CA1199291A - A41030 antibiotics - Google Patents

Abstract

Abstract of the Invention Antibiotic A41030, a complex of 7 individual factors, I is produced by submerged, aerobic fermentation of new Streptomyces virqiniae NRRL 12525, and Strepto-myces virginiae NRRL 15156. The antibiotic factors are separated and possess antibacterial activity against Staphylococcus and Streptococcus species which are penicillin resistant. In addition, the antibiotic factors have shown inhibition of Streptococcus pneumonia Park I. The complex and the individual factors enhance feed efficiency in ruminant animals, and are growth promoters in chickens and swine, and are especially valuable in milk production in dairy cattle.

Description

X--59~

Summary of the Invention This invention relates to antibiotic A~1030 complex comprising several factors, includiny indi-vidual factors A, B~ C~ D, E, F, and G. This complex is producecl by culturing either one of ~wo hitherto undescribed microorganisms, namely, Streptomyces virginiae NRRL 12525~ and Streptomyces vi1.giniae NXRL
1~ 15156, or an ~41030 producing mutarlt or variant o each microorganism, under su~merged aerobic fermentation conditions.
The A41030 antibiotics inhibit the g:rowth of certain pathoser.ic microorganisms, in partic~la , those wi-thin the gram-po~itive genara Staphylococcus and Streptococcus which are resistant to penicillin. The antibiotics of this invention act to promote yrowth and improve ~eed efficiency in ruminant animals, poultrv and swine, and other livestoc.~/ and to improve rnilk ~: production in ruminant animals, This inventlon also r~late~ to a biologi-cally-pure culture o StreptomycQs vi.rginiae NRRL
15156, usefu1 for the production of A410~0 anti~io~ics.

~-~9~3~ -2~

Description oE the Drawings Infrared absorption spec~ra of A41030 factors A, B, C, D, ~, ~', and G are presen~d in the drawings as follows;
Figure 1 - A41030 factor A (in KBr pelle-t) Figure 2 - A41030 ~ac~or B (in ~r pell t) ~igure ~ ~ A41030 factor C (i~ K~r pellet) Figur~ 4 - A~1030 factor D (in.KBr pellet) 1~ Figure 5 - A4103C fac.or E ~in K~r pellet~
Figu~e 6 - A41030 ~actor F (in KBr pellet) Figure 7 - A41030 actor ~ (in KB~ pellat) Detailed Descrlption of the Invention This invention ~elates to antibiotic su~-lS stances. In particular, it r~la~es to an antibiotic complex cQmpriSing save al factors, includinq indi-vidual ractors A, B, C, D, E, F, a~d G. This complex i5 produced by culturing hitherto undescribed micro-~rgarlisin, Streptomyces vir~iniae NRRh 12525 / or hitherto undescribed microorganism, Streptomvce~ vlr~iniae N~L 15156. Fo~ convenience in our laboratoxies, cul-ture NRRL 1515~ has been de~ignated ~s culture ~41030, and culture NRRL 12525 h~s been designated as culkure ~41030.~, This in~entlon also relates to a biologically-pure culture O r Streptomyces virgi~iae NRRL 15156.
The te~ "ccmple~", as used i~ the ferm~n~
tation art, ~nd ir~ tn.is speci'ication, r~fers to a mixture of co~rodued individual an~ibio~lc factors.
.~s will ~e recogniæed by those famlli2r with ar.tibiotic S953~ -3- .

production by fermentation, the n~mber and r2t~0 of the individual factors produced in an antibiotic comple~
will vary, depending upon ~he fe~entation conditions and the strain used.
Culture A41030, iden~ified as a strain o Stre~tomyces virsiniae, was initially isolated from a soil sample collected in Indianapolis, Indiana, and has been deposi~ed and made a part or the s~ock culture collection o~ the Northern ~egional Research Center, U.S. Department of ~griculture, ~gricultural Research Service, Peoria, Illinois 61604, from which it is available to the public under the number ~RRL 151~.
Culture A41030.4, which is a chemically-i~duced mutant of ~ strain of the Streptomvces virginiae culture ~41Q30, has also ~een deposited and made a part o~ the ~toc.~ culture collection of thP Northern P~egional Researcn Ce.nter, U . S . Department of Agricultur2, Agricultural Research Service, Peoria, Illinois 61604 rrom which it is avaiiable to the p~l~lic under the number NRRL 12525. Cultuxe A41030.~ is cl~lmed in companion application Serial ~o. 424,055, filed March 21, 1983.
Cultuxe A41030 is classi-ied 2S a str in o~
Streptom~ces vir~iniae, and culture A41030.4 is classl ~ied as a chemically-induced mutant of a s~r~in or Strept~myces virginiae, ~ased lpon a s~mul~aneous culturin~ of ~1030 ~nd A~1030.4; S~epto!nyces avidinii ~CC 2741~; S~-eotomyces columbiensis ~TCC 27~25;
Str~pt~my~es aos~ikiensis ~TCC 23914; Streptomvces ~ri3eolavendlls ~TCC 25457; S~re~tom~ces lavendulae ~TCC

~' ~5953~ -4-8664; Str~ptomyce~ toxytricini ATCC lg813; and Str~pto-m~ vi.rginiae, Grundy, Whi~man, Rd~ok, Hanes and Sylvester 1952, Arrc~ 19817. The method~ and media xecommended by Shirling and Gottlieb ["M~thods or Characterizatio~ of Streptomyces species/" Int. J.
Syst. Bacteriol. 16(3), 313-340 (1966)], along with certain sup?lementary tes~s were used. Culture A41030 and cul~ure ~41030.4 were al30 compared wlth published descriptions Q~ the above-named strains appearing in 1'3ergey's Manual of Dc~terminati~e Bac~riology" (8-th Edition, edited by R.~. 3uchanan and ~.E. Gibbons, Th~
Williams and Wilkins CO. ~ Bal~imore, .~aryland); and bv Shirling and Gottlieb, "Cooperati~e ~escription of Type Strains OL Streptomyces", I~-t. J. ~ . 3acteriolO
1~(2), 178 (19~8).
.
CHARACTE~IZATION OF A~1030 CULTURE AND OF A41030.i~ CU~TUR~
Morphology A~1030 produces well-develope~ aeria mycelia ~ a~d ~porophores which are both coile~ and exhi~i~
hooks and loops. ~41030 is placed ir the Spi~ales ~S) section oE Pxidh~ et ~ Guide for the Class,-fication of Streptomycet2s According -to SelPcted Groups", Appl. ~icro'~iol~ 6, 52-79 (1957)/ as a primari~ morpnology type, ar.d in the Retinaculum-Apertum (L~) section o~
~ri~h~m _ al. as a secondary morp;~olo~ type.
~ 41030.4 produces no aeri~l mycelia and no spores~

g 5953A ~5-Cu1tura1 Characteri~tics Th~ growth characteristics of culture A~1030, cu1ture A~1030~4, and S. vïrginia~ ~TCC 19317 on var1Ous med1a are presented in the fo11Owing Table 1.
Color name~ wer~ as~3igned accord1ng ~o th~
ISCC-NBS Centroid Co1Or Chart~ Standard Samp1e No. 2106 (National ~ureau o~ Standards, U.S. ~epartment o~ Com-merce, 1958), and the Colox Hanmony 1`5anua1, 4th Edition (Color Standaxds Depa~tment, Con-ta1n~_ Corporation of America, Chicago, Illi~ois, 1958).

~ 5

2~

~5

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~ ul ~ ~ 0 x TABLE 1 w Cultural Characteris~ics of A41030, A41030.4, and ATCC 19817 Medium A41030 A41030~4 ATCC 19817 Abundant GQod Abundant ~SP ~ 72.d.0Y 90.gy.Y 75.deep y~r No~ 2 Am Ahun;63.1.brGY Ncne Abun:63.1,brGY
Sp Mone Nonr~ None G Good Poor Yair ISP R 93.yGray 93.yGray 90.gy.Y
Mo. 3 A~ Good:63.1.brGY l~lone Fair:63.1.brGY
Sp None ~one None G Abundant Poor Good IS~? R ~39.p.Y 89.p.Y 91.d.gy.Y r~
No. 4 Am A~un:63.1.brGY None Good:63.1.brGY ~ ~a Sp None None None G AbuIldant Poor Good ISP R 89.P.Y 89.p.Y 89.p.Y
~lo. 5 Am Abun:22.rGY None Good:63.1.br~tY
Sp None None None G Fair Not grown Poor C~apek's R 264.1.Gray - 2&4.1.Gray agar 2~1 Poor:lO.pKGY ~ Poor:lO.pKGY
Sp None - None G Abundant Good Abundan~
~PO R 72.d.0Y 54 . brO 7 5 . deep yBr Am ~bun:63.1.brGY None Abun:63.1.brGY
Sp None None None G = growlh R - reverse ~m - aerial mycelia Sp = soluble pignient ~-5953A -7-A comparison ~f the carbon utilization pat-terns o culture A41030, of culture A410300 4, and of Streptomyces ~rirginiae A~CC l9al7 was conducted using ISP No. 9 basal medi~n to which filter-steriii~ed 5 carbon sources were added ~o ~qual a f inal concen ~
txa~ion o~ 1. 0% . P1 ~s were read a.~ter ourteen days incubation at 30C. The results are set forth ir.
Table 2, which follows:

1~ .

~ .

2~

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~-5953~ -8-TABL~ 2 CARBON UTILIZ.~TION PATTERNS OF A41030, OF
A410~0~4, ~ND STREPTOMYCES VI~GINIAE ATCC 19817 5Carbon Source A41030 A41030.4 ATCC 1981'7 ~cetate-Na ~ - -D-Arabinose - - -L-Arabinose - - -Cellobiose ~ D-~ructose +
D-Gala~tose + ~ _ D~~lucose i-Inositol ~ - -Lactose D-.~altos2 ' D-~annitol Melibiose ~af~inose - - -Rhamnose - - -20 D_~ibose Salicin ~ + +
Succinate-Na + ~ ~
Sucrose _ _ D-Xylose ~ _ R.ev: - - no utili~ation a utilizatio~
~ a parti~l utiliza~ion 31) ~-5g53~

Cell wall composition Usirlg hydroly~ed whole oell5 of the novel microorganisms, ~he isomers of diaminoplmelic acid were determined according to the method of Becker et al~, ~ . Microbiol. ll, 421-423 (1964). The results of this study are set forth below.
T _ R2sult Obser~ed ~somers of 2,6- LL i~omer 1~ diaminopimelic acid The similaritles and di~ferences of culture A41030 and culture A~1030O ~ ~ as compared to Strepto-myceq virginiae ATCC 19817, are set forth in Table 3, ~_..
which follows. Color names are assi~ned as described above, and the morphology as set forth in Pridh~ et al~ su~ra.
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i X~$353A -10 rJo rJ~ u.i t~,~
r~ ~
~ ~ ~ O ~ r~
V ~ ~ I + + ~ + I ~ I r~ U~ i + + I ~ ~ I I
~ R ~n c r- ~ -- C ) rj c~ ~

C~
O E~~r ~r t~. o a~
ar~
r; o o ~ + I I I ~ i o ~ I I ~ ~ I O O I o ~ t~ r~~:
' J 1) t~
IX rl a O
~ ~ s . r~
O ~ + + ~ + ~ + ~ + + I C 1 , `~ P; S, o s:
r S O r~i r ~

~! S ~ O ;) r O O. r~
~ ~ . r 1~

C C l O ~t) r ' U~ I) -O ~ /~ J
51 t,~ r ~ ~ tJ r I ~ 1'~ ~ ~ ~ ~ ;
U ~ ~ h " 3 t) o r~ r ~ o ~ o ~ ~ o ~ r~
r ~ C~ ~ r 1 ~ Z ,~ ~ 5~ O
S l ~ I ~ ~ ~ rJ r ~ rJ~ r.~ ,~, C4 n ~ o r~
~ rn ~ O ~ n r ~ . ri I--l ~ O ~ J rn Ei r~ rl r l O O r~
~1) i~ ~ il o ,;~ .~, n ~J ~, O ~ ~ ~ E-'~ ~ O ~ 'J~ rJ~ rQ tn U~

The antibiotic substances of this invention are arbikrarily designated herein as A41030 an~ibiotics. The A41030 complex con~ains several individual factors which are designated A41030 factors At B~ C, ~, E, F, and G. In discussions of utility, the term "A41030 antibiotic" will be used~ for the sake of brevity, to denote a member selected from the group consisting of A41030 complex, and A41030 factors ~, Br C, D, E, ~, and G.
The seven antibiotic factors are recovered from the fermentation and are ohtained as a mixture, the A41030 complex. It will be recognized that the ratio of the factors in the A41030 complex will vary, depending upon the fermen~ation conditions used. The individual factors A, B, C, D, E, F, and G are separated and isolated as individual compounds, as hereinafter described. The A41030 complex is soluble in water, dilute aqueous acid, dilute aqueous base, methanol-water mixtures, ethanol-water mixtures, dimethylformamide and dimethylformamide-water mixtures, dimethylsulfoxide, dimethylsulfoxide-water mixtures, acetonitrile, acetone, ethyl acetate, tetra-hydrofuran, methylene chloridet and khe like.
The following paragraphs describe the physical and spectral properties of the A41030 factors which have thus far been characterized.

.

X-5953~ 12-A41030 FACl'OR A
Antibiotic A41030 factor A is a white, crystalline solid. Elemental analysis of A41030 factor A lndic?tes that it ha~ the following approx-imate percentage composition: 56.44~ carbon, 3.58~, hydrogen, 8.11~ nitrogenl 23.20~ oxygen, and 8.~9~
chlorine. A~ det~rmined by field desorp~ion and plasma desorption mass spectrometry, A41030 f~ctor A h~s a mol~cular weight of 1231. Bas~d on the ~lemental analysis and the m~lecular weight, an empirical ~ormula o C58H~C13N7O18 is assigned to factor A. Elec~ro-metric titration of factor A in 66% dimethyl~orma~ide in water indicated the presence of three titratable ~ groups ha~ing PKa values of about 5.53, 7.~0 a~d 10.37, ~with possikl~ additional ?Ka's ~ 10.5 ~ini~ial pH
7.83). Antibiotic A41030 factor A has the followin~
sp~cifi~ rotation: ~a~25 -19.6 (c, 9.0 i~ dlmethy~.-sulfoxide).
The infrared a~sorption spectrum o A41030 actor A i~ XBr pellPt is shown in the accompanying drawings as ~ig. 1. The ollowing distinguis~.able - a~sorption ma~ima are observed: 344~-322O (strong, broad3, 1653 (strong), 1610 (weak), 1587 (medium), 1515 ~strong), 1488 (~eak), 14~9 (medium), 1227 (strong), 1139 ~medlum), 1064 (st~ong), and 1010 (strong) cm ~.
The ul~raviolet absorp~lon maxima of A41030 factor A i~ methanol.water (1:1) under acid, neutral, and basic condit.ions are ~eeorded in Ta'~le 4.
3~

~-5953A -13-Antibiot~c A41030 factor A i~ soluble in alcohol-wat~r mixtures, in dimethylsulo~ide, in dimethylformamide, in dimethylsulfoxide~water mixtures, in dimethylfGrmamide-water mi.x~ures, in ~ilute aqueous acid, and in dilute aqueous base.
On the ~asis of the observed physical chem-ical data, th~ following s~ruc~ure has been asslgned to A41030 factor A. QH

f ~ / \CI `~ / C 1/ ~ / \CH~

UO-C .H fH ~ CH

h~ OH .~ hO ~

Usir.g ~ biolo~ical assay a~.d high performance liqui~ c~omatog;-aoh~i anal~sls, lt has beon found that ~acto~ A accounls for from about ~4 to abou~ 96~ by t~lght of th~ antl~ otiC ~actG-5 produc~d by culture 3~ ~'030.4, -~lrr~ fac~ors 3, C, D, E, F, and G accoun~in~
i-.or the r~maining abollt ~ to about 6~-~ b~,r weigh~ o the .ac~.ors producsd.

