anaerobi non sporigeni era di l01u/g di feci. I clostridi inv1~ce ;;ono risultati molto variabili da soggetto a soggetto, infatti mentre in alcuni individui le concentrazioni erano 105-106/g di feci in altri la loro presenza Pra talmente massiva da impedirne la conta.

Sulle 720 colonie esaminate predominanti tra i germi at•robi sono risul­tati i Coli e gli Enterococchi mentre tra gli anaerobi i Bacteroides ed i Bifido. (Tah. 2 e Tab. 3). Questi 4 generi sono gli unici ad essere risultati presl'nti eostantemrnte in tutti i campioni di feci esaminati.

Invece è da sottolineare una certa variabilità nella pn'St>nza degli altri gt>neri batterici sia aerobi che an.al'robi non solo tra i vari individui ma anfhe nt>i tre campioni ottenuti in giorni successivi dallo stesso individuo. Mentrl' le differenze osservate nella composizione della flora in individui diversi. "econdo una analisi della vari.anza compiuta da Moore e Coli., [4] sembra riflettano rt>ali differenze esistenti tra individuo ed indiYiduo, per qut>lle osservate nei tre campioni succt>ssivi dello stesso soggetto abbiamo ipotiz· zato si trattasse di differenze dovute a prelievi t>ft'ettuati in punti divf'f~i

della massa fecale.

Inoltre abbiamo potuto rilevare dalla analisi dei risultati una llora batterica intestinale simile nelle uniche due coppie di soggetti appartenenti allo stesso nucleo famigliare. Infatti 2 individui (moglie-marito) presenta· vano entrambi una massa di entt-rococchi predominante tra tutti gli aProbi •~ altri 2 individui (madre e figlia) cv avano entrambi una presenza massi' a di clostridi. Considerando i limiti dovuti al numero di soggetti esaminati. non abbiamo fatto ipotesi ma ci siamo proposti di approfondire il prohlt'uw •·nn analisi succt'ssive !IU ><oggetti convivt'nti.

J risultati di questo lavoro, seppure pr~'liminare, conferme.rehlwro dunque i dati ottenuti da altri AA., in altri PaPBi, fornt'ndo l'indicazione •lt·lla sovracolonizzazione dt'gli anaerobi rispetto agli aerobi nel colon, eon un rapporto di l: 100 e la predominanza dei Bacteroides rispl:'tto a tutti gli altri generi pr('S('nti [5-9].

Dalla Tab. 4 risulta inoltre evidente che tra gli anaerobi i generi mag· giormente rappresentati oltre ai Bact.-rnides ed i Bifido sono gli Eubattl'ri •·d i Lattobacilli. Seeondo lavori di Pt'aeh e Coli. [10], popolazioni che ~~nn· .~umano dit•te di tipo occidentale ad l'levato contenuto di grassi (Jn!!hil-

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tPrra. USA. Scozia) rm:l'wntanu uua flura ft•calt· rieca di auat•rohi (;nuu

JH•gatÌ\ i (Bactt•roidt·~. Fu~nlutttt·ri) mt·uLrl' tjUt>llt• popolazioni clw l'on;,u­

mauo dif'll' ric:eh{' di earlwidrati (ludia. (;iappoue) prt'I"'{'Jltanu un mag:j:o!;iur nm1ww tli anat•ruhi (;ram JIV~ÌtÌ\ i.

J uo~<tri dati st•mhn•rl'hlwru tlnnqul' indieart· m•lk fei'Ì t•saminatt· una

Jll't•dominanza di anal'rnbi Gram 111:gati\i eomt• IJU!'IIa ri~eontrata in popo· l azioni con diete rit·ch•· tli gra~~i. an eh t· ~<t' il 'alorl' t• l t'\ alt• di LattoLaeilli

f'd Euhattf'ri 1;(-'lll~m·rehlw eollocarei in una pnsiziom· intl'ftnf'dia. dH' i11

dfi·tti }lt'nt• si atldict· ad 1111a pupolaziont• qual{' la nostra eh!' ctmsuma una

dit•ta rieca sia di gra~!:'i che di carlmidrati. PN i Clostridi inoltri:' si sono otttmuti d1:i ,-alori chi' si discostano dai

risultati di altri AA. dal monwnto che 4 soggetti sui I2 ''-~a minati a\ l'vano

una pre<wnza di clostridi massi,·a tanto da impedirm• la conta. Questo dato puìJ es!Wft' molto int{'r!".,~antt-' t' quindi degno di approfondimento data la

importan?.a di'i Clo~tridi quali dtgradatori degli steroidi nell'intestino. Infatti studi rt~centi hanJHI dirnostratu che il (:ancro del colmi può essere il risultato

d••ll'aziont• di careinogeni prodotti da steruidi intestinali (-' dal momento eh•·

qm·sti possono esst"ft' dt-"gradati da molti Ciostridi, il più rapprcsentatiYo di tutti npJia produziont-" di steruido-dcidrog•·nasi è- il CI. parapll.tr~(intm.