X-5953A ~14-A41030 FACTOR a Antibiotic A~1030 factor B i~ a white solid, having a~l approximate elemental analysis as follows:
58.54~ carbon/ ~.21~ hydrogan, 8.63~ nitrogen, 5.95~
chlorine, and by di:Eerence, :22.66% oxy~en. Electro-metric titration o~ ~actor B in 66% dimethylormamide in wat~r indicated the pr~encQ of two titra-table groups at PKa valuP~ ~f about 5~6 and 7.5~ respectivel~
with possibly additional pKa' >10 (inltial pH 6~2~.
An ~bserved ~.olecular weight of about 1197 was obtained using fast atom ~ombardment mas3 spectrometry. Ba~ed on ale~ental analysis and the observed molecular weigi~t, an empiriGal fonmula of C58H45C1~7018 is a~signed ~o fackor ~. ~
~ he infrared absorption sp'ectrum of anti-~iotic ~1030 factor B in KBr pellet is sho-~n in the accompan~ing drawing~ a~ Fig. 2. The follGwing dis-tinguishable absoxption maxima are observed: 3'48-3226 (strong, ~road), 1653 (strong), 1610 (medium), 15$7 (weak), 1515 (strong~, 14~8 (weak), 14~9 (medium), 1290 (weak), 12~7 (stron~), 1139 (mQdium)~ 10~4 ~st~ona~ r and 1010 (strong) cm 1.
The ul~raviolet absorption ~im~ of A41030 factor B in nautr~l, acidic, and basic methanol:water (1:1) are re~orded in Table ~.
Antibi~tic A41030 factor 3 is soluble i~ the S~mQ solvents as acto~ A.
On tn~ ~3S' s of t:r.e obs~vea ph~sical chemlcal 3~ data, ~he followi~g structure has been assigned to A41030 fa_tor B~

X-5953 -15~

~1 T~ '3~ I~`~/ \l `

,~ ~ H f f ~ ~ H~

15 H~ ~/ OH ~h' A41030 ~ACTO~ C
Antibioti~ A41030 factor C is a white solid having an approximate elemental analysis as ~oilows:
48.87~ carbon, 4~39s hydroge~, 6~16~ nltrogen, 6.96%
chlorlne t and 33.81~ ox~gen. Electrometric titration of factor C in 66~ dimethylformamid2 in water indic~ted ~S the pres~nce o:E ~o titratable sroups at PKa values of about 5.~ ~nd 7.1, r~spectively~ wlth ~ossiblv addi-tional pRa~s ~10 ~ini~ial pH 6.5). An observed molecular weight of about 1393 was obtained u5ins fas~ atom oom~a~-dment mass spectrometry. Based sn elem~tal 3~ analysis and t;.e ob~er~ed mole~ular weight, an empirical o~ c6~4cl3~37o23 iS assign~d to fac, or C
.

~-59S3A ~16-The inrrared absorption spectrum of anti-biotic P~dlO30 factor C in KBr pellet is shown in the accompanying drawir~gs as Fig. 3~ The rollowing dis tinguishable absorptiorl maxima are ob.ser~ad: 3448-3~26 (strong , broad), 1653 (strons), 1610 (medium~, 1S87 (wea3c~, 1504 (strong) ~ 1481 ~weak~, 142g ~n~edium~, 1220 (strong), 1136 (strons), 10~4 (weak), 1053 (medium), and 10 0 5 ( s trony ) cm The ultraviolet abso:rption maxima of A41030 ractor C iIl neutral, acidic, and basic metharlol :wa~er (1 :1) ar~ rf~corded in Tabl~ 4 .
Antibiotic A~1030 factor C is soluble in the same ~olvents as factor A.
on the basis of the. o}:ser~ed ph~sical chem-ical data, the structure o~ A41~30 fac~or C is believed to ~e as follows~

q \f`i~ IH
H'~'~CH
~ I t Ha ~

3 HC/ ~ `Oh ~H 33 1 3CT05~3 ~-5953A -17-A~1030 FACTOR D
Antibiotic A41~30 falctor D is a white, amor-phous solid having an approximate elemental analysis a follows: 54.46% carbon, ~.35~i hydrogen, 7.58% nitrogen,

4.~7% chlorina, and by ~ifference, 29.34% oxygen.
Elsctrometric titr~tion of rac-tor D in 66~ dimethyl-form~nide in water indicated tha presence of two titratable groups at P7~a values of about 5.5 and 7.6, xes2ectively, with possibl~ additional pK 's ~10 10 (initial p~ 6.~3)~ An observed molecular weight of about 1326 wa5 obtained using fast atom bom~ard~ent mass spectrometxy.
The infrared absorption spectrum, of anti-biotic ~41030 factor D in K~r pellet is shown in ~e accompanylng drawings as Fis. ~. The ollowing dis--tinguisha~le absorptien maxima are observed: 3~48-3226 (strong, bro~d), 2959 (weak), 16~1 (strong3, 1592 (strong), 1511 (strong), 1~9 (wea}c), lZ90 (weaX~, 1227 (weak), 1212 (~edi~), 1163 ~weak), 1143 (wea]c), 1053 (medium), a~d 1010 (strong) cm 1.
The ultravlolet absorption maxima o~ A41030 factor D in neutral, acidic, and basic methanol:wa~er are recoxded in Ta~le ~.
An~ibiotic A~1030 .~actor ~ is soluble in the sama sol~ants as factor A.
On the bas.~s or the o~served ~hysical che~-ical data, the struc~ur9 c- ~11030 ractor ~ is believed -to be as rollows .5~53A ~18-QH

l~ 0~ ~ ~C~O ~ ~

H ~H ~ ,~ \ N'rl2 f 3 Ha ~

plus one or more n butyl groups.

Ant~biotic A41030 Eactor E ls a w~ite solid ha~ing a~ ~pproximate elemental analysis as follows:

5~.06~ carbon, 4.06~ hydrogen, 8.53~ nitrogen, 3.50%
chloxi~e, and by diffarence, 27.35~ oxygQn. ~lectro-m~tric ~ltration o actor ~ ~n 56~ dimethyl,orm~midQ
~5 ln water i~lcated the preser.ce of t~o titrata~le g~oups at ~Ka values o~ about ~.8 an~ 7.7~ respec-tiv~ly, wit:n possibly additional pKa's >10 (i~itial pH

6.57). A~ obs~r~Jed molecular we ght of about 11~3 was o~t~i~e~ uslng 'ast atom ~ombar~ment mass s~ec~xometry.
~ ~en.atl~J~ empi_ic~l ~~ormula o~ Cj8H~5Ci~7Ol~ is as~igned to CaC~O~ ~.

X-5953~ ~19-The infrared absorpt:ion spectrum of anti biotic A41030 factor E in ~Br pellet is shown in the accompanying drawings as Fig. 5. The ollowing dis-tinguishable absorp~ion maxima are observed: 3448-3226 (.~trong, broad), 1653 (st:rong), 1600 (medium), 1504 (strong), 1429 (weaX), 1290 (weak), 1198 (medium), 1136 (WQa~) ~ 1064 ~weak), and 1010 (strong) cm 1.
The ultra~iolet absorption maxim~ o~ A41030 factor E in neutral, acidic r and basic methanol:~ater (1:1) are recorded in Table 4.
~ ntibiotic A41030 fac~or E .is solu~le in the same sol~ire~ts as fac~or A.
On the basis of ~he obser~ired physical chem-ical data, the ~ollowing structu~e has be~n assigned to A41030.Lactor E.
4/Q\f~, ~J/ \CI ~ \C~H~
~ IIH '\`C ~' 2~ ~C~ fH~ 2 \0/ ~ I

. 3~ ' b~

Antibiotic A41030 factor F is a white solid hav-ing an approximate elemental analysis as follows: 51.39 carbon; 3.96~ hydrogen~ 6.45% chlorine, 6.45% nitrogen, and 28.65% oxygenS Electrometric titration of factor F in 66~ dimethylformamide in water indicated the presence of two titratable groups at PKa values of about 5.4 and

7.1t respectively, with possibly additional pKa's >10 ~initial pH 5.93). An observed molecular weight of about 1555 was obtained using fast atom bombardment mass spectrometry. A tentative empirical formula of C70H64C13N7O28 is assigned to factor Fo The molecular weight data suggest that factor F
differs from factor A by the addition of a disaccharide substituent comprised of two galactose moieties attached through the phenol group corresponding to the phenol group of factor C to which phenol group a single galactose moiety is attached.
The infrared absorption spectrum of antibiotic A41030 ~actor F in KBr pellet is shown in the accompanying drawings as Fig. 6~ The following distinguishable absorp-tion maxima are observed: 3448-3226 (strong, broad), 1653 (strong), 1600 ~medium), 1504 (strong), 1429 (weak), 1258 (weak), 1227 tstrong), 1136 (strong), lQ75 (strong), 1053 (strong), and 1010 (strong) cm 1.
The ultraviolet absorption maxima of A41030 factor F in neutral, acidic, and basic methanol:water (1~1) are recorded in Table 4.
Antibiotic A41030 factor F is soluble in the same solvents as factor A.

,~

On the basis of the observed physical chelni~
cal data, the following structure has 'oeen assigned to A41030 Factor F:

~H

f; ~, \C I ~ , C ~ CH2 lfO ~\C ~ ~ \C~j~ C

~o~ f ~ ~ NH~

HO~ ~9/ OH ~5H 5a 1 ~ct osy 1 ~'a I ac-'os ~21-Antibiotic A41030 factor G is a white solid hav-ing an approximate elemental analysis as follows: 50.02%
carbon, 4.61~ hydrogen, 4.74~ chlorine, 6.11% nitrogen, and 30.70~ oxygen. Electrometric titration of factor G in 66%
dimethylformamide in water indicated the presence of titra~
table groups at PKa values of about 5.4 and 7.0, respec-tively, with possi~ly~additional pKa's >10.5 ~initial pH
6.3~. An observed molecular weight of about 1684 was obtained using fast atom bombardment mass spectrometry.
Factor G has a disaccharide substituent comprised of two galactose moieties attached through a phenol group in the same manner and in the same location in the molecule as de~cribed above for factor F~
Factor G also has two equivalent n-butyl groups attached to the nucleus at an as yet undetermined loca~ion.
A tentative empirical formula o~ C7~183C13N8O
is assigned to factor G.
The infrared absorption spectrum of antibiotic A41030 factor G in KBr pellet is shown in the accompanying drawings as Fig. 7. The following distinguishable absorp-tion maxima are observed~ 3320 (very broad, strong) r 2975 (sharp, weak), 2920 (sharp, weak3, 1659 (normal, strong), 1594 (broad, strong), 1512 (sharp, strong) t 1492 (shoulder), 1430 (sharp, weak), 1386 tbroad, weak), 1337 (broad, weak), 1308 (sharp, weak)~ 1264 Isharp, weak), 1230 ~broad, mediumj, 1145 (broad, medium), 1077 ~sharp~
medium), 1062 (sharp, medium), 1014 (sharp, medium), and 846 (broad, medium) cm 1.

X-59S3~ -2~-The ultraviolet absoxp~ion rlAx;m;~ of A41030 Eactor G in neutral, acidic, and basic methanol :water (1:1) axe recorded in Table ~L.
Anti~iotic A41030 f.actor G is soluble i n the 5 same solvents as is factor Ac The ultraviol~t absorption charactexi ~ics of the A41030 factors are presented in the following Ta}: le 4 .
TABI,E 4 1~
UV Spectropho1:ometr~f oE A41030 E~actoxs Factor Acidic or Neu~ralBasic:
max nm ( c )max nm ( c ) t~S r 278 (11~100) 298 (17~200) ~3 ~!7~ 600) . 298 ~16~800) C 27~3 ~8~400) ~!98 114~000~
1:) 27~3 (10,60~) 298 (19~900) E 278 (8"00) 298 (15,500) F ~78 (9,3~0) ~8 (7L4,5~0) G 278 (15,000) 2g8 (la,000) Factors A, ~, C, D, E, ~, and G or t:~le ~41030 complex can be separated and distinguished from one ano~her by employirl~ silica-gel thin~ er ch.romatog-~5 raphy (TLC) and paper chroina.ography ~ i3acill us su~ a~ the organism used or t;~e bi oautography.
The ratio of movemerl~ (Rx~, expre~sacL relative to that OL A4].030 factcr A, which was ~i~ren a value of 1. 00, is set forth in Table S, which îollows.

~.

X-5953A o23 Rx Solvent System Factor A B
A 1~ 00 1. ûO
:13 0.76 0.75 .
C 0 . 6 8 0 ~
D 0. ~5 û. 91 E 0049 0.63 lQ F 0.21 0.2 G 0.21 0. 2S
Sy~t~m A
Paper : W;hatman No. 1 (untreate~).
Sol-~2~t: n-~uta~nol saturated with w~tex:methanol ( 1 System B
Sorbent: Me~ck~Darmstadt Silica Gel 60.
Sol~ent: Acetonitrile:ethanol:w2te~ (8 1 lo ~) o 2~ The high perfor~ance li~id chromatography (HPIC) retentlon tim2s o ~1030 'actors ~ th:rcu~h G, inclusi~e, were de~er:~nined using a st2inless steel colum~ havin~ 10 micron LiChrosorb ~P-18 as th~ packing, with a sol~ent consisting or wat~r:acetonitrile-dibutyl-~
amine (82:13:0.03~1) adjusted to pH 2.5 with phosphoric ac~d. ~he solvent was applied at a 'low rate o~
0.75 ml./minO The eluate was monitored b~ UV absorption at 2?.5 nm. T;~e relative re~en~ion values, which are the ratio o~ t~e retention ti~e for each factor relative to that or AdlO30 fact~r ~, are set forth in Table o, ;~hich ~vllows.

X-5953A -2~--Relative Factor Cm.M . Retention A 6.4l9o 2 1~ 00 B 4.112.3 0.64 C 5.416.2 0.84 D 3.811.4 0.59 E ~.78.1 OD42 F 4.513.5 0.70 1~ G 4.513.5 0.70 Since the several factors of antiblotic A41G30 are amphoteric, containing both an amino group and a carboxyl-,c acid runction, they are capable or formin~ salts r~Jith suitable acids,and bases. ~he 1~ pharmaceutically acceptable salts so formed are a]so part of this in~ention. "Pharmaceutically-acceptable"
salts are salts which are useful in the chemotherapy of warm-blood~d animals. Representative and suitable salts of A41030 factors A, B, C, D, E, F, and G include those acid acldition salts rormed ~y standard reaction with both oryanic and inorganic acid~s such as, for example, sulfuric, phosphoric, hydrochloric, acetic, succinic, citric, lactic, maleic, fumaric, p~lmitic, cholic, pamoic, mucic, D-glutamic, d~camphoric, glu-taric, glycolic, ~hthalic, tartaric, lauric, stearic, sallcycliG, methanesulfonic~ benzenesulfonic, sorbic, picric, benzoic, cinnamic and like acidsl as well as salts Eormed with the carboxylic acid function with such bas~s as sodium hydroxide, sodium carbonate, 3~

~-5953~ -~5-potassium carbonate, calcium hydro~ide/ potassium hydroxide, trimethyl~mine, ~Immonium hydroxide, di-e~llanolamine, and like bases.
Antibiotic A41030 co~plex and factoxs are 5 active against gxam positiv~ microorganism~, including Staphylococcus a~ Streptococcus.species. Thes~ anti-biotics al50 show ac~ivity for ~xowth promotion and improving feed ~ff icient y in poultry, swine, and catti~
The in vit~o an~imi.crobial activity of the A41030 comple~ and the individual ~ctors has been demonstrated by a n~mbar of tests which are described hereina~ter.
AGAR-DILUTION AS5AY PROCEDU~

Th~ agar-dilution procedure described b~ the Internatiollal Collaborative study (ICS) group ~as u~ed to de~ermine the m~nimum inhibitory concentrations IC's).
The results obtained from tests of anti~iotic 2~ A41030 factor A in th~ agar-dilution assay procedure zre gi~n in Table 7, w~ich follows~
TA~LE 7 ACTIVIT~- OF A~1030 FACTOR A
~IC -~
Test Organism (~Ig~mi~) Sta~h~lococcus aure1~s 30~5* ~0.

Staphylococcl~s aureus 307~** ~0~-Streptococcus ~aecalis X~ ~0.5 3~ *~en.~.Ylpenicillin-suscQ~ti~ie ~en2ylpenicillin-r~si~ani X-5953~ -26~

DISC-PLATE SENSITIVITY PROCEDURE
Agar platesf inoculate~ with ~he t~s~ organism, were used; 6 mm. disc~ (0.02 ml. capacity) were saturAted from log ~ dilutio~s of the antibiotic solution~ Disc 5 content was 1/5 or l/SOth of t:~e concentra~ion of th~
solution used, iOe.l disc content of 100 ~g. or 10 ~g.
obtained from a solution o 500 ~g.~mlO concent~ation.
Tha siz~ of the zon~ of i~hibition produced by the A41030 factor A a~tibiotic for each disc content is 1~ reported in Ta~le 8, which follo~sO

ACTIVITY OF A41030 F~CTOR A
Zone aiameter (mm) (at ~Ig~/disc~
Test Org~ni.sm - 100 10 Sta~hylococcus aureus 3055* 20.8 17.2 Staphylococcu5 aureus 3074** 19.~ 16.7 Staphylococcus aureus 3130**~ 21.4 17.4 Streptocsccus pyogenes C203 15.0 12.0 St~eDtococcus s (Group D) 9960 ~0~5 16.4 S~.reptococcus pneumoniae P~r~ I 17.0 15.0 Escherichia coli EC14 8.0 0 *benzyl~enicilli~-susc~ptible ~-**~enzylpeniciliin-resistant *~benzylpenicillin-resi3tant, mQthicillin-resistant 3~

~5953~ -27-Antibiotic .A41030 factor A ha~ showm activity against a num~er o~ s~rains Q~ Hemophilus inf.luenza~, a~ determi~ed b~ the aga.r-~ilu~ion method. The results of the tests are recorded in Table 9, which follows~

ACTIVITY OF A4103û FACTOR A A&~I~ST
HEMOPHILUS INFLUEMZAE ST~INS
Hemophilus ~IIC
10influer~za~ (ug~/ml. ) ~272 16 ~274 16

8 4 2 7 5-9~3 23 16 P. Wylie 16 G . Newton 3 2 Bruno 1 6 S. Ford 16 A. ~all 16 C. S~eele 16 Miller 16 ~oward 1 6 ~5-312 8 75 313 lG

75-46a 3 3a Antibiotic A41030 fac'cox A is active against Neis~eria ~, as determined ~y ~ha agar-dilution method. The t~st results are recorde~ in Table 10, which ollowr~.
s AGAIN~T NEISSERIA S

~ MIC
Nelsseria sp. (~g./ml.) L. Na~ce ~.0 Woods 4-0 Schultz 8.0 ~litchell . 4.0 4 Sandex~ 1.0 The activlty of antibiotic ~41030 accor A
aga1~nst a vari~ty of ~acterla, as determin~d b~ ~he agar~dilution method~ i5 reported in Table ll, which ollows, TA~LE ll ~CTI~JITY OF A41030 FACTOR A
AGAI~T A VARIET~ OF BACTERIA
: Baoteria ~5 Staph~lococcus aureus ;~lIC ~u~ nl.) 30~,1 0.13 307~* <0.06 3131** C0.05 -3~ 313~** <0O06 H43* <0.05 ; ~7S,* <~.06 7, O ~ O . O ~i ~-5953A -29--TABLE 11 continued ~acteria MIC ~g . /ml . ) Streptococcus pyogerles C203 0 . 25 99~ 0. 25 1038~ 0. 25 12344 0. ~
M-6517 0. 25 10 Str~ptococcus pneumoniae Par}; I 0 . 25 T~pe 1~ 0. 25 27~ 0. 25 6301 0. 25 BI~343 0. 25 Staphylococcus epiderrni~is LittoIl 0. 13 Mencher <0. 0~
Britton 0.13 ~obley ~ 0 . 06 irid.ans stxeptococcus S~1~1134 0. S
SSI-glO o . ?5 SSII 895 0. 25 streptococcuC sp. ~Grou~
23~ 0 . ~5 2~2 0 . 2S
gclol 0. 2 12253F 0. 25 Guze 0. :25 ~9~z~

~-5953A -30-TA~LE 11 continued ~actexia MIC (~g./ml.) Shisella flexneri *Penicillin xesistant **Penicillin, met~icillin, and erythromycin resistant 1~ The name or nu~bex under the named oxganism ls the strain designation.
The A41030 antibiocics, factors A, B, and C, ~re acti~Je against a genus o anaerobic bacteria iden-tified as Bacter~ides sp., tne 24-hour MIC values being 15 deter~ined by the aga~dilution method, and are se c forth in Table 12, which follows.

3~

~-5953~ -31-AGAINST BACTEROIDES S~.
MIC (~Ig./ml.) Test Organism ~ B C
B. fragilis 187~ 32 32 32 B. fragilis 103 32 32 32 B. ~ragilis 104 32 32 32 B. fragilis 106 64 32 ~2 __ B. frasilis 107 32 32 32 B. fra~ills 108, 32 32 64 B. fragilis 110 64 64 64 ~B. ~ragilis 111 . 3~ 32 32 ~. fra~ilis 11~ 6~ 32 32 , B. fragilis 113 32 32 32 B. fragills 1451 64 64 6 _ ~ragllis 1470 6~ 64 ~4 B. fragilis 2 64 32 32 ~n ~
B. fragilis 9 64 32 32 3. fragilis 9032 ~4 ~2 32 B. corrodens 1874 32 32 32 __ B vulgatis 1563 3~ 32 32 B. thetaiotaomicron 1438 64 32 32 3. thetaictao~icron 1900A 12~ 1~8 128 ~ ibiotic A~1030 factors A, ~ ~nd C ha~e al~o been test d and foun~ to be activ~ against a senus o anaerobic bacteria identified as Pro~ioni~acterium ~cnes. ~he .~rc valu~s were determined by the 24-hour ~gar-dilution method, and a~e set -_orth in Table 13, , ~

TABLE :L3 AG~INST PROPIONIBACTERIUM ACN~S

` Strain of MIC (~g./ml.3 P. acnes A B C
44 0.1~5 0.06 0.125 79 0.1~5 0.06 0~125 101 0.125 0.06 0~125 103 0.1~5 0.06 0~125 104 0.2S 0.25 0.25 105 0.125 0.06 0.125 106 0.125 0.06 0.125 lQ~. 0.0~ 0.0~ 0.125 lS 529~ 0.06 0.06 0.06 5170 <0.03 <0.03 <0.03 5176 ~0.03 0.06 <0.03 5187 ~0.03 0.06 0.C~
51gl 0.125 0.06 0.125 ~Q 5197 ~.03 0.05 <0.03 5226 0.5 0.5 0.1~5 ~2~7 _0.03 0.06 0.06 522~ 1.0 0.5 1.0 522~ 0.~ 0.25 0.5 -.
52~6 0.06 0.125 0.0~
An~ibiotic A41030 f~cto~s A, 3, C, D, E, ~, and G have been tested and round to be acti~Je agains~ a nl~m~er of anaerobic bzcteria, a~ recorded in Ta~le 14, which ollows r the ~lIC ~alues having been determined b~
the ac;ar diiu~ion ~ hod.

o In o , . ~ ~ o ~ ~ ~ ~ ~ ~ o:
V I ~ ~ ~ ~9 ~ ~ ~D

L~
oooo~ oo o oo o .~ J ~-1 ~4 V I (~) ~) ~) ~) (r) ~D ~ ~1 t~
A

Ll~
o u~
o O, r~ O O O ~1 ~ ~ O ~ ~ ~ ~ ~ N ~1 d ' H 0 ` V I ~ ~\ /~) ~ ~ ~D ~ ~I ~ ~ ~
a ~_ ~ u~ In 15 ......... -~ ~ O O O O O O O ~ O ~ ~ ~ ~ ~ O ~ ~ O O
_, V I ~ r~ D (~ ~ V I
C~
C H ~) Ll~
R ~ o V
O O O O o o~ ~7 ~ ~ CO
a~ ~ vl vl vl vl A ~ /~
Cl:
Lr~ ~ In o In . . . . .
CO O ~ O O ~D ~ ~ ~ ~ C~ ~ ~ ~ 1--l CO
:~ ~ m ~ o ~ ~ vl vl v~
r~ A
U~
~) M Ln ~ u~
~1 ~ ~ o ~ o ~ o~ o o o ~ o co O ~ ~ ~ ~ ~ V I V I V I V I ~ ~D
^
~r ~ oo O ~ ~ ~ ) o ~r ~ ~ ~D
~ u~
> u~
o v u~
u ~ ~ ~ o~ <~ a u~ ~ E c L ~ ~ ~ a) ~1 ~1 ~1 0 ~1~ 5 0 O ~ ~ U Ul '' ~ C~ L C rl j U~ U~ U~ U G ~ u~ - O
U~ ~ Ll ~_l ~ C O ~ F ,1 ~ O F
~-I ~ ~ JJ C C~ ~ J _1 3 c~ c-, c~ ~ ~ b~
c ~ ~~ ~ a~ a a) ~ ~ ~ ~ u~ ~:
QJ U. ~ J _~ ~ ~ Ll 5~ O
c ~ ~ 3 J a) ~1 ~1 ~1 ~ O O
o e ~ E ~ u l v ~ ~ u~ u~ u~ u~
P Q~ al L Ll O O O O O O O O U O
E-~ Ll ~ Ll ~ '.3 J U~ U~ O ~ ~ ~ L I Ll ~; ~
~ ~ ~ v o C o o ~ a) ~ a a a, a a ~ ~ ~
.rl u~ Q~ C ~) I O O O ~ ~ O ~ O ~ C~ r~) U U~
P~ m m ~ L r~ P~ m ~ ~

9S3.~ 3~

~ ntibiotic A41030 factors ~, B, and C ha~e shown acti~ity again~t a num~er o. species o' two genara oE anaexobic cocci id2ntified as Peptococcus and Peptostreptococcus, respectively. The MIC values were Z~termined by the agar~dilution ~e~hod, and are re-cord~d in Table 15, which follows.

1~
.

Y~--5953A --350 Lr L~ Ln U Ln ~ Ln U~
~ o Ln ~ ~ ~ o Ln o ~ ~ ~ O
~ o O r--i O G O O rJI O r,--I O ~ O O C:~ O
f.. V I

Ln Ln ~
~ ~ r~J Ln Ln Ln ~n ~ Ln Ln Lr) 1~ rJ~ ~ (~ '~) C`~ L~ ~J L~) ~ O r~ r,~J
O C:l O O C:~ ~ O O C~ O O O O C:~ O
_ L''l ~n y ~ r1 c~ ~ o ~n ~ ~ ~, Ln u~ o o u~ Ln Lr~ o n V .... ~ ........ .
C) o O C~l r~ l O C _I ~ O O O O ~a C
~, C.) vl `~I
~) o C rJI ~1 :~
~ O ~Z;
r,: r~l~ _~
,~r~
I~

U
_) ~ O
C;~ r-l C r--l 4 ~r ~~1 ~; u ~ ~t r~ U~ U r~~
e ~ J r~ O U~
O r~ O ~ J. .~' ~J ul ;r t~ ~~ rc ~r ~ ~ ~r J r~ '~ ~ J
r; ~r-- r~ l r--i ~ r~ ~ r ~
CJ ~r; r r~ ,5 ~ r ,~ S
tJ .S~ J ~ 0 C 0 0 C
J~J r' t, J~ r~ ~ ~ ri S~ S ~- S
rn a, t, r" ~ S~ a Q Q a Q~ r~ ~ S~ 3 V ;3 S' ~ t r r.l 1~ U. t~ r~. S _ S: S r~
rc V ~ 5 ~ ~ r~ r; fr. ~, r I ~
~ 4 ~ G ~ ~ ¦ G l r~

w ~ 1~

TABI.E 15, continued MIC (~g./ml.) Tes~ Org~nism A B C
l~sanaero~ius 1477 G.5 0.125 0~25 PS. inter~edius 1264 c0.03 ~0~03c0.03 l~s~ inteLIriedius 1524 0.5 0~250.5 Ps. illter~edius 1624 1.0 0.5 1~0 ~C = Peptococcus ~s - Pep~ostreptococcus ~ t .

The A41030 antl~ioti.c factors A, B~ C, D, E, F, and G, are also active agai.nst a rlumber of stxairls of Clostrldiurn difficile, as c'ete:rmined l~y the agar-dilution method~ The results of t}le tests are recorde~
in Table 16 j which follow~.