Ì• ovvia la loro importanza i11 qul'sto tipo di rict·rche [L 2. Il J. L't-"vidf'nza del rapporto quantitati"\o tra germi a!'robi l'ti ana{'rohi

lwll"intt>Stino di ~nggetti normali può f'f>!-ol'n' utili' inoltrf' nt•gli studi sul meta·

lwlismn umano. Infatti è stato aeecrtato (Gall l' Coll. [3]) clw gli anaf'rohi

suno implicati in molti dt•i proces!<i assoeiati alla tligestimw. COlDJireso il metabolismo di et-"rti carboidrati, gras.~i t' protcÌilf' e nella produziont· di vitamina H2 • B 1z. aeido pantott-"nieo. folico. o in produzioni di t<ostanzt· par·

ticolari quali ad t'H. il colt>sterolo n la formazionr- eli amint• tossieht· do"\ulo· alla d1•carLossilaziOJw di certi aminuao:idi.

R quindi necessario tl'nPr conto nell'"o:culogia d!'lla flora intt~stinalt• ddJ.. ulazioni tra i vari microorganismi anaerobi ed a1•rohi c dl'llt: int('ra­

zioni tra flora intestinalt· f'd ospitf' w·l mantcninwnto di uno stato di

equilihrio.

Infine i dati ottenuti potranno servin· qualt~ ha>'t' 1wr la valutazinnt• ,. rint!!rprl'taziom· di camhiamf-'nti iu questa microflora in situazioni patn·

lugich('.

Riassunto. - Al fin(' di valutar" il rapporto quantitativo tra germi ao•rohi, anaerobi non formanti spor{' p clol'!tridi nelle feci di soggetti sani l'!Onn

>itati esaminati in qua6ta fase preliminare del no~trn lavoro I2 individui sani di età compresa tra i 20 ed i 60 anni. che prl'sentavano regolari funzioni

df'll'ah·o, mf'diante prelil'vo di trt" campioni di feci ottenute in giorni succe~-

GHNFRILJ.l YIASTHANTONIO E ALTRI 27

~ 1v1 • L'identificazione del genere batterico veniva t'st>guita con prove bio­

chimiche e gas-cromatografiche. I risultati hanno dimostrato che la concentrazione dei germi at1rohi

è di 109/g di feci mentre quella dei germi anaerobi non Aporigeni è di 1010/g di feci. I Clostridi sono risultati variabili tla soggetto a soggetto, infatti mentre in alcuni individui le concentrazioni sono 105-106/g di feci, in altri la loro presenza è talmente massiva da impedirne la conta. Tra i germi aerobi predominanti sono risultati essere i Coli e gli Enterococchi, mentre tra gli anaerobi i Bacteroides ed i Bifido.

I risultati di questo lavoro confermerebbero dunque i dati ottenuti da altri AA. in altri Paesi, fornendo l'indicazione della sovracolonizzazione t!egli anaerobi rispetto agli aerobi nel colon con un rapporto di l : 100 e la predominanza dei Bacteroides rispetto a tutti gli altri generi presenti.

Summary (Inle-~tinal bacterial fl•Jra in normal atlults). - In this preli­minary study we ha ve examint>d 12 healthy men aged betwe!'n 20- fiO yPars,

by three specimens each. All the isolated could be assigned to genera by hiochemical tests and gas-liquid chromatography. The results showed a l()K/g concentration of aerobe hacteria in facces and a l01n/g concentration of anaerohe non sporing bacteria.

The results of the count of Clostridia showed a variability among the

cxami.ned individuals. The predominant groups of aerobes in the faeces were found to he E. coli and S. faecali.~ spp. and the predominant group of non sporing anaerobes were Bacteroides and Bifidobacteria. These results were similar to the data obtained from foreign AA., showing the predomi­nance of anaerobes in the faeces and particularly of Bacteroides.

BIBLIOGRAFI.\.

l. HILL, :\f. J. 1974. Colon canr.-r; a <ii•~ase of tibre deJ>letion of or dietary exresst f)ige­

~tion, n. 289 .. JUO.

~. liiLT-. \1. J. 1974. Racteria and the f'tiology of rolonie cancer. C,.nc~r. :U: 81::>-R18.

:1. GuL. L. S. 1970. Nornml fecul llora of man. Am. J. Clin . .V1•lr. 23: l·l~7-·146S.

k HoLDMAN, L .. Gono. J. & \foore, W. 1976. Human fecal flora; variation in bactcrial compo~ition witbin individuals and a pos•iblt- f'fff'ct of emotional otreRs. Appl. Enviromn . . Hirrobiffl. 31: 359-:ns.

·•· GOHBARCH. S. L. 1971. Inte,tinal mkrotlora. G,.slruenteroloey. 60: 1110·-ll22.

IJ. HII.L, M . .l. & Dra~ar, B. S. 197.~. The normal colonie hact<'rial fiora. Gut. 8: 318-323.

IlRA~AIL B. S., !:>IU!'.ER. ~"1. &. :\lci,EUU. C. M. 1969. Studi~' in the intestina! fiura. (;,..

S/rOPff!('TQ/rJI!.l. 56: JJ--i\1.

8. GORBACII. S. L.. PJ,ALl", A. C .. i\ AllA~. J.., \\ t;ll'iSTF.II'\, L., SPANIC'I/J;Bt:J., (,. & Ll•:\"1·

TA"'- H. 196~. Studi .. ~ un iUll'~tinal microflon1. Il", Mieroorj!:anism of tlw ~nulli Ìnte~tiru· and tlrt·ir relation, to ora] aud faecal flora. Gaslmmtl'rolo}~Y· 53: 8S6--Rt>7.

'J, 'lUURf:. \\. 8.. HuLl>F.M.~"i L. l9i·L Human ft'cal flora: lht' norma] 1\ura of 211 .lapaul'~•·

Hawaiiau,. Appl. Mi<·n.o!>io/, 27.: 91>1-Y/'.1.

10. PE-ACII, S .. l't:H"'A"'IJF.Z. F. JonNsol\, K. & URASAR H. S. l \li-L Tlw •um-~porml'-" ,. .. ,,...

rohk hactt>ria in humanf~wce~ .{. JHPd. Mirrt)bio/. 7: 2B-22l.

Il. J)HA!'>AR. B. S., (~ol>IJARD, 1' .. lhaTO!'., S. I'EAeJJ. S. & \\-t:~T. B. l<t71J. C!u~uidia i~o],.

tl"d from fae""'· .1. Med. Mi~robiol. '; (.::1-71.

Abnormal intestina[ Oora

S. TABAQCHALI

Department af Mfdical Micrabialazy, St. HflrllwlameWJ's Hospital, /,rmJon.

Conditions associated with abnormal bacterial overgn1wth

Thr~e main types of gastrointestinal aLnormality encourage hacterial on-rgrowth, The first group consists of gastric ahnormalitie11 such as achlorhydria and partial gastrectomy and the second group consists of I'On<litions which cause stasis, such as surgical euteroanastomosis; strictures. eongenital or infective; ~mali intestina! diverticulosis, multiple or single; and conditions with abnormal motility as in scleroderma. The third group <'onsists of conditions in which there is free communication hetween the ~mali and large bowel as in gastrocolic or enterocolic fistulas or following massi ve intestina! resection with the remo val of the ilt',ocaecal valvf', [l, 2].

Thl\ microflora colonising the .~mali intestine umler abnormal conditioru

Recent studies have t'mphasized th~ importance of investigating the­whole nf the small intestine from duodenum to ileum in order to under8tand the relationship between the microtlora and the host, Furthermore, impro­vt•d anaerobic techniques have Leen used which bave demonstrated that the small intestina! Rora under these conditions consists predominantly of anat•robic hact~ria.

The micro-organisms eolnni1'1ing the small intestine in the ahove ronditions are different from the norma! flora; they are ' faecal' in typt•. Tlu· hacteria isolated are the aerohic Enterocneci. Enterohaeteria. aerobi(' Laetnbacilli an<l yeasts, and the anaerobic micrn-organisms such as Bacte­roidt·s. Bifidobactf"ria, Clostrida and Veillondla. The euneentrations and

~li~tributions are eomplex and dilfer fro01 patient. depending on the sitf" and f"xtent of the causative lesino. The concentratinns range from 105-10~

ur!!anisms per mi of intestina! flui d (l J.

.lu !'>;!'EZIO'>;! l>A BATTERI A'\A~:ROHI NO'> ~POHIG}::\1

Conditiomo asl!lociated with an abnormal bacterial flora

l. .o\huormalitic.; of p;aotric functiou:

Polya partial !(a~trectomy - Aff~rcnt loop ~yndronH'! Malfunctiouinp; ga~trojejunodom\:

Peruicious anaemia.

:!. - l.:ondition~ causing ~taoi>:

o) Surgical hlind loop•:

Entero-ana•tomosi•:

b) Stricturc~:

Con!(enital: l.:rohn'o dihcllse;

Tuhrrrulou~:

c) Adhesiom·. X-ray irradiation:

d) Sma!l intestina! divuticu]o~ih:

e) Ahnormal motilit~·:

Scleroderma; Tropical spnrc: Diahetic nf'uropathy:

Vagotomy; Gangliou blocking ll.j!:enb: lnt,.stinal pseudo .. obMruction:

f) Partial hiliary ohstruction "'ith cholangiti, .

. l. 'Frec communications h~t,.,een lar)l:<' anù smallloow~]: Gastrocolic fistula:

Ent.-rocolie listula; M assive intcstinal r,.o,.ction:

The e.lfect of gastric acid on tlu- intestina/ .flora.

'fABI.E i

The presenc(~ of acid appean; to limit the growth of colonie micro­organisms in hoth ~tomach and duodenum in most cases, hut does not alway.~ exclude it. ]u the ahsence of gastric acid howen~r, the hacterial count.~ can go up a~ high as llF-108 organisms/ml.

The r.ffel'l of the anatomica/ [t>.~ion on tht> barterial jlom

The distrihution of two types of organisms, coliformA and Bacteroidt·s in four conditions is shown in order to illustrate how the si te and extent of the lesion determine thc microhial flora of th(' small intestine (Fig. l and 2).

IILE SAli DECONIUGA110

•

,.. ,.. ,. ,.