ACTIVITY OF A4103G FACTORS AG~I~S~
CLOSTRIDIIJM DIFFICILE STR~
Closl:rid~llm MIC (~g. /ml . ) .~d:lfficile A 3 C D F F G
__ _ _ .
8484 1.0 1.0 1.0<0.25 0.5 1.0 l~d 6~0 1.0 1.0 2 0.5 0.5 1.0 1.0 2634 1.0 1.0 2 0.5 1.0 2 i.O
73 1.0 0.5 1.0<0.?5 0.5 l.C 1.0 15~-194 1.0 1.5 ~ <0.2~ 0.5 1.0 1.0 ~-195 1.0 1.0 1.0<0.~5 0.5 1.0 1.0 A-1~6 1.0 l,O 2 0.5 1.0 2 1.0 A-27~ 1.0 1.0 2 <0.25 0.5 1.0 1.0 A 2 80 1 . O O . 5 1 . O ~ O . 2~ 0 .; 1 . 0 ~ . 0 .4-281 l.O 1.0 ~ 0.5 1.0 2 1.0 T.~AL--~112 1. 01 . O <O . 25 0 . 5 1. 0 1. 0 WAL~3657 1.0 1.0 2 ~0.25 O. 5 1.0 1.0 WAL-4268 1.0 0.5 1.0<0.25 0.5 1.0 1.0 2~107B 1~0 0.5 1.0~0.25 0.5 1.0 1.0 lllF l.O 1.0 2 ~0.25 O.S 7 l.O
1153 1.0 loO 2 l~O 1~0 ~\ 1.0 3h24-53 1.0 l.O 1.0 O.S 0.5 l.G 1.0 33'~ ~.0 1.0 2 0.j 0.5 1.0 1.0 ~03950D '.0 1.0 2 0.5 0.~ 1.0 1.0 The in vitro acti~it~ of antibiotic A41030 factors A, B, C, D, E, F, ~nd G against a number of aexobic bac texla has been determin~d u~ing a standard agar~dilution assay . The results af ter reading the 5 end point a~ter 24 ~ours are recorded in Table 17 f which f ollows .

1~

~5 O ~ N ~ o O ~I N ~ ~ I I N
tO I i ~) t~) N N N
A A A A
N
O O ~ ~1 0 N N ~ ~ ~ I I N N ~ OC) ~
I I r~ ') N t'l (~ N

U~
~! ~ ~ N ~
o o o o o ~ o o ~1 ~1 1 1 oo oo c~ ~o co a ~ I I ~ N

NN N N ~ 11~ LO
~ .. o O ~ O r~ O O ~ ~ I I 0~ G~ Ct) C~ 0~ ~
I I N N N ~9 a: r-l ~1 ~1 A A A
C.) H

P o o ~o o ~ ~ ~ ~ ~ ~D ~r I I co ~ a~ o~
I I N ~ N N
C ~ ~
U
r~ N ~I r~ 1 N Lll o ¢ O O O O O O O O r-l ~I CO N I I CO CC~ CO a:
I I N N N N
rn ~1 u~
N N 1~ N
o O O O ~1 0 0 r-I N cr~ N I I CO CX) ~
I I N N N
O I~
A A A
o ~ ~ ~ O
> ~ Y ~ ~D
~ ~ X Ci~
O ~ ~ r I ~ N C) r~
e ~ U ~ a, a~
U~ 0 U~ a~ o o ~ ~ ~ ~r r- O)~ N O
Q~ .. ~
C. Q Q ~ 1 ~ ~-1 C - ) ! ~ U~U~~ ~ ~1 ~~ 1 ~_1 ~' rc~'~J ~~7, U U ~ 0 O O
u ~ ~,~
~ J J 'J : 3 : ~
Ot~cJr~ W ~ 7J 1,) J rJ_~0 0 0 0 C
J ~ ~ 3 ~I n J ~ J _~_ J J ~ 1 ~ ~ S S
U~O O(~ O OO J J
~~I '~ ~ ~1 ~~ O (~ O C ., ~-, ~ ~ ,-.,1 .~ .
E-'::"~ ~ ~Y ~~ J J ~ ~ ~ . 4 ~ ~ ~ L Ll L
~ ~Cr --~~
N ~~JrcJ
V ~-~ ~ ~ ~ ~ r r ~ r,~rl n u:
U~ U~ n U~ r~ U~
.~

TABLE 17l continued MTC ~g./mlO) Test Organism A B C D E F G

KLebsiella pneumoniae X26 >128>128 >128128 >128 >128 64 Klebsiella pneumoniae KAE ~128>128 >128>128>128 >128 >128 Klebsiella pneumoniae X68 >128128 >128>128>128 >128 >128 Enterobacter aerogenes C32 >128 64 >128>128>128 ~128 ~128 Enterobacter aerogenes EB17 >128>128 >128>128~128 >128 ~128 Enterobacter cloacae EB5 >128128 >128>128~128 >128 ~128 Enterobacter cloacae 265A >128128 ~128>128~128 ~128 >128 c Salmone~la typhi X514 >128>128 ~128~128~128 ~128 >128 Salmonella typhi 1335 >128>128 >128>128>128 >128 >128 Pseudomonas aeruginosa X528 >128>128 >128>128>128 ~128 >128 Pseudomonas aeruginosa X239 >128~128 >128>128>128 >128 >128 Pseudomonas aeruginosa Ps18 ~128>128 ~128~128>128 ~128 ~128 Pseudomonas aeruginosa Ps72 ~ 128 ~128 >128 ~128 Serratia marcescens X99 >1~.8128 >128>128>128 >128 ~128 Serratia marcescens SE3 >128>128 >128>128~128 >128 >128 Proteus morganii PR15 >128>128 ~128~128>128 >128 ~128 Proteus inconstans PR33 >128>128 >128>128>128 >128 >128 Proteus rettqeri PR7 >128>128 >128 -- -~

TABLE 17~ continued MIC (~g./m~
Test Organism A B C D E F

Proteus rettqeri C 4 ~128 ~128 ~128 ~128 >128 >128 >128 CitrQbacter freundii CF17>128 ~128 >128 >128 >128 >128 >128 Bordetella bronchiseptica 16 ~128 ~128 >128 --Acin~tobacter calcoaceticus AC12 -- -- -- >128 >128 >128 >128 - = not tested '~, The in vitro activit.y of antibiotic A41.030 .
f actors A and B against a number of reprosentative aerobic, gram-positive bacteri.a, including St~epto-coccus sp. Gxoup D, has ~een dQtermined using a starldard S agar-dilution a~say. The results, determined by reading th~ end point af ter 2 ~ hours, are recorded in Table 18, which follows~

lû

, 2~

~5 :~0 ~ll9~d~l X-5953A -4.3-ACTI~ITY OF A41030 FACTORS A AND B
AGAINST AERO~IC BACTERIA
MIC (~g.~ml.) Test Organism A B
Staphylococcus aureus 305~ 0.06 0.~
X4~0 0.1~ 0.1~5 ~138 ~.06 0.06 V140 ~.125 0,125 1~ V102 0.125 0 A 125 Staphylococ~us epidermidis ~22 0.1~5 0.12 27~ 0.06 0.06 285 0.06 0.03 219 0.06 ~.0~
269 0.06 0.03 Streptococcus pyogene's C203 0.25 0.125 Streptococcus s~ ATCC 10389 0.125 0.25 Stre?tococcus s~. Group B

.1~ , 0.06 ~.125 2~ Stre~tococcus sp. Group D
~6 0.25 ~.~5 99~0 0.5 0.
2041 0~5 0~5 ~3043 0.25 0.25 1~253F 0.25 0.5 ~-2S~ 16~ 0.25 0.25 205S 0.5 0.5 Streptococcus pneumoniae Pr'.c I 0.125 0.125 Streptococcus pneumQni~e ~ 38 0.~5 TABLE 18, continued MIC (~g.~1.) Test Organism A
~aemophilus parain1uenzae Haemophilus parainflusnzae 97~5l~ 16 Haemophilus in~luen~ae C.L. ~ a ,~x366 2 4 Mx371 2 4 7~ 4 4 Bond 2 31~ 2 4 The activity OL antiblo~ic A~1030 comp~ex against a number o~ anlmal pa'hogens was determined by a standard in ~itro antimicroDial broth microtiter ~est, and the results are set forth in ~able 19, which ~ollows~ -~5 ACTIVIq'Y OF A41Q30 C:OMPLEX AG~INST
SEVERAL ANIM~ P~TH~&ENS
Test Or~anlsm MIC (~g./ml.) Staphylococcus s~. 1130 ~0.78 Strep~ococcus s 80 ~0.78 Pa~teurella multocida (~ovine) 3.1~
Pasteuxella hemolytica 6.~5 10 Bordetella bronchiseptica (Swit~er~ 50.00 Esch~richia coli 50.00 , Mycoplasma s~noviae 50.00 Mycoplasma hvorhinis 50.00 PseudQmonas -f ish < O . ?~
1~ Aeromon~s li~uefaciens 50.00 All of the A41030 ~accors tested have sho~,m in vivo an.tmicrcbial activity against experimental bacterial inections. When t~o doses o~ test compound 2Q were adminLscered subcutaneously to mice ~n lllustrative inections J th2 activity observed is measu_2d as a~ '~D50 va'ue ~effective dose in mg./kg. to protect fl,ty percent o the test anlmals: See Warren ~7ick, et a_., . BactQriol. 81, 233W235 (1961)]. The ~D values ~0 ~.
o~s2rved ~or A41030 f~ctors A, 3, C, 3, E, an~ F are given ln T~le 20, w~ich -ollows~

Staph. aureus S. pyogenes SO pneumoniae AntibioticED50 ED50 ED~o A41030A 1.4 2.8 1.68 A41030B ~0.4~ 1.4 1.4 A41030C <0.43 10.4 6.~
A41030D 0.339 3.24 2.21 A41030E <0.31 3.54 3.11 A~1030F <0.31 >5.0 >5.0 1~
The acute toxicity of each of the antibiotic A41030 factors A, B, and C, has been determined in mice and has been found to be >300 mg~/kg., administered intraperitoneally.
The~LD5~ of each of the antibiotic A41030 ~actors A, ~, and C, has been determined in mice as being >300 my.~kg., when administered intraperit:oneall~.
The ln vivo oral activity of each of -the antibiotic A410~0 factors A, B, and C, as determined against S. pyogenes in mice, is >300 mg./kg. X ~.
In one of its aspects this invention provides A method for trea~ing lnec-tions in a wann-blooded animal which comprises administering to said al~imAI a chemotherapeutically-effective amount; for ex~mple, between about 25 mg. and about 2,000 mg. of the A41030 ~5 antibiotic complex or ~actox th*reot, or a pharmaceu-tically-acceptable sal~ thereof.
Fac~or A, or a pharmaceutically-acceptable, salt thereof, can be used in the treAtment oE infec-tions i~ man, but in general che comple~s and oth~r 3~ factors~ arld salt~ thereof are best suited ~or use in the treatment of infections in other warm-blooded animal s .

X-5953A ~47-In the treatment of infections in man the antibiotic factor, preferably factor A, can be admin-istered by the parenteral route, e.g., by i.rn. injec-tion, or iAV. infusion. For injection, the antibiotic or a pharmaceutically-acceptable salt thereof is dis solved in a physiologically-acceptable diluent at the desired concentration al~d administered. ~uitable diluents include for example, Water-for-Injection, 0.9%
saline, 5~ dextrose, Ringer's solution, or o~her com-monly employed diluen~ ~or administration by i.v infusion, the antibiotic or salt thereof can be made up in a pnysiological fluid or dilute nutrient at a suit-able conentration; for e~ampler at a concen~ration ~etween about s% and about 10~, and slowly in used with the flui~. Alterna-tiv~ly, the antibiotic may be admin-i~tered by the "piggtl-back" method.
The indlvidual factors, combinations of the factors, or the whole complex of factors and the pharma-ceutically-acceptable salts thereo can be made up in dosage unit formulations in hermetically sealed vlals, sterile, rubber-stoppered vials, or in plastic pouches.
Such uni-t dosage forms can contain e~cipients such as antio~idants, solubilizing agents, dlspersing agents, buffers/ and the like. One such dosage unlt formula- -tion comprises 100 mg. o~ ~actor A, or a pharmaceu-~5 tically acceptable salt thereof, in a rubber (butylrubber) stoppered vlal. Another dosage uni~ formula-tion comprises 2~0 mg of factor A, or a salt thereof, in a sterile, ~ealed vial. For i.v. in~usion a dosage unit ormulation oE this invention compri~es 5 ~. of 3~ ,actor A, or a pharmaceutical ~7-ac_ep-table salt there-of, in a plastic pouch.

X~5953~ -4~-When A41030 complex or an A41030 factor is used a~ an anti~acterial agent:, it may be administered either orally or parenterally. A~ will be appreciated b~ those skilled in the art, the A41030 complex or 5 factor is commonly administered together with a pharma-ceutically-accepta~le carrier or diluent. The dosagP
of A41030 complex or factor will depe~d upon a variety o considexations, such as, for example, the nature and severity of the particular infsction to be t.reated.
10 Those skilled in th~ art ~ill recognize that appro-priate dosage ran~e3 and/or dosage units for admin-istration may be determined by considering the MIC and D50 values arld toxicity data herein provided, together with factors such as tne patient or host and the infectiny orga~.is~. D
Th~ A~1030 antibiotics axe useful inter alia for suppressing the gxowth of Staphylococcus, St~epto-coccus and Propioni~acterium acnes orsanisms, ar.d the an~l~iotics could therefore be USef~, for example, in 20 the treatment of acne. The A~1030 individual ~actors, or mixtures thereo~ in the puri ied state, can ~e formulated in ~har~naceuticall~I-acceptable diluents such as isopropyl alcohol for applicat.ion to the sk~n. Such solutions c~n be made up with antl~iotic concentrations ~;
~5 of rGm a~cut 1 to a~ou~ 15 ~erce~t weight ~e. vol~me.
Alternati~JQly,. _he antibiotics can be ~ad2 ~ into creams or _otions for appllca~ion to the skin.
~ he A4103Q antibiot~cs are also use~ul ~or sup~ressing th^~ growt:~ o~ ~lostridlum diflcile cr~anis~, whi-h cause ~seu~omembranous colitis in t'n~ l~testin~. Th~ ~1030 indi~r~dual actors or ml~c-tures thereof could be used i~ the treatment of pseudo-membranous colitis by the o.ral ~m ~ istration of a therapeutically e~fective dose of sald antibiotics or a pharmaceutica].ly-acceptable, non-toxic salt thereof, prepared i~ a pharmaceutically-acceptable dosage form.
For such use the antibiotic can be administered i~
gelatin capsules or in liquid suspension.
The antibiotics of this invention also can be used ir~ veterinaxy medicine in th~ treat~nerlt of in-fectious dis2ases in domestic and rarm ~n;mals, ~heyare useful also in animal husbandry, e.g., in enhancing the srowth of beer cattle and other r~minznts~ An ~specially valuable use for tne antibiokics of this invention resides in their ability to ir,c.ease the production of milk in dairy cattle.
The A41030 complex has shown activity against infectious canine hepatitis virus ln vitro at 40 mcg./ml~
The A41030 complex has also shown activity ln vitro against pseudorabies ~t 20 mcg./ml; and A41030 factor .
~0 has shown ac~ivity in vitro a~ainst pseudora~les at 20 mcg./ml.
Antibi~tic A4103~ comple~ h~s shown activil:~
as a growth ~romot~ in chickens, th~ test ~el.lg carried out as follows:
Ch cks, 8-day old Penobscot broilers, were utilized in t~is test. A tot~l o~ _60 cllic~s wer~
used, di~ided in groups of 7 ~irds each. T~.er~ w~re 35 groups acting as controls, anc ~ groups were treated with th~ antLbiotic added to ~he standard chic~ ration 3~

~-S9~3~ . -S0~

at t~e rate o 20 g. of antibio~ic ~41030 complex per ton of feed. Feed and water were available to all group~ ad libitum for 21 daysO ~wo time replica~es were run~ The criteria or activi~y: 3~ increase in 5 weight gain and/or 2~ improvement in feed efficiency ln one or ~oth time replicates. The results a~e se-t ~orth in Tabl~ 21, which ~ollows.

lû

lS

~J~ o ul Ul Taible 2 1 Weight Gain Feed Ef f iciency Conc .
1'~eatlli~n t ~3 . ~Jm . ~ Impr . F/G ~ Impr .
~on~Lol - 433 - 1. 671 A41030 2(~ 4514 . :3 6 1. 6û1 4 .19 Co ~ 4 5 1 - 1. . 6 7 3 ~4:~030 ~ ~6~ 2.8~ - 1.651 ~1.32 F/~ - ~otal feed consumed divided by total weight gain.

~-5953A -52- .

~ ccordingl~, this invention provides a method of enhancing th~ growth of ch:ickens which compri~es administering to the chickens in their diet betw~en about 20 and about 30 gO o~ an A41030 antibiotic factor or the A41030 a~tibiotic complex, or a pharmaceutically-acceptable sal~ thereof, per ton of feed. Alternatively, the antibiotlc factors or the complex, in the form of a phanr.aceutically~acceptable, non-toxlc salt can be administered in the d~inking water c~f the chickens.
10Antibiotic A41030 also acts as a growth promoter when ~ml~; stered to wear.ling pig9~ The antibiotic was t~sted in young pigs at several dosage levels~ as hereinafter describe~'.
The antib~ctic A41030 complex wa~ tested at 15le~els of 5, ~0, 50 and 100 ppm~ in the diet of pigs initially ~eighing about 21 pounds (5-7 weeks of age~O
The e~periment was conducted in an environmentally controlled nursery facility. Ther~ were five raplicates per treatment and ive pigs per replicate ~or the ~ 27~day e~periment. The results appear in Table ~2, which ollows:

~'~

X
o ~ O Ul O U~

Ta~le 2 2 I.eve~ ADG % ~0~ % 96 eatIilent ppm_lbs. Increase lbs.~ncrease F~G Improvemerlt Basal -- 0 . 67 1. 25 1 . 87 }~11030 5 D~71~.5 1.33 6.4 1.89 -1.0 .~4~ 0~(~ 0.~7~.~ 1.25 0.0 1.93 -3.2 A41û30 50 0.71~6.U l.~S 0.,0 1078 4.8 A~1.03010~3 0.77+14.9 1~34 702 1~74 7~4 ~b Al)~ -- avel-age daily gain .~3E' = averag~ daily feed conswnPtiOr ~'~G = ratio o~ f eed consurllption to gain ~-5953~ -S4-In this trial the ~ntibio~ic elicited a dose-related improvement in growth perrorman.ce in weanling ~iss, a~ indicated by the response at i pp~
and at lO0 ppm.
The antibiotic A4103G complex was ~urther tested in weanling pigs (17 pounds, 4-6 weeks of age) at levals of 25, 50 and 100 gm./ton of feed for 35 days. There wexe four ~eplicates of six pigs per treatment.
10. Antibiotic A~1030 complex when a~ministered to these weanling pigs at the rates ~aught, increased the rate of gai~ by 5.6 percent, 8.5 percent, and 7.0 percent; and improved t~ feed conversion e~ficiency by 6.6 perc~nt, 9.~ percent, and 3.1 percent, when added 15- to the dlet at 25, 50 and lO0 gm./tcn, respectively.
These r~3ults are recorded in Table 23, which follows.

Table 23 Level ADG % ADF % %
Treatmentgm~/ton lbs.Increase lbs. Increase ~ Improvement Basal - 0.71 - 1.39 - 1.96 A41030 25 0~75 5.6 1.38 -0~7 1.83 6.6 A41030 50 0.77 8.5 1.38 ~0.7 1~78 9.2 A41030 100 0.76 7.0 1.44 3.6 l.90 3.1 u X-5953~ -5~;~

Thus in another aspect~ this invention provides a method ~or promoting the growth of weanling pigs which comprises administering to the pigs in their diet a gEowth-promoting amount of the A41030 an~i~iotic complex, or an A41030 antibiotic actor, or a pha~a-ceutically~acceptable, non-~toxic salt thereof. The me~hod ~s preerably carried out by administ~ring to the pigs between about 25 g~ and about 200 gO of the ~41030 ankibiotic complex, or a pharmaceutically-acceptable, non-toxic sal-t thereo~ per ton of Eeed.
The antibiotic A41030 comple~, or the individual factors, in the form of a pharmaceutically-acceptable, non-toxic salt, can'also be administered to the pigs in the d.rinking water.
The A~1030 antiblotics aæe also useful ror increasing the e~ficienc~i of feed utilization in r~inant animals. It is known that the efficienc~ o~
carbohydrate u'ciliza.ion in ruminants is increased ~y treat~ents which s~imulate the ani.mals' rumen ~lora to 2~ produce pxopionat2 compounds rather han acetate or but~rate compounds ( for a more complete discussion, see Cl1l1xch _ al. i~ "Dige~tlve P~ysiclog~t and Nutritlon of m;nants," VolO 2, 1911, pp~ 622 and 625).
The e-fecti~eness o antibiotic A41030 com-2~ plex and A41030 factor ~ ,~or modif-~ing the ratio of volatile rat-Ly acids (~JFA) produced in the rumen is shown bv ~eans of in ~ tro t9sts according to the ~roc~dure set Eorth ~ereinb210w.

3~

Rumen fluid was obtained from a steer having a surgically-installed ~istula opening into the rumenO
The steer was maintained on a high-grain ration, the composition of which follows:
5 69.~5~ coarse grou~d corn 10% ground corncobs 8 % soybean meal ( 5096 prot_in~
5 % al~alfa meal 5 % molasses 0.6 ~ urea 0.5 ~ dicalcium phosphate O . 5 96 calcium carbonata O ~ 3 ~ sait O . 07 ~ Vitamin A and 3~ premi~
O, 05g~ Vitamin E premi;~
O ~ 03% trace mlneral premi~
A sample of rumen fluid was stxained through 4 layers of ch~3eseclcth arld the filtra~e was collected ;2~ in a vacuum bottle. The part~ c~late matter retain~od by th~ ch~s~cloth was resuspanded in enough physio-log~cal bur^er to xeturn i~ to the origln21 volume or the rumen 1uid, and the suspension was again strained through chees~loth. The buf~r used is described balow.
~5 'O

-5~-0.316 g./liter Na2HPO4 0.152 g./lit~r KH2PO4 2.260 g./liter NaHCO3 0.375 gO/liter KCl 0.375 g./liter NaCl 0.112 g./liter MgS04 0.038 g./liter CaC12 0.008 g./liter FeSO4 a 7H20 0.004 g~/liter MnSO4 0.004 g./liter ZnS4~7H2 0.002 g./liter CuSO4.5H2O
0.001 g./liter CoC12 Cheng et al., J~ Diary Sci. 38, 1225~ (1955).
The two filtrates were pooled in a separatory funnel and allowed to stand till particulate matter rose to the top. The clear layer was then separated and diluted 1:1 with the same buffer, and adjusted to pH 7Ø
Ten ml~ of the diluted rumen fluid thus prepared was placed in a 25 ml. flask with 90 mg. of finely~
powdered high-grain ration, the composition of which is described above. The compound to be tested was weighed out and dissolved ln ~he appropriate solvent, supraO The solution was placed on the finely-powdered ration in each test 1ask and dried~
Two sets of our untreated control flasks each ~ were also prepared. One set of our untreated control ; 30 flasks was incubated for 16 hours at 38C~ with the test 1asks. The other set of four untreated 0~

~5953A ~59-control flasks were zero~~ime controls, ~he fermen-tation in which was stopped as SOOIl as the flasks were prepared by addition of 2 ml. of 25 percent meta-phosphoric acid to each 1ask.
Fermentation in the incu~ated test and con-trol flasks was stopped at the end of 15 hours by addit~on of 2 ml. of 25 percent metaphosphoric acid to each flaskO
All of the samples were allowed to settla, a~.d the supernatant was analyzed by gas chromatographic me~hods for acetate, propiona~e, and bu~yrate.
The al~alysis Eor each volatile f atty acid found in the ~ero-time controls was substracted from the analyses of the untreated con~rols and o the tes~
15 fla~ks. ~he resultin~ values reflect the amount of , each ~FA produced during `.~e 16-hour fermentation period.
The data below are re~orted as the ratlo of 's produced in treated flasks to VFA's produced in 20 untreated control flasks. This method of reporti~g the dat~ s'nows mos~ clearly the results of Lhe chan~es in tha chemistry of the r~men ~rou~ht a~out ~y the present novel method of feed ut~,lizat on improve~ent. S~e Ta~le 24, which follows~

TablP 24 Acetat~Propionate Butyrate pOU~ ate A41Q30 5 n~cg./ml. 0.94 1.23 0.72 A41Q30 5 mcg./ml. 1.03 1~33 .~103~ 5 ~Itc~ l. 094 1 33 0 94 A4103(3A 2 mcg./ml. 1.03 1 31 0~66 A4 10 3 ûA 1 mcg . /ml . 1 . 01 '~

The data tabulated above shows that the anti-biotics are effective in increasing propionate production in the rumen.
Administration of the antibiotic compounds useful in this method prevents and treats ketosis as well as improves feed utilization. The causative mechanism of ketosis is a deficient production of propionate ~ompounds.
A presently recommended treatment is administration of propionic acld or feeds which preferentially produce propionates. It is obvious that the method disclosed in this application, which method encourages propionate production from ordinary feedsl will reduce incidence of ketosis.
It has been found that the antibiotic compounds disclosed herein increase the efficiency of feed utili-zation in ruminant animals when administered in a propionate-increasing dose. The propionate-increasing dose can range from about 10 to about 120 g. per ton, preferably in the range of from about 40 to about 80 9O
per ton. The antibiotics, individually, or as the whole complex, or as an economical~ less-purified whole complexl are suitably administered by incorporation in the animal's feed.
However, the antibiotic compounds can be usefully administered in other ways. For example, they can be incorporated into tablets, drenches, boluses, or capsules, and dosed to the animals~ Formulation of the antibiotic compounds in such dosage forms can be accomplished by means of methods well known in the veterinary pharmaceuti-cal art. Each individual dosage unit should contain aquantity of the feed-efficiellcy-improving compound which has a direct relation to the proper daily dose for the animal to be treated.
Capsule~ are readily produced by filling gelatin capsules with any desired form of the desired antibiotic.
I~ desired, the antibiotic can be diluted with an inert powered diluent, such as a sugar, starch, or purified crystalline cellulose in order to increase its volume for convenience in filling capsules.
Tablets of the antibiotics are made by conven-tional pharmaceutical processes In addition to theactive ingredient, a tablet usually contains a base, a disintegrator, an absorbent, a binder, and a lubricant.
Typical hases include lactose, fine icing sugar, sodium chloride, starch and mannitol. Starch is also a good disintegrator, as is alginic acid. Surface active agents such as sodium lauryl sulfate and dioctyl sodium sulpho-succinate are also sometimes used. Commonly used absor~
bents again include starch and lactose, while magnesium carbonate is also useful for oily substances. ~requently-used binders are gelatin, gums, starch, dextrin and variouscellulose derivatives. Among the commonly-used lubricants are magnesium stearate, talc, paraffin wax, various metallic soaps, and polyethylene glycol.
The antibiotic compound can also be administered as a slow-payout bolus. Such boluses are made in the manner tablets are made t except that a means to slow the dissolving of the an~ibiotic is provided. Boluses are made to release for lengthy perioc~s, and the slow dissolution is assisted by choosing a higllly water-insoluble form of the antibiotic. A substance such as iron Eilings is added '.'~;

to raise the density of the bolus and Iceep it static on the bottom of the rumen~
Dissolution of the antibiotic is delayed by use of a matrix of insoluble materials in which the drug is embedded. For example, substances such as vegetable waxes, purified mineral waxes, and water-insoluble poly-meric materials are useful.
Drenches of the antibiotics are prepared most easily by choosing a water-soluble form of the antibio-tic. If an insoluble form is desired for some reason, asuspension may be made. Alternatively, a drench may be formulated as a solution in a physiologically-acceptable solvent such as a polyethylene glycol.
Suspensions of insoluble forms of the antibiotics can be prepared in nonsolvents such as vegetable oils such as peanut, corn, or sesame oil; in a glycol such as propylene glycol or a polyethylene glycol; or in water, depending on the form of the antibiotic chosen.
Suitable physiologically-acceptable adjuvants are necessary in order to keep the antibiotic suspended. The adjuvants can be chosen from among the thickeners, such as carbGxymethylcellulose, polyvinylpyrrolidone, gelatin, and the alginates. Many classes of surfactants also serve to suspend antibiotics. For example, lecithin, alkylphenol polyethylene oxide adducts~ naphthalenesulfonates, alkyl-henzenesulfonates~ and the polyoxyethylene sorbitan esters are useful for making suspensions in li~uid nonsolvents.

In addi-tion, many substarlces which affect the hydrophilicity, density, and surface tension o~ the liquid can assist in making suspensions in individual cases. For example, silicone antifoams) glycols, 5 sorhitol, and suga.rs can be useful suspending agen~s.
The suspendable antibiotic may be offered to the animal grower as a suspension, or as a dry n~ixture of the antibiotic and adju-vants to be diluted before use.
1~ The antibiotic~ may also ~e administered in the drinking water of the ruminants. Incorporation into drinkiny water is performed by adding a water-soluble or water-suspendable form of the desired antibio-tic to th~ water ln the proper amount. Formu-1~ lation of the antibiotic for addition to drinking water follows the same principles as formulation`of drenches.
The most practical way to treat animals with these antibiotic compounds is by the formulation of the compounds into the feed supply. Any type of feed may be medicated with the antibio~i.c compounds, including common dry ~eeds, liquid feeds, and pe~leted feeds.
The methods of formulating d..uys in-to animal feeds are well known. It is usual to make a concen~
~ra-ted drug premix as a ra.w material for meclicated feeds. For example, typical drug premixes may conta:ln from ~bout one to about 400 grams of drug per pound of premix~ The wide range r~sults fror.l the wide range of concentration of drug whlch may be desired in the final feed. Premixes may be either liquid or solid.