POLU PARTIAL G&S1AEtTDMY

o o o

-· -· ----,_

r~BAQLHALI

IINGlE OUOOEMAL OIHHICUlUM

t--

-u •

o o

-· -· --·­,_

STAitTUH Of l~E ILEUM

. . •

"

o

.i

Fi~. l. - The eoncentration ;md di~tribution of two microor~anisms, the a.-robie <"llli­

forms •---------- and the anaerobie Baeteroides O O at dilferent sites ut" th~ ga~tro--intestinal tract of 3 patients (~olya partial gastr..-etomy, oingle duodenal

divertieulum and ~trieture oi" the ileum) together with the pr<"~<"nCe or abo,.nce of bile ;;alt deeonjugation iu each ~ampie.

E

~

> ' z < ,, " o o o u >

COLI FORMS

IO'-

>O'

,. •o l

STREPTOCOCCI IO ~ l !

IO'~-------------- A y' l v \il

•o L

8ACTEROIOES

STAPHYLOCOCCI

LACTOBACILLI

Anaerobrc

• /' '

l ,, ,. .! l /-'

r----------<f, (

CLOSTRIDA

Fii!. ~- Tlw <"one~ntration •111d di~tribution of Of)!;IIH!Orn~ at t!ilf,.r<'nt !t""vds of thc ~!""

~rro-intestinul tract in a patient with lllnltìple <luodenul HllÙ j~junal ùiHrti­.:nln•i•.

INPEZIO!';I H~ BATTERI A"'iAièROHI NO:\ SPURIG.tNI

Jn uneomplicatNI polya partial gastrectom~ wht•r•• tiH:~re 1~ IW Sla!<i.~

coliforms may be pres•~nt throughout tht· small int~stint·. increasing iu con· centration in th~ ileum. hut no Bacteroidel'- wcrt• isolatcJ.

lu tht• patient with a sing:lt• duodenal diverticulum hoth coliform~ anJ Bacteroides art• pre;;e-nt in the proximal part of tht• small inte.stine. wher•· tht• lesion is, Lut the.'w micro--organisms art> ahst'nt lower down the intf'· stiut·. In contra~L hov.f'.\'t'f. in a paticnt with di~tal ~tasi~ coliform~ wert· prt~sent throughout the srnall intN>tint·, hut tlw anaerohic flora such as Bactl·­roidt•;; only appt~an·d al the art·<l of stasi~. di~tally in tht· ileun1.

Tlu· distrihution of hacteria in a patit>.nt with f'Xtensi\·t~ proximal sta· si.'- a~ may Lt• cause-d hy rnultiph· duorlenal a111l jejunal divcrticulosi~< i~

~hown in :Fig. 2. Both at'rohic and anaerobic microflora are present in higl1 eoncentratiom throu~thout tht• small intestine frorn stomach to lower ileum.

Th•· aerohic flora thf'feforf', and in particular tht• coliforms, can coln­ni"'f' tht• small intf'~tint> in a variet~- of conditions. hut tiH' anaerohic flora re-prt'sPntt>d hy Bactt>roidt'!< only apJwar i11 areas of stagnation {l].

TIH: CLii\ICAL CO~SE(IUE:\CE~ OF AB!'òOHMAJ. BACTEIHAL COLONISATIO~

Then• are numerou~< mt>taholie activities associateci with tbc intestina! hacteria colonisin~t thl:' small intestine in patientl< with small intestina! hactf·· riai on·rgrowtb and thesf' havf'. been rf'vie-wed previously [2J. In thil' papf'r l would like to consider thf' eJI'ects of hacteria on tht' bile acid metaLolism. 'itamin B 1 ~ and protein metaholism an d thf' clinica! consequenct's which rna,- df'velop in thl-' host.

Th1• rmtaboli.m1 of bill' acid.,·

Primary bilt• acid~. cholie acid~o (h. 77 12~) and chenodeoxycholie ar.id (3a:, 7~.~:) are synthesis('(l in tht• live-r from cholt>sterol anrl conjugated with glycint~ or tauriru~ antl st•crett~d in the bile aci d(*) dt'oxycbolic aci d (31)' .. 121)';) which is formed hy bacterial action on cholic acid. Thesc conjugatell hiil· acids contrihutt' to fat digt'stion and ab110rption in thf' proximal small howf'l and are largely n·ahsorbed by actin· transport mecbanism in tht• ilf'um lo return to th1• livf'r via the portai vein. thus forming tbc enteroht·· patir circulatioiJ. A small amount of unahsorhM hilf' acids pass into thf' e:wcum and cnlon wht·rt• tht·y ar(' mt'taholised by hacteriu.

(*)A~ conjugat('d bii~ af'id,. Anotb~r comtilul'nt of th•• norma! hilf' i~ thrsecondar~

bile a•·id.

TABAQCHALl

Tht> main rt~actions earried nut by bacteria are:

i) llydrolysis: Bacteria hydrolyst> tht> amide bond to rt>lease the free hile acids from their taurine or glycine eonjugates. Tht> hacterial t>nzy­mt' responsihle is cholylglycine peptide hydrolase.

ii) 71'f.. dehdroxylation: Bacteria OH-group at C7-po~;~ition to form the an• 7x dehydroxylases.

dehydroxylate by se~ondary bile acids.

removing thf' The enzymrs

iii) Oxid(J-rcduction and deh)"dragenation: Bacteria oxidise and further n•dnce the bile acid moleculc to form ketoacids and o: and ~ rpimers. This ean necur at the C1, C7 ami C12 position, thl:' t•nzymes responsible bring the d t> h yrl rogena8f'S.

Tht-se bactf'rial mt~taholites of bile acids ha ve lwen identifit~d in the faect·~ .

. \1icr(J-arganisms associated with bile m:id mctabnlism

Varinus ~u·ruhic and anaerohic bact~>ria ha\"t' lu~en :;hown to be rapable uf 1·arrying nut these metabolic activities in vitro [3. -l-] but due to their n·la­LÌ\ l' high concentrations. i t is tbc anaecobic bactf'fia which are hy far the most important. Similarly in L'ivo studics of patients with bacterial ovt'"r· ;::nmth, there was a good correlation hetween the presencc of free bile acids ami the Bacteroides sp. in ~amples ohtained from differ1·nt levt""!s of the ~mali intestine {l].

l11testinal bile llfÌiis

rn patÌt'lllS with iJa1~teriuJ OVI!tgruwth Ìn the ~mali Ìnte~tÌUI:'. hiJe salt degradation may occur, leading to an abnormal bile acid patt1•rn,

Fig. 3 eompares the bile acid concentration in fasting jejunal Huid from 5 contro! subjects (open enlumns) and in 7 patients with the stagnant loop ~yndrome (hatched columns). The mean and range of the bile acids are I'Xpres:;ed as mmoles.:"! of intestina! aspiratt""s. The conjugated bile acids are presented as total and Hub-divided into glycline and taurine conjugate;~. The frf'f' IJile acids dt""tectetl \tt're eholic. chenodeox.y-cholic and deoxy­dwlic acids. In the jejunum of contro! ~uhjt>cts there are only eonjugated hile aeids detected, In th1l patients \\"ith the bacterical overgrowth (:;la· ;..:uant loop syndromc) the pattcrn is difl'erent. Firstly. there an~ large amounts .,f fn•c hilt! aeids present (cholic. chenodeoxyeholic and deoxyrholic acids). :-;!'("tondly. tht'" taurine eonjugah•s are much 1mlf!: rt•duccd than the glycinf'". and. thirdly. as a result of hactt•rial formation of tht•se free hilc acids. the [Pvl'l of tht' ennjugatf'd hile salts falll'l. ali lwing under .'i mmolt•s/1.

5

l

l~ l

Ocontrol subjects (S)

m Stagnalt loop synct-o.ne(7)

J.'ij!". 3. - Tht' concrntration of hik ocids in tbc fa~ting UJ.lJ'~r jcjunal Huid (mcau aud ran~t' in mrnole~/litre) from 5 control subjech (op<"ll auas) and i patienh with the stagnant loop syndrome (hatched areao). Tuta! conce-ntration of conju!(at.-.<1 bile acids and their suhdivision into glycìnr and taurinf' coJlju~~:ate~ an· fl"l•r•·­sented. The free bil(acid" are· rr:prese-nted b) C~-cholic acid, CV•-,cht-llndl"n")· cholic acid and D ~deoxycholie adJ.

Consequences of bacterio/ degradatimi of bile aritl.~

l) Steatorrhoea. Thr• smalJ intestirw normally contain:. conju~atecJ

Lilc salts in sufficient concentrations to promote the dispersion and ah!:>Ol"Jl· tion of lipids through the formati011 of mixed micelles. Undt'r ahnormal conditions when•, thert• Ìlo; colonisation of the small intestint'. bile "alt df'conju~ gation and dehydroxylation may occur and thus lead to a rcductiou in tbe level of the conjugated hik salts sufficiently to impairt~ micelle fornu•­tion, therehy causing malabsorption of fat [5, 6J. TJH' fre•· hilf' acid~. 1111

the other hand, at the pH of inte8tinal contcnts, appear to )J(' eitlu~r ah~nr­

hed by non-ionic diffusion or prccipitated. However. th•· merc pre~('IJ('r·

of ahnormal hactl'rial overgrowth or th•· presence of frf'e hill· add~< in the small intf'stinf' does not necessarily lead to steatorrhoea. Limitt>d lr­sions of the jejunum or ileum may not he assoeiated with sh~atorrho('a if there is insuffwient degradatÌ011 of bilt· salts [1]. This suhj••ct has hecn I'f'vil'­Wt~d in detail clsewhere (2].

TADAQCHALI

Heduction in the concentratinn of intraluminal conjugated bile salts may also lead to malabsorption of fat soluhle vitamins (A, D, K) with cli­nical ~onsequencf'S.

The effect of antibiotics on the faecal fat excretion and the jejunal hile acids are shown in Fig. 4. Before antibiotics, the faecal fat was 22 gjday and there were low levels of conjugated bile acids and high lcvds of free hilf' acids. On antibiotics not only ()id the free hile acids tli~appear bu

COHIUGATES m M/lìtre

IO TAl COHJUGATES

FREE mM/l1!re

FAECAl FAT G/OAY

TC

TCO

TOC

G OC

Belare Alter

179 1l77

O·JI

Fig. ~. ·- ~~ffect nf antibiotic on bile salt nmcentration in upper jejunum and faecal fat <"'<<"r<•tion. TC: taurocholate, TCD: taurochenodeoxycholate, TDC: taurodeoxy­cholate, GC: ~lycocholate, GCD: ,!!:lycoheudeoxycbolate, GDC: ~lycodeoxy­•·holate. C: cholic acid. CD: rh~nodt'ox.ycholic acid, DC: deox.ycholìc acid.