X-~953A -65~

The formulation of anlmal feeds containing the proper amounts of the ant.ibiotic compounds for use~ul treatment i~ mainly a matter o arithmetio.
It i~ n~cessary o~ly to calculate the amount o~ com~
5 pou~d which it i~ desired to ~;n;ster to each animal~
to take into account the amount or ~eed per day which the ~n;~l eats~ and the concentration of antibiotic compound in the premix to be u~ed, and calculate the proper concentxation o~ antibiotic compound in the feed.
All o~ the metnods of ormulating, mi~ing, a~d pell ting feeds which are no~mally us~d in the ruminant or nonruminant feed art ~re entirely appro priate for manufacturing ~eeds containing the antibiotic compounds usable in this method.
lS I~ ha~ also been ound that antibiotic A41030 complex not only increases the efficiency of feed utilization in rll~; n~n S utili7.~d for meat production (as de~c~ibed hereinbefore~, but also causes a ~urprising impro~ement in milk production without an adverse effect on milk quality when admini~tered to lactating animal3 having a developed rumen process.
The requirements and obiec~ives of feed utilization o~ lactating rllmin~nts such as dalry cows dif~er considerably fro~ those o~ ruminants raised ~or m~a~ production. ~llm;~l volatile ~atty acid ~VF~) production is oE course of primary importanc~, since it relates directly to the normal mainten~nce o th~
animal, ~s well as to the quality and quantity of ~he milk produced by th~ animal. In the lactating rllm; na~t, ~Q however, energy or lactatlon i~ the most limi lng factor in milk production. ~cetate is required or X~5953~ -66--milk fat sy~thesls, while propionate is utilized to produc~ glucose, which i.n turn is r~quired for lactose synthQsls, and also has a minor role in milX fat pro-ductionO Butyrate is more g:Lycogenic than lipogenic, the lipogenic aspect being indirect since butyrate must irst be degraded to ac~ta~e unit~ before i~ can be utilized for long chai~ fatty acid ~ynthe5is~ -Oe., milk fatO
Accordingly, in order to incr~ase milk pro-duction in lactating rllm;n~ts, it is n~cessary toincrease propionate productiQn, but no~ at a large expense o~ acetate and butyrate production~ Signifi-cantly reduced acetate and butyrate levels result in drastically reduced milk fat content, thereby : 15 rendering milk production less efficient wlth respect to both quality and economically (bulk milk price~
are determined in part by miik fat content).
The present improvement in milk productior~
is manifested in increased protein content of the ~0 milk, withou~ appreciable change of fat content. The m~thod of accomplishing the improvement comprises orally administering to a lacta~ing rll~;n~nt a propionate-incxeasing ~mount o antibiotic ~41030 complex.
The antibiotic A41030 complex can be formu~
lated for convenien~ oral administration to a rl~m1n~nt, the formulation bei~g as a eed pre-mix, a faed additive, a lick, a water additive, or if desired, the antibiotic A~1030 oompl~ can be formulated ~or slow r21ea~e over a prolonged period of time following a single 3~ administration.

2~

~-5953~ -67-Th~ antlblotlc A41030 complex and factors A9 B, C, D~ E, F, and G can be i.solated for use in the ~ feeds described hereinbefore by th~ methods disclosed in the Examples ~et forth ~ereinafterO It S is al~o possibl~, if desired, after the production of A41030 ~ntlbiotic actlvity, to simply dry the whole fermentation broth a~d mix th dried medium directly into the feed or f~ed premix.
Furthex, it is possible the hereindisclo~ed 1~ A41030 antibiotic complex and factors can be combined with a synthetic ~n~; ~g agent which is a sul~ited phenol fo~maldehyde syntan, such as is sold by A.J.
0.3. Pilar Inc. of Newark, N.J., under th~ tradename TruTan RT Regular. The combination o~ the an~ibiotic lS and the syn~hetic ~nn;ng agent, the antibiotic syntan complex, can be used without separation of t~e con-~tituents, in the a~imal feed supplement compositions described above.
As has b2e~ shown, antibiotic A41030 complex ~ a~d A41030 factor A bene~icially alter the production of propionates xelative to the production o acetates in the rumen. The same treabment al 50 benefita ~ono~
gast~ic ~nimA~s which fenment fibrous vegetaole ma~ter ; in the cecum~ The monogastric ~ni~l S here referred to ~5 are thoss which co~sume ibrous vegetable food and digest at least part o~ it by microbiological fermen-tation in the cecum~ The cecal fermentation follows a chemical pathway similar to rur.len e~me~tation.
Horses, ~wine, and rabbit~ are axemplary 3~ ~;m~ls which digest a part of their food by cecal f X 5953A -68-~

fermentat.ionn The overall feed utilization of such animals is improved ~y the oral administration of these antibio~ics which cause a benleficial change in the propionate/ace~ate ratio. Horses and rabbits are exemplary of animals in which cecal fermentation is a major par~ of the total digestive process, and in which these antibiotics are accordingly particularly bene-ficial.
The A41030 complex can be produced by cultur ing the hitherto undescribed microorganism Streptomyces virqiniae NRRL 125~5, or by culturing the hitherto undescribed microorganism Streptom~ces virginiae NRRL
1515h, or an A41030-producing mutant ox variant of either microorganism, in a culture medium containing lS - assimilable sources of carbon, nitrogen, and inorsani~
salts, under submerged aerobic fermentation conditions until a substantial level of antibiotic activity ls produced. Most of the antibiotic activi~y is generally found in the b.roth, while minor amounts of antibiotic 2~ activity may be associated with the mycelia. The A41030 comple~ is most readily separa~ed from the ermentation mixture by removal of the myceliat i.e., the ~iomass, ~y filtration. The mycelia are generally discarded~ The antibiotic complex is then isolated from the filtered fermentation Drotn prefer~bly by column chromato~raphy, over a suitable adsorbent usir.g methanol:wat~r (1:1) as the elu~ing agent.
Suitable adsorbents include carbon, alumina, anion and cation exchange resins, sili_a gel, poly-~mide J carboxymethylcelluloses, highly porous copoly-: 3~
: mers o st~ren~ and di~inyl~ienzene such as Diaion HP-20, z~

the Amberlite~ XAD resins, and the Duolite~ resins such as ES~865 and the like, as well as Sephadex~
resins~ the hydrophilic, insoluble, molecular-sieve chromatographic mediums made hy cross-linking dextran, and also TS ~ Gels. The Diaion~ resins are a product of Mitsubishi Chemical Industries, Limited, Tokyo, Japan.
~he Amberlite XAD resins are produced by Rohm and Haas, Philadelphia, Pennsylvania. The Duolite resins are products of Diamond Shamrock~ Redwood City/ California.
Sephadex resins are manufactured by Pharmacia Fine Chemicals ABI Uppsala, Sweden. The TSK Gels are available from E. Merck, Darmstadt, and from ~io Rad, 2200 Wright Ave., Richmond, California~ 94804.
The A41030 antibiotic complex can be further purified and separated into its individual factors by chromatographic techni~ues.
A number of different media may be used with Streptomyces virginiae NRRL 12525, or Streptomyces virginiae NRRL 1515~, to produce the A41030 complex. For economy in production, optimal yield, and ease of product isolation, however, certain culture media are preferred.
These media should contain assimilable sources of carbon~
nitrogen, and inorganic salts. Suitable carbon sources include dextrin, s~arch, mannose, glycerol, and cottonseed oil. Optimum levels o~ carbon sources are from about 2 to about 3 percent by weighta Preferred nitrogen sources include soybean grits, soybean flour, peanut meal, fish meal, meat peptone, and pork blood meal.
Essential trace elements necessary for the growth and development of the organism may occur as impurities in other constituents of the media in amounts sufficient to meet the growth and biosynthetic requirements of the organism. However, it may be beneficial to incorporate in ~he culture media additional soluble nutrient inorganic salts capable of yieldiny sodium, potassium, magnesium, calcium, ammonium, chloride, carbonate, phosphate, sulfate, nitrate and like ions.
Addition ~o the fermentation medium of Tween 80~ (oily liquid polyoxyethylene sorbitan monooleate, a product of ICI Americas, Inc~, Wilmington, Del.), at a level of 2-4% serves to increase the yield by about 300%.
However, difficulty is experienced in isolating the A41030 antibiotic under these conditions.
Although small quantities of the A41030 anti-biotic may be obtained by shake-flask culture, submerged aerobic ferm ntation in tanks is preferred for producing substantial quantities of the A41030 antibiotic. For tank fermentation, it is pre~erable to use a vegetative inocu-lum. The vegetative inoculum is prepared by inoculating a small volume of culture medium with the spore form, or mycelial fragments, to ohtain a fresh, actively growing culture of the organism. The vegetative inoculum is then transferred to a larger tank where, after a suitable incubatiorl time, the A41030 antibiotic is produced in optimal yield.
An alternate method of providing inoculum for the vegetative medium consists of substituting a lyophilized pellet for the aqueous spore suspension. Lyophilized pellets are prepared in a manner known in the art.

Preparation of the spore suspension for lyophilization is similar to the preparation of the aqueous spore suspen-sion, except that sterile calf serum is substituted for sterile distilled water.
The A41030-producing organism can be grown over a broad temperature range of from about 10 ~o about 34C.
Optimum production of A41030 antibiotic complex appears to occur at a temperature of about 30C.
As is customary in aerobic submerged culture processes, sterile air is dispersed through the culture medium~ For efficient growth of the organism, the volume of the air used in tank production is in the range of from about 0.1 to about 0.5 volumes of air per volume of culture medium per minute (v/v/m), with from about 100 to about 300 RPM agitation. ~n optimum rate in a 165-liter vessel containing 100 liters of fermentation medium is about 0.25 v/v/m, with agitation provided by an impeller rotating at about 200 RPM.
Antibiotic activity is generally present after about 48 hours and remains present for at least 144 hours during the fermentation period. Peak antibiotic produc-tion occurs at from about 96 hours to about 120 hours fermentation time.
Production of the A41030 antibiotic can be moni tored during the fexmentation by either agar diffusion using B~ subtilis~ or a turbidimetric method using Staphylococcus aureus ATCC 9114.
The antibiotic complex, and individual factors thereof, provided by this invention are produced by either S. virginiae NRRL 15156 or S. virginiae NRRL 12525 under ~..,~
. ~ ..

fermentation condi~ions of temperature, duration, and media ingredients which are substantially equivalent.
However~ it appears that under such conditions, S.
virginiae NRRL 12525 produces the antibiotic complex in somewhat greater abundance.
In order to illustrate more fully the operation of this invention, the following Examples are provided.

Example 1 Preparation of First Stage Inoculum The ~ollowing medium was prepared for use in the agar slant culture of both Streptomyces virginiae NRRL
12525, and Streptomyces virginiae NRRL 15156:

Ingredient Amount (g/L.) Dextrinl 10 . O
Yeast extract 2 l.o Enzyme-hydrolyzed casein 2.0 Beef extract 1.0 CoC12 . 6H20 0 . 01 Agar 20.0 Deionized water q.s~ to 1 liter lMatheson ~oleman & Bell r Norwood, Ohio 45212 2N-~-Amine A(Humko Sheffield Chemical COr~ Memphis, Tenn.).

~.
J'\
", .i ~

2~

The pH of the medium as prepared was 5.5, and was adjusted to 7.3 using 5 N aqueous sodium hydroxide before autoclaving. After autoclaving, the pH of the medium was 6.g.
In the case of each microorganism, tile spores were inoculated on an agar slant made up of the above-identified ingredients, and the thus-inoculated slant was incubated for about six days at a ~emperature of about 30C. The mature slant culture was then covered with sterile distilled water and scraped with a sterile tool to loosen the spores and the mycelium. One milliliter of the resulting spore suspension was used to inoculate 50 ml. of vegetative medium. An alternate method of providing inoculum for the vegetative medium consisted of substituting a lyophillzed pellet for the aqueous spore suspension. The composition of the vegetative medium for NRRL 15156 was as follows:

Ingredient Amount (g/L.) Glucose 15 n O
Dextrin 20.0 Soybean grits (or soybean flour) 15.0 Corn steep liquor 10.0 CaCO3 2.0 Tap water q.s. to 1 liter The unadjusted pH of the medium was 5.5, which was adjusted to pH 6.5 with 5 N aqueous sodium hydroxide before autoclavingu The pH of the medium after auto~
claving was 7 n O

.~

The composition of the vegetative medium for NRRL 12525 was as follows:

Ingredient ~mount (g./L.) Glucose 20~0 Soybean grits (or soybean flour) 15.0 Corn steep liquor 10.0 CaCO3 2.0 10 Tap water q.s. to 1 liter The unadjusted pH of the medium was 5.5, which was adjusted to pH 6.5 with 5 N aqueous sodium hydroxide before autoclaving. The pH of the medium after auto-claving was 7Ø
In the case of each microorganisml the vegetativeinoculum was incubated in a 250 ml. wide-mouth Erlenmeyer flask containing 50 ml. of medium at about 30C. for about 48 hours on a shaker rotating through an arc 2 inches in diameter at 250 RPM. This incubated medium is used either to inoculate small fermenters (the inoculum ~eing approxi-mately 1~ per volume of fermenter medium~ or to inoculate a second stage medium having the same composition as the vegetative medi~m for the production of a larger volume of culture.

Fermentation of NRRL 15156 Fifty mllliliters of a production medium was inoculated with 1% (0O5 ml.) of the incubated vegetative medium from above. The production medium had the Following composition:

Ingredient Amount (y/L~) Dextrin 30.0 Soybean grits 6~0 K2HPO~ 1.0 FeSO4.7H2O 0.005 MgSO4.7~2O 1.0 NaNO~ 1.0 CaCO3 2.0 10 Deionized water q.s. to 1 liter The dextrin may be either tapioca or potato dextrin.

For the fermentation of NRRL 12525, the concentration of CaCO3 was 4.0 g./L.
The K2HPO~ was dissolved in water, the solution sterilized separately, and the requisite amount of the solution added to the other ingredients of the medium that had been autoclaved.
The inoculated fermentation medium, 50 ml., was incubated in a 250-ml. Erlenmeyer flask at about 30C.
for about 4-5 days on a shaker rotating through an arc 2 inches in diameter at 253 RPM.
The Streptomyces virginiae N~RL 12525 was also incubated in a fermentation carried out on a larger scale in 165-liter and 1600 gallon tanks using the production medium described immediately hereinabove.
The inocula~ed production medium was allowed to ferment in a 165-liter fermentation tank containing 100 liters of medium for about 210 hours (8.75 days) at a temperature of a~u~ 32C. The fermentation medium was 2~

aerated with sterile air at a rate of 0.25 v/v~m and was stirred with conventional agi~ators at abou~ 200 RPM.
The large scale fermentation of NRRL 12525 was the source of the broth from which the A41030 antibiotics were separated, as described hereinafter.

Example 2 Separation of A41030 Antibiotics Whole fermentation hroth (4215 liters), obtained as described in Example 1, was filtered using a filter aid (Hyflo Supercel~, a diatomaceous earth, ~ohns-Manville Products Corporation), in a filter press The filtered broth was applied to a column containing 100 L. of Diaion HP-20~ (a highly porous styrene-divinylbenzene copolymer in bead form, Mitsubishi Chemical Industries, Limited, Tokyo, Japan) at a flow rate of 4 L./min. The column was washed successively with 300 L. of water and 1000 L. of methanol:water (1:3) at a rate of 4 L./minO Elution was performed with methanol:water (1:1) at the rate of 6 L./min., collecting 100-L. fractions. Each fraction was analyzed for biological activity. The bioassay was performed by a paper disc assay on agar plates seeded with Bacillus subtilis. Fraction 1 was discarded. Fractions 2~15, inclusive, were combined, concentrated under reduced pressure, and the concentrate lyophilized to give 220 g.
of crude antibiotic complex.
A portion of this complex, 110 g.~ was dissolved in 5 L. of methanol water (1:1), by adjustment to pH 10 with aqueous sodium hydroxide, and the mixture was filtered. The filtrate was applied a~ 50 ml./min. to a 30~L. column (0.2 x 1 m.) of coarse Sephadex G-50 (a hydrophilic, insoluble, molecular-sieve chromatographic medium~ made by cross linking dextran, and sold by Pharmacia Fine Chemicals, Piscataway, NJ 08854~, pre-viously equilibrated with methanol:water (1:1). The column was eluted with methanol:water (1 1) at 50 ml./min.~ collecting 3-L. fractions. Fractions 1-12, inclusive, were discarded. Fractions 13-24, inclusive, which contained activity against B. subtilis, were combined, concentrated under reduced pressure, and lyophilized to give 35.7 g. of the A41030 antibiotic complex.
Example 3 Isolation of A41030 Factor A

An 8 9. portion of the A41030 complex from Example 2 was dissolved in 200 ml. of a solvent consisting o~ water:acetonitrile:sodium chloride ~84:16:2 g.j'L.) and filtered. The filtrate was applied to a stainless steel column ~8 x 100 cm.) packed with 4 L. of 10-20 micron LP-l/C18 reversed-phase silica gel which was prepared in our laboratories by a special procedure described in Examples 6 and 7 of U.S. ~atent No. 4,299,763. Th~ column was part of a Chromatospac Prep-100 unit tJobin ~von, 16-18 Rue du Canal 91160 Longjumeau, France). The column was eluted at 60 ml./min. with water:acetonitrile:sodium chlorLde (84:16:2 g o/L ~ ) collecting 480-ml. fractions. The '`'~;
..~

~78-eluate was monitored at 254 nm using an ISCO Model UA-5 W monitor with a Type 6 optical unit (Instrumentation Specialties Co., Lincoln, NE 68505). Selected fractions were analyzed for the presence of Factor A by analytical high performance liquid chromatography (HPLC) on a 4.6 x 250 mm. stainless steel column packed in our laboratories with 10 micron LP~l/Cl~ which was prepared in our laboratories by the special procedure described above The sample was applied with a Rheodyne Model 7120 injec-tion valve (Rheodyne Inc., Berkeley, CA 94710). The solvent, consisting of water:acetonitrile:sodium acetate (81:19:0~03M) adjusted to pH 6 with glacial acetic acid, was supplied at 1 ml./min. ~1200 psi) by a ~ilton Roy Duplex Minipump (Laboratory Data Control, Division of Milton Roy Co~, Rivera Beach, FL 33404). Factor A was detected at 254 nm using an ISCO Model UA-5 W detector.
Fractions 1-51, inclusive, were discarded. F'ractions 52-79, inclusive, rich in factor A were combined and concentrated under reduce~ pressure to a volume of 500 ml. The concentrate was adjusted to pH 8.2 with aqueous sodium hydroxide and filtered. The filtrate was applied at 15 ml./min. to 100 ml~ of ~iaion HP-20 resin in a column (2.3 x 22 cm.), previously equilibrated with water.
The column was washed with water (400 ml. adjusted to pH
2.5 with formic acid) until no chloride was detected in the wash by precipitation as silver chloride. Elution was performed with water:acetonitrile (8:2) at 15 ml./min., collecting 1 L. fractions. Fractions were analyzed for activity against B. subtilis. Crystalline factor A, i~.

which formed in fraction 2 upon refrigeration, was recovered by filtration (389.6 mg.). Fraction 1 and the filtrate from fraction 2 were each concentrated under reduced pressure and lyophilized to give 731.8 mg. and 514 mg. of factor AJ respectively.

Example 4 Isolation of A41030 Factor B
A 1.0 g. portion of the A41030 complex was dis-solved in 35 ml. o~ a solvent consisting of wa~er:acetoni-trile:sodium chloride (85:15-2 g./L.) and the solution was applied to a 4.7 x 45 cm. Michel-Miller high-performance-low-pressure-liquid-chromatography (HPLPLC) glass column (~ce Glass, Inc., Vineland, NJ 08360) packed in our laboratories with 25-40 micron LiChroprep RP-18 [hydro-carbon phase (Cl~) chemically bonded to silica gel, from MC/B Manufacturing Chemists, Inc~, Cincinnati, OH]. An FMI valveless piston pump (E'luid Metering Inc., Oyster Bay, NY 11771) was used to elute the column at 21 ml./min.
(100 psi), with the same solvent combination used for sample dissolution, collecting 21-ml. fractions. The eluate was monitored at 280 nm using an ISCO Model UA-5 W
detector. Fractions 1 183, inclusive, were discarded.
Fractions 184-245, inclusive, rich in factor B, were combined and concentrated under reduced pressure to 25 ml. Concentrates from seven similar purifications were combined, diluted to 1.4 L. with water, and applied at 8-10 ml./min. to 100 ml. of Diaion HP-20 resin in a column, previously equilibrated with water. The column was washed with water (600 ml.) until no chloride was - ~o -detected in the wash by precipitation as silver chloride.
Elution was performed with water.methanol (1:1) at 8-10 ml./min., collecting 300 mlO fractions. Fractions were analyzed for activity against B. subtilis~ Fractions 1-5 were combined, concentrated under reduced pressure, and lyophilized to give 523 mg. of crude factor B.
A 550 mg. portion of two combined crude pre-parations of factor ~ was dissolved in 10 ml. of a solvent consisting of water.acetonitrile:dibutylamine ~75:25:0.03M, which solvent had been adjusted to pH 7.8 with phosphoric acid3 by addition of tetrabutylammonium hydroxide until solution had been accomplished. The solution was applied to a 2.8 x 59 cm. Michel-Miller HPLPLC glass column packed with 25-40 micron LiChroprep RP-8 [hydrocarbon phase (C8) chemically bonded to silica gel, from MC/B Manufacturing Chemists, Inc.~ Cincinnati, OH]. Using an FMI pump, the column was eluted at 5 ml./min. (35 psi~ with the same solvent combination used for sample dissolution. The eluate was monitored at 254 nm using an ISCO Model UA-5 W detector. Selected 27-ml fractions were analyzed for the presence of factor B by analytical HPLC on a 4.6 x 250 mm. stainless steel column packed with 10 micron LiChrosorb RP-18 (a commercially available, reversed-phase silica gel, manufactured by E.
Merck, Oarmstadt, Germany). The sample was applied using a Rheodyne Model 7120 injection valve The solvent, con-sisting of water:acetonitrile:dibutylamine (82-18:0.03M) adjusted to pH 2.5 with phosphoric acid, was supplied ~' ~-5953~

at 1 ml./mi~. (750 psi) by a Constametric IrI pump (LDC-Laboratory Data Control, Division of Milton Roy Co., Riviera Beach, FL 33404). Factor B was detQcted at 225 nm usiny an LDC Spectro Monitor III varlable wavelength UV detactor. The por~ion o the RP~8 column eluate from 99g-1296 ml., rich in factor B, was con~entrated to a volume of ~OO ml. T~e concen-trate was dilut~d to a vol~e of 500 ~.1., adjusted to pH 2.0 with phosphoric ~cid, and sodiu~ chloride 10 ~1 mg. /ml . ) wa~ added as an ionic mar~cer . This solution was applied at 20 ml./min. to 100 ml. o Diaion ~-20 resin in ~ column (2.8 x 22 c~.), pr~viously equili~rated with water . The column was washed ~h water (500 ml.) adjusted to pH ~.5 with a~ueous fo~nic acid, ur.til no 15 chlo~ide ~,7as detected in the wash by precl2itation as s ilver chloride . The column was ~hen eluted wi th 1 L .
of watex:acetonitxile (6:4) at 30 ml./min. The eluate was concentrated ~nder raduced pressure and lyophili~ed to gi~e 295.6 ~g. of crude factor B.
~0 A 285 mg~ portion of this prepara~ion was dissolved in 30 ml~ dimethylf3~nma~ide:water (1:6) by heating, cooled to room temperatu~e, and refrigerated, resulting in preci~ita~ion of ~actor B. The precip-it~te was xecovered by filLratlon, washed ~ith acetone, ~A
Z5 and ~ried urder vacuu~, to ~ield ~4 mg. of Eactor B.
Exampl2 5 Isolation Oc ~1030 ~actor C
~ g~0 ~. portlon of th~ A~1030 com~le~. was 3~ dissolved in ~00 .nl. OL a aolvent COIlS~ stin~ cf water:

X-S953A -82~

acetonitrile:sodium chloride (83:11:2 g./L.) and the solution was filtered. The il~rate was applied to an 8 x lO0 cm. stainless steel colum~ pac'~ed with 4 ~. of 10-20 micron LP-l~Cl~ reversed-phase silica gel which was prepared in our laboratories by the special pro~
cedure described in Example 3. The column r part of a Chromatospac Prep-lO0 unit, was eluted at 60 ml~/min.
with the same solvent combination used for sa~ple dissolution, and 480-ml. frac~ions were collected. The eluate was monitoxed at 254 nm us.ing an ISC0 l~odel UA-5 W detector. Sel~.cted fractions were analy2ed for the presence of ractor C by analytical HPL~LC on an 0.~ X
30 cm. ~ichel-~liller glass colur.~n packed in our lab oratories with 25-40 micron LiChropre~ P~-8. The aOl~
l~ vent, water:acetonltrile:sodiuI~. chloride (~4:l6:2 g./L.), - ~as supplied at ~ ml./min. by an E'~I pump. Factor C
was detected at 254 nm uslng an ISCO.Model UA-5 UV
det~ctor. Fr~c'ion~ 1-27, incLusi~e, were disc2rded.
~ractions 28-52j inclu~ive, rich in fa~tor C, were combined and concentrates unc~er reduced ~ressur~ to a volume o~ 500 m7.
Concentrates ~rom twc similar purifica~ ons were combinsd, filtered, and applied at lO ml.,min. to lO0 ml. of Diaion HP-20 resin in ~ column ~2.8 x 22 cm.)~
pre~JiGusl~ equilibra~ed wi~h water. r~he cclumn was washed ~,~it~ w,~ter (2 L~) until no chlori~e was d-~tected in th~ wash '.oy pxeciPitation as siiver ch'orlde~
Elution was pe~o~med ~ith l L. o~ iater:acetonitrils ~ 15 ml.Jm~n. The eluate was concent~ated undex ~educe-~d pressure and iyophili~e~ to give ~ g. o a .. acto~ C-e~rich.e~ rnl,~ture o factor~ 5 g. ~or~ion

9~

X-5953.~ -~3-o this mixture was dissolved in 25 ml. of a solvent consi~ting of water:ac~tonit~ile:dibutylamine (80.20:0~0 3L~ r which solvent had ~een adjusted to pH 7.8 ~i~h phosphoric acid) by addition o tetrabutyla~monium hydroxide until solu~ion had been accomplished. The sample was applied to a 2O8 x 59 cm. Michel-Mill~r ~lass column packed with 25 4n micron LiChroprep RP-8 and the column was elu~ed a~ 4 ml.~mi.~., using an FMI pump, with the same solvent com~ination used for s~mple dissolution. The eluate was monitored at 254 nm using an ISCO Mod~l UA-5 UV detector. Selected 28-ml, frac~ions were a~alyzed for the presence o~ factor C by analytical HPLC on a 4.6 ~ 150 ~m. 5tainles3 steel column packed in our la~or2tories t7ith lO micron hucleosil Cl~ (a commercially availab'e, .~e~ersed-pnase sillca gel, ~anu actured by Rainin Instrument Cc., Inc., Woburn, L~A
01801). The s~mpl~ T.~as applied using a Rheodyne ~lodel 7120 i~jection valve. The solvent, consisting o water:acetonitrile:sodiu~ ac.tate (81:19:2 g.fL.) adjusted t~ pH 6 with glacial acetic acid, ~as supplied at l ml.~mi~. by a ~ilton .~ov Duplex Minipump. Factor C
was de~ec~ed at ~25 nm usiny ar~ ISCQ Model 1~00 variable wavelengt;~ UV detector. The portion o, t~e elllate rrom 4.2-5.1 L., rich in factor C, was concentrated unde~
Z5 xeduced pressure to a volume o~ ~00 ml.
Concentr,~tes ~rsm th-ee slmilar puriricatlorls T~ere combined and diss~l~e~ by add~tiQn ~r phosphoric acid to pH 1.7. Sodlum chloride ~ mg~/Tnl.) ~.~a~ added as an ionic marker~ The s~mple was applied at 2G ml~,~mi.n~
33 to 100 ~l. of ~iaior ~20 resi~ in a col~mn (2.~ ~ 22 c~

X-5953~ -84-previously equilibrated with water~ The column was washed with aqueous f~r~ic acid of p~ 2.5 (300 mlO), until no chloride was detected in the wash by precip~
itation as silvar chloride. ~h~ column ~.~as eluted with l L. of watar:acetonitrile (6:4) at 30 ml./min. The eluata was collected, concentrated under raduced pressure, and lyophilized to give 0.87 g. of partlally purified ~actor C. This preparation w~s dissolved in 20 ml. o~ a solvent consisting of water:acetonitrile:
l~ di~utylamine (80:20:0.03M, whicn solvent ~ad been adjusted to pH 7.8 with phosphoric acid) ~v addition of tetr~butylammoni,~m hydroxide until solution had occurred.
T~e sample was chromatogra2hed on 25-40 micron LiChroprep . RP-~ in a 2.3 ~ 59 cm. ~ichel-Miller glass column, ~s previousl~ ~escribed. ~he ~ortion of the eluate from 2.~-3.20 L. was concentrate~ under reduced pre~sure to a volume of 500 ml.
Concentrates from two similar p~rifications w~re com~lned and desalted on a column containing ~0 ~iaion HP-20 resi~ in the fashion previously ~esc~i~ed.
Tha eluate was concentra~ed under r~duce~ pre3sure and lyophilized to g.ive 688 mg. oE Eactor C. ~ 678 mg.
portion of this preparation was dlssolved in 50 ml.
watQr acetsnitrile (604) b~ heatir.gO The ~olution was ~5 cool ed arld factor C precipltated u~on rerigeration.
The ~recipitat ~.~as recot,Tered by filtration, washed wlth ac3tone, and dried under vacu~ to glve 428 mg. of fac~or C~