A11n. t.t. Super. 8a>!il< 11~09) 15, "\l--t2

also thl" conjugatcd hik acid.~ incn'a'H'tl and tlw ftwcal fat no;cretitm rt•tur­ned to norma!.

2) Diarrhoea. Tht• mechani~m of tlw diarrhot".a u~.sociatt-tl with JJactt'­rial overgrowth in the small intestifw antl after intt~.,;tinal re~t"ction i~ not

weU understood. The absorption of frl"e hill" acid~ ha, e bet•n shuv. 11 t" lead to a disturhance of water antl electrolyte ahsorption in patif'nt~ [7) aml in ex:perimental ani mals [B].

3) Monosaccharide malabsorptùm. (;Jueust· transport h~ jejunal mu­co~a was impaired by the presenee of deconjugated bik acid." in vitrn [9) and in experimental animals in vit'O [8, 10-12] denwnstratetl revcr~ihlt·

inhibition of active intestina! sugar transport hy deconjugated hiJ.· ~alt~

and suggested tbat this may explain the tempurary monosaccharide malah­sorption in infanq:.

DeU>ctirm of bile acid deconju~atirm.

The presenct- of bill" acid deconjugation in the small intestint• can Jw detected without intestina! intuhation by two methodE:

l) The Breatb Test: Thf' mcasurement of 14C0 2 in t>xpirt>d a1r fol­lowing on oral dos(' of 11C-glycocholic acid. Bacteria Ùt•eoujugatf" th~· }Jil~·

acid" and further metaLolise the glycinr, liberating 14C02 [13].

2) The Demonstration of Free Bile Acids in thf" Serum: The SPrlllu bile acid paltern rellects tbe changes which occur in the small intl'.~tin.­

[14, 15]. The tt"St~. however, are no t SJlecific for patient~ with Lactt•rial o' cr­

p-owth in the smaU intestinr Lut art• a]so positivi' in conditiouJ< with hil~·

salL malaLsorption or hilliary tral't inf't"ction~<.

Vitamin B12 ab$orption.

lt i~ weU estaLJishPd that haeteria can take up fret· vitamin B1z ;, t•i­trtJ, hut Linding of vitamin B 12 to intrinsic factor (IF), protect~ agaiu1<t its uptake [16]. ln vir,·o. however, therr i~ malahsurpiton of lF-Lound ùta­min B12 in paticnts with the stagnant Joop syndromf' [17]. Thi~ di!-1cre­pancy was investigated hy in t•itro and in viNJ t>xperimcnt~.

i) In vitro 'uptakr ' of vùamin B 12.

Varinm intestina! hacteria isolated from patil'nt~ with tht· stagnant lonp syndrome werc incuLated with 51Co-ùtamin B 1z. free and IF-hound

(Fig. 5). The hacteria used were Enterobacteria, Bacteroides, Bifidohactrria and Clostridia. Ali tbe bacteria ested were capable of taking up free vitamin 8 12 whereas only certain .'ltrains of the anaerobic Bacteroidt"s were eapalllr

nf taking up IF-bound vitamin Bu in vitro [18].

"{, UPTAKE o

r

o 80 o

o 6'

~ o o o 60

l ' o

r

o o o

40 o o

• ~ o

lO o 00

l --'--o -T l

~ 00- ---Entero- Bactero<de5 Bifìdo- Cio>tnd,a bacteri a bacteria

100 "o 6,9 ng s7CoB12 o Uptake of unbound o7C,JB12

• Uptake of bound 07CoB1:!

Fi!!:. S. - l:ptake of 57f:oB 1 ~ hy ha<:tl'ria ~ubtulturl'd from ~mali int•·~tinf" a~pirat•••.

ii) In vivo ' uptake ' l)f t•itamiu R12 •

Patients with the stagnant lnop syndrome in w horn then~ W li.;! \ itamin B12 malahsorption. patients with bacteri al nvergrowth with norma! \ ita­min 8 12 ahsorption. and contro! subjects. Wt"re intuhatrd to the ileum: after an overnight fast a test meal was administeretl eontaining l mirrngram of 57f:o-vitamin Bu Lound to human JF, lleal a;;piratf's were nhtainetl at different times for 5 hours aud eentrifuged. The pt"rct"utage radioacti­vity in the tleposit was measun~d and tOund to be on avNage t'rom -i6 to 74 "/0 in the patients with the stagnant lonp syndrome in whom th,~re wa~ vitamin B 12 malahsorption, whert>as it was less than 10 °';, in the othf'f twn

groups (Fig. b) [19].

':',57 CoBJz

in depos1t 40%

20%

INFEZI0!'\1 DA BATTERI A~AEIIOIII NO~ SPORIGE:\l

• •

Jejunal d!Vert'lculosJS and s12 malabsorption Jejunal diverticulosis and norma! B 12 absorption Contro! subjects

p

•

--C

:--

1 3 ' HOURS AFTER TEST MEAl

.. ~

--"

5

Fig. 6. - AmoÙnt of S7Co812 in eentrifuged deposiu ofupper ileal aspirate~ after feeding a test-meal with !.tg S7CoBl2 bound to human gastric juict·.

Thus, thP evidence so far suggests that bacteria interfere with vitamiu B 12 aLsorption in the stagnant loop syndrome by competitive uptake of tbc vitamin within the lumen of the small intestine, even when the vitamin is bound to IF.

Malabsorption of vitamin B 12 can lead to vitamin B 12 deficiency and mcgalohlastic anaemia in thc host.

METABOLJSM 01' AMINOACIDS

Intestina} hacteria metabolise various amino acids and in particular L--tryptophan. L-trytophan is degraded by bacteria to form indoles which are absorbed, conjugated in the liver and excreted as indoxyl sulphate (indican), in tbc urine. The normal rangc is < lO mg/24 hour urine.

The urinary indican excretion is usually raised in tbc stagnant loop syndrome but this is not specific as it may also be raised in other conditions where there is protein malahsorption.

How much of the dietary trytophan is degraded by bacteria is uncer­tain; in some patients the urinary indican excretion may be as high as 500 mg per day, suggesting that a major proportion of the dietary trypto­phan, is metabolised by bacteria. Other amino acids are metabolised by bacteria such as tryrosine, phenylalanine and their metaholites can be detected in the urine of patients with bacterial overgrowth and in experi-

..ifin. llt. Su~r. Sanità (11170) 11, 2~2

T,I.HAQCdALl

nwntal animals. The metabolites include hippuric acid and volatile and non-volatile phenols (20, 21].

The degradation of amìnoacids by hacteria may deprive the host nf ess~ntial aminoacids, which in tum may le:td to protein-calorie ma1nu­trition. If this occurs in adults, a situation resembling kwashiorkor may f!~velop [22).

In cbildren, on the other band, the long continued calorie under-nu­trìtion can lead to intestina} infantilism, w bere there may be failure of growth :md sexual development [23].

f:ONCLUSION

There are numerous conditions which can lead to abnormal bacterial over-growth in the small intestine. The bacteria which colonise the small howel are qualitatively similar to the faecal dora and these bactcria can if·ad to malabsorption of fat, fat soluble vitamins, vitamin B12 and disturh protein metabolism, an d if the malabsorption is severe, i t c an le ad t o mul­tiple nutritional deficiencies in the host.

S111D1111lr)'. - The normal small intestine in ~parse microflora consisting of Gram-positive from the or()-pharynx.

man usually harbours a microorganims derived

The concentrations in the upper jfljunum are approximately 10~-104

nrganismsjml, whereas in the ileum the concentrations may be higher and faecal type organisms may also be present, such as Enterobacteria, Bifì­dobaeteria, and Bacteroides in concentrations of 105-106 organisms per mi of intestinal aspirate [24, 25]. This situation is well maintained unless the intcgrity of the small intestine is deranged. There are however, certain t'tmditions in which bacterial proliferation in the lumen of the small inte· ~ti ne may occur and give rise to various metabolic abnormalities (2]. In this paper I would like to list some of these conditions, then describe the type nf microflora which colonizes the small intestine, and finally discuss a ff'w

aspects of the metabolic consequences of these hacteria.

Rill88unto (Flora intestinale anormale). - L'intestino tenue normal­mente è colonizzato da batteri Gram-positivi derivanti daJI'orofaringe. T ,e concentrazioni nel tratto superiore del digiuno sono approssimativamente l0~-101 organismi/mi, mentre nell'ileo queste concentrazioni sono più ele­Yate e possono essere presenti anche organismi di tipo fecale come Entero­batteri, Bifidohatteri e Bacteroides in concentrazioni di 10~-106 organismi/mi

•

-lO INFEZIONI DA BATTUH ANAEROBI NO!'i SPOIIIGE'\1

di aspirato intestinale [24, 25]. Questa situazione si mantiene normalml'nte costantt' senza alterare l'integrità dell'intestino tenue. Tuttavia yj sono delle condizioni in cui può avvenire una moltiplicazione batterica nl'l lume intestinale che di consequenza determinano alterazioni metaboliche [2].

In questo lavoro ho voluto citare alcune di que3te condizioni descri­vendo il tipo di microflora che colonizza l'intestino tenue ed infine discutt'n

qualche aspetto delle consequenze metaboliche di questi batteri.

RE.FERENCES

J. GoRBACH. S. L. & TABAQCHALI, S. 196SI. Bacteria, bile and thl' small intestiue. Go<!.

IO: 963-972.

2. TABAQCH.U.I, S. 1970. The pathophysiolof!;ical role of small intl."stinal hacterial flora. Scanà . .1. Ga5lroeraurol. Suppl. 6: 139.

3. MtDVEDT, T. & NoB!IIAN, S. 1967. Bile acid transformations by mierobial strains helun· ging to genera found in intestinQ] contents. Acta Pmhol. Microbiol. Scand. 71: 629-638.

4. HILL M. J. & llRASAB B. S. Degradation of bile salt• by human inteatinal bartl'· teria. Gul. 9: 22-27.

S. KrMM, Y. S., SJUTs, N., BLUM, M., TEBZ, J. & Sut;BLOCK, P. 1966. The role of the alte­red bile acid metabolism in the steatorrhoea ol experimental blind Ioop. J. Clin. lnvfll. fS: 956-962.

6. TAIIAQCHALI, 5,, HATZIAONNOU, J. & 800TH, C. C. }968. Bile salt deconjugation and steatorrhoea in patients with the stagnant loop syndrome. Lancel. ii: 12-16.

7. MEKHJIAN, H. S., PBILLIPS, S. F. & HOFHAN, A. f'. 1971. Colonie secretion of water

and eleetrolytes induood by bill.". aeid8: perfusion studies in man. ). Clin. lnves1. 50: H69-1.)77.

8. HARRIJ;;S, J. T. & SLADt;N, C. E. 1972. The effeets of different bile salts on the ahsorl'· tion of lluid, eleetrolytes and mono-saceharides in the lllllall intestine of the rat in t•ivo. Gul. 13: 596-603.

9. PoPE, J. L., PARKIN~O:<o:, T. M. & 0LSON, J. A. 1966. Action of bile salts on the meta­bolism and transport of water-soluhle nutrients by perfused rat jejunum in vilro. Bio· chim. Biophys. Acta. 130: 218-232.

IO. TABAQCHALI, S. & BooTH, C. C. 1966. Alteration of bile salt metaholism in the stagnsnt loop syndroml'. Gul. 7: 712.

Il. GRACEY, M .• BuRKE, Y. & ANDERSON, C. M. 1969. Association of mono-saccbaride malahso-rption with abnormal small intestina! flora. Lam:el. ii: 384~385.

12. GRACKll, M., BuliKE, V. &: OsHI,.,-, A. 1971. Reversible inhibitiun of intestina) actiw sugar transpart by deconjugated bile ijalts in t'ill'o. Biochim. Biophys. Acta. 225: 308 · 314-.

.A nn. l1!. Suptt. Sanitti (197ìl) 15, 2J-i~

13. F•o-, H. & HoFIJANN, A. F. 1971. Breath test for altered bile acid metaholism. Lan­fel, ii,: 621-625.

14.. LEWIS, B., PANVELLIW:ELLA, D., TABAQCHALI, S. & WooTTON, l. D. P. 1969. Serum bile aeids in the stagnant loop syndrome. Lancel. i: 219-220.

15. PANVELLIWELLA, D., TABAQCHALI, S., WOOTTON, l. D, P. & LEWIS, B. 1969. Bile acid metaboliem in the stagnant loop syndrome. In: Proe. 81h lruernational Congreu on 1Vutrilion, Prague. Amsterdam, E1~rpta Medica (Int. Congress Serie& No. 213, -J.-1.8.)

16. DoNALDSON, R. M. ]R. 1962. Malabsorhption of Co60 labeled cyanocobam in rah witb intestina! divenieula: I. Evaluation of possible mecbanisms. Ga.rtromlerology, 43: 271-281.

17. MoLLIN, D. L., BoOT&, C. C. & BAX:ER, S. J. 1957. The ah!!Orption of vitamin 8 12 in contro! subject& in Addiasonian Pemicious Anaemia and in the malab!!Orption syn· drome. Brit. ]. Ha.!malol. S: 412-428.

18. SCBJONSBY, H. & TABAQCHALI, S. 1971. Uptake of rat gastrie--juice-bound vitam.in 812 by inte&tinal bl'UIIh borden ieolated from hlind-loop ratl, Etl'ect of small inteBtinal bacterial on intrinsic factor and the vitamin B1t- intrinsic factor complex. Scand'. ]. GaalrMnurol. 6: 515-521 and 707-713.

19 SCHJONSBY, H., DIIASAR, 8. S., TABAQCHALI, S. & BoOT&, C. C. 1973. ScanJ. ]. Go-61rtffllltMOI. 8: 41-47. Uptake of vitamine 812 by intestina! hacteria in the stagnant loop syndrome.

20. MILLEII, 8., MITCJUSON, R., TABAQCHALI, S. & NEALE, G. 1971. The effect of excessive bacterial prolileration on protein metabolism in rah with self filling jejunal sacs. Europ. ]. Clin. lnt>f!3t, 2: 23-31.

21. BoNE, E., TAKII, A. & HILL, M. J., 1976, The production of urinary phenols by gut bacteria and their possible role in the causation of large bowel cancer. Amer. ]. Clin. Nulr. 29: 14.48-1454.

22. NEALE, G., ANTCLIFFE, A. C., WELBOUIINE, R. G .. MOT,I.IN, D. L. & 800TH, C. C. 1967. Quom. J. M~d. 36: ·1.69.

23. NEALE, G. 1968. Protein deficiency in tempt'ratt' zones. Pror. Roy . .'\oe. Jftd. 60: 1069-1071.

24.. GoiiBACH, S. L., PLAUT, A. G., .'/AliAS, L. & W EINSTEIN, L. 1967. Studies of intestina! microllora. faecal flora.

Il. Microorgani8m~ of the smaU G.u1roenterolo!O'. 53: 856-867.

intestine and tht>ir relations to ora! and

25. DRASAB. 8. S., 5HJINEB. M. & McLEOD, G, }1, 1967. Studies on intestina! llora. l. Elfects of diet, age and periodic ~ampling on numben of fae,.al micro-organisms in man. G.utroemerolo!Q'. 53: 84S~855.