~0 Example 6 Isolation of A4lO30 Factor D

A 6.0 g. portion of the A41030 complex was dis solved in 200 ml. of a solvent consisting of water:ace-tonitrileosodium chloride (83:17:2 g./L~) and the solu-tion filtered. The filtrate was applied to an 8 x lO0 cm.
stainless steel column packed with 4 L. of 10-20 micron LP-l/Cl8 reversed-phase silica gel which was prepared in our laboratories by the special procedure described in Example 3. The column, part of a Chromatospac Prep-lO0 unit, was eluted at 60 ml./min., with the same solvent combination used for sample dissolution, and 480-ml.
fractions were collected~ The eluate was monitored at 254 nm using an ISCO Model UA-5 W detector. Selected fractions were a~alyzed for the presence of factor D by analytical HPLPLC on an 0.8 x 30 cm. Michel-Miller glass column packed in our laboratories with 25-40 micron ~iChroprep RP-8. The solvent, water:acetonitrile:sodium chloride (84-16.2 g./L~), was supplied at 4 ml./min. using an FMI pump. Factor D was detected at 254 nm using an ISCO Model UA-5 W detector. Fractions 1-34, inclusive, were discarded. Fractions 35-53, inclusive, rich in factor D, were combined and concentrated under reduced pressure to a volume of about 500 ml.
Concentrations from two s~milar purifications were combined, diluted to 3 L. with water r and applied at 8-lO ml./min~ to lO0 ml. of Diaion HP-20 resin in a column ~2.8 x 22 cm.), previously equilibrated with water. The column was washed with water (300 ml.) ~19~

until no chloride was detected in the wash by precipita-tion as silver chloride. ~lution was performed with 1 L.
of a solvent consisting of water:acetonitrile (6:4) at 8-10 ml./min. The eluate was concentrated under reduced pressure and lyophilized to give 2.33 g. of a factor D-enriched mixture of factors.
A 1.15 g. portion of this mixture was dissolved in 25 ml. of a solvent consisting of water:acetonitrile:di~
butylamine (80:20:0.03M, which solvent had been adjusted to pH 7.8 with phosphoric acid) by addition of tetrabutyl-ammonium hydroxide until solution occurred. The sample was applied to a 2.8 x 59 cm. Michel-Miller glass column packed with 25-40 micron LiChroprep RP-8, and the column was elu~ed at 5 ml./min., using an FMI pump, with the same solvent combination used for sample dissolution. The eluate was monitored at 254 nm using an ISCO Model UA-5 W
detector. Selected 25 ml~ fractions were analyzed for the presence of factor D by analytical HPLC on a 4.6 x 25 mm.
stainless steel column packed with 10 micron LiChrosorb RP-18 (a commercially available, reversed-phase silica gel, manufactured by E. Merck, Darmstadt, Germany). The sample was applied using a Rheodyne Model 7120 injection valve. The solvent, consisting of water:acetonitrile:di-butylamine (80:20:0.03M) adjusted to pH 2.5 with phos-phoric acidl was supplied at 0.75 ml /min~ using a Milton Roy Duplex Minipump. Factor D was detected at 225 nm using an ISCO Model 1300 variable wavelength W detector.
The portion of the eluate from 2.6-3.4 L~, rich in factor D, was concentrated under reduced pressure to a volume of 300 ml.

;~

21~

~87-Concentrates from three similar purifications were combined and dissolved by adclition of phosphoric acid to pH 7.7. Sodium chloride (1 mg./ml,) was added to an ionic marker. The sample was applied a~ 20 ml./min.
to 100 ml. of Diaion HP-~0 resin in a column ~2.8 x 22 cm.), previously equilibrated with water. The column was washed with water (300 ml.) ad]usted to pH 2.5 with aqueous formic acid, until no chloride was detected in the wash by precipitation as silver chloride~ The column was eluted with 1 L. of wa~er:acetonitrile (6:4) at 30 ml./min.
The eluate was concentrated under reduced pressure and lyo-philized to give 0.63 g. of partially puriied factor D.
This preparation was dissolved in 15 ml. of a solvent consisting of water:acetonitrile:dibutylamine (80:20:0.03M, which solvent had been adjusted to pH 7.8 with phosphoric acid) by addition of tetrabutylammonium hydroxide until solution occurred. The solution was chromatographed on 25-40 micron LiChroprep RP-8 in a 2.8 x 59 cm. Michel-Miller glass column, in the manner previously described.
The portion of the eluate from 2.5-3.0 L. was concentrated under reduced pressure to a volume of about 200 ml. This concentrate was desalted on a column containing Diaion HP~20 resin in the fashion previously described. The eluate was concentrated under reduced pressure and lyo philized to give 193 mgO of partially-puriied factor D.
A 259 mg. portion of two combined partially purified actor D preparations was dissolved in 6 ml.
of a solvent consisting of waterOacetonitrile:dibasic sodi~ phosphate ~82:18:0.03M, which solvent had been ~,.

-~8-adjusted to pH 7.8 with phosphoric acid) and adjusted to pH 10 by addition of aqueous 5N NaOH. The solution was applied to a 2.8 x 59 cm. Michel-Miller glass column packed with 25 40 micron LiChroprep RP-8, and the column was eluted at 4 ml./min., using an FMI pump~ with the same solvent combination used for sample dissolution. The eluate was moni tored at 254 nm using an ISCO Model UA-5 W
detector~ Selected 27-ml. fractions were analyzed for the presence of factor D by analytical HPLC on a 4.6 x 150 mm.
stainless steel column packed in our laboratories with l0 micron Nucleosil C18. The sample was applied using a Rheodyne Model 7120 injection valve. The same solvent combination used for the preparative elution was supplied at 0~6 ml./min. by a Milton Roy Duplex Minipump. Factor D
was detected at 225 nm using an ISCO Model 1800 variable wavelength W detector. The portion of the eluate from 405-1134 ml. was concentrated under reduced pressure to a volume of 500 mlO, and desalted on a column containing Diaion HP-20 resin in the fashion previously described.
The eluate was concentrated under reduced pressure and lyophilized to give 120 mg. of factor D.

Example 7 Isolation of A41030 Factor E

A 0.3 g. portion of the A41030 compl~x was dissolved in 30 mla of a solvent consisting of water:
acetonitrile:sodium chloride (85:15:2 g./L.~, and applied to a 2.8 x 59 cm. Michel-Miller glass column packed in our Laboratories with 25-40 micron LiChroprep RP-8. An FMI pump was used to elute the column a~ 12 ml,/min. (~5 psi), with the same solvent combination used for s~mple dissolution, collecting 240 ml~ fractionsO The eluate was monitored at 254 nm using an ISCO Model UA-5 W detector. Fractions 1-54, inclusive, were discarded, Fractions 55-122, inclusive, rich in factor E, were combined and concentrated under reduced pressure to a volume of 50 ml.
Concentrates from 13 similar purifications were combined, diluted to 1,5 L. with water, and applied at 5 ml./min~ to 100-ml. of ~iaion ~P-20 resin in a column (2.8 x 22 cm.), previously equilibrated with water~ The column was washed with water (900 ml.) until no chloride was detected in the wash by precipitation as silver chloride, Elution was then performed with water:methanol (1:1) at 10 ml./min,, collecting 300-mlO fractions.
Fractions were analyzed for activity against B. subtilis.
Fractions 1-8, inclusive, were combinedl concentrated under reduced pressure, and lyophilized to give 1.04 g.
of a factor E-enriched mixture of factors. A 0.5 g.
portion of this mixture was dissolved in 10 ml. of a solvent consisting of water:acetonitrile:sodium chloride (84:14:2 g./L.), and applied to a 2.8 x 59 cm. Michel-Miller glass column packed with 25-40 micron LiChroprep RP-8. An FMI pump was used to elute the column at 5 ml./min., with the same solvent combination used for sample dissolution, and 25-ml. fractions were collected, The eluate was moni~ored at 254 nm using an ISCO Model UA~5 W detectorO Selected fractions were analyzed for the presence of factor E by analytical HPLC on a 4.6 x ~.

~i99~D~

150 mm. stainless steel column packed in our laboratories with 5 micron ODS-llyperspheres (Shandon Southern Products, Ltd., Cheshire, England). The sarnple was applied using a Rheodyne Model 7120 injection valve. The solvent, consisting of water:acetonitrileosodium acetate (81:19:2 g./L.) adjusted to pH 6 with glacial acetic acid, was supplied at 0.65 ml./min. by a Milton Roy Duplex Minipump. Factor E was detected at 225 nm using an ISCO
Model 1800 variable wavelength W detector. The portion of the eluate from 1520-1780 ml. ~as concentrated under reduced pressure to a volume of 50 ml.
Concentrates from three similar purifications were combined, diluted to 1 L. with water~ and applied at 10 ml~/min. to 100-ml. of Diaion HP-20 resin in a column (2.8 x 22 cm.), previously equilibrated with water. The column was washed with water (200 ml.) adjusted with aqueous formic acid to pH 2.5, until no chloride was detected in the wash by precipitation as silver chloride. Elution was performed with 0.5 L. of water:acetonitrile (6:4) at 15 ml~/min. The eluate was concentrated under reduced pressure and lyophilized to give 202.2 mg. of partially purified factor Eo This preparation was dissolved in 4 ml. of a solvent con-sisting of water:acetonitrile:sodium chloride (86O14:2 g~/L.) and chromatographed at 4 ml~/min. on a 2.8 x 59 cm. Michel-Miller glass column, packed with 25~40 micron LiChroprep RP-8, as previously describedO The portion of the eluate from 2060 2480 ml., rich in fac~
tor E, was concentrated under reduced pressure to a ~ ,~

volume of 50 ml. Concentrates from three similar puri-fications were combined and desalted on 100-ml~ of ~iaion HP-20 resin in a column, as previously described. The eluate was concentrated under reduced pressure and lyo-philized to give 242 mg. of fac~or E.

Example 8 Isolation of A41030 Factor F
A 9.0 g. por~ion of the A41030 complex was dis-solved in 200 ml. of a solvent consisting of water:ace-tonitrile sodium chloride (83:17:2 g./L.) and the solution was filtered. The filtrate was applied to an 8 x 100 cm.
stainless steel column packed with 4 L. of 10-20 micron LP-l/C18 reversed-phase silica gel which was prepared in our laboratories by the special procedure described in ~xample 3. The column, part of a Chromatospac Prep-100 unit, was eluted at 60 ml./min., with the same solvent combination used ~or sample dissolution, and 4S0-ml. frac-tions were collected. The eluate was monitored at 254 nm using an ISCO ~odel UA-5 W detector. Selected fractions were analyzed for the presence of factor F by analytical HPLPLC on an 0.8 x 30 cm. Michel-Miller glass column packed in our ]aboratories with 25-40 micron LiChroprep RP-8. The solvent, wateroacetonitrile:sodium chloride t34:16:2 g./L.)/ was supplied at 4 ml./min. by an FMI
pump. Factor F was detected at 254 nm using an ISCO Model UA-5 W detector. Fractions 1 25, inclusive, were dis-carded. Fractions 26-36, inclusive, rich in factor F, were combined and concentrated under reduced pressure to a volusne of about 500 ml.

-.r Concentrates from three similar purifications were combined, filtered, and the filtrate applied at 10 ml~/min. to 100-ml. of ~iaion HP-20 resin in a column (2.8 x 22 cm.), previously equilibrated with water. The column was washed with water (900 ml.) until no chloride was detected in the wash by precipitation as silver chloride. Elution was performed with 1 Lo of water:ace-tonitrile (6:4) at 15 ml~/min. The eluate was concen-trated under reduced pressure and lyophilized to give 2.6 9. of partially purified factor F. A 500 mg. por-tion of this preparation was dissolved in 10 ml. of a solvent consisting of water:acetonitrile:sodium chloride (34:16:2 g./L.), by adjustment to pH 7.0 with aqueous sodium hydroxide. The solution was applied to a 4.7 x 45 cm. Michel-Miller glass column packed in our labora-tories with 25-40 micron LiChroprep ~P-18. An F~I pump was used to elute the column at 6 ml./min., with the same solvent combination used for sample dissolution, and 24-ml. fractions wexe collected. The eluate was moni-tored at 254 nm using an ISCO Model UA-5 W detector.
Selected fractions were analyzed for the presence of factor F, using the analytical HPLPLC system previously described. The portion of the eluate from 1940-2520 ml.
rich in factor F was concentrated under reduced pressure to a volume of about 300 ml.
Concentrates from two similar purifications wexe combined and applied at 10 ml./min. to 100-ml. of Diaion HP 20 resin in a column (2~8 x 22 cm.), pre-viously equilibrated with water. Tlle column was washed with water (300 ml.) adjusted to pH 2.5 wlth formic acid, .~, -~3-until no chloride was detected in the wash by precipi-tation as silver chloride. Elution was performed with 0.75 L. of water:acetonitrile (6:4). The eluate was concentrated under reduced pressure and lyophili~ed to give 299 mgO of factor F.

Example 9 Isolation of A41030 Factor G
An 8 g. portion of the A41030 complex from Example 2 was dissolved in 200 ml. of a solvent consist-ing of water:acetonitrile:sodium chloride (84:16:2 g./L.) and filtered. The filtrate was applied to a stainless steel column (8 x 100 cm.) packed with 4 L. of 10-20 micron LP-l/C18 reversed-phase silica gel which was prepared in our laboratories by the special procedure described in Example 3. The column was part of a Chromatospac Prep-100 unit (see Example 3). The column was eluted at 60 ml./min. with water:acetonitrile~sodium chloride ~84:16:2 g./L.), collecting 480~ml. fractions.
The eluate was monitored at 2S4 nm as described in Example 3. Selected fractions were analyzed for the presence of factor G by an analytical high perormance liquid chromatography ~HPLC) procedure described in preceding Examples.
Eractions 22 35, inclusive, rich in factor G~ were combined and concentrated under reduced pres-sure to a volume of 500 ml. Concentrates from three similar purifications were combined, adjusted to pH
8.5 with aqueous sodium hydroxide, and filtered. The filtrate was applied at 10 ml~/min~ to 100 ml. of ~ .h Diaion E~P-20 resin in a column (2.8 x 22 cm), previously equilibrated with water. The column was washed with water (400 ml. adjusted to pH 2.5 with formic acid) until no chloride was detected in the wash by precipita~ion as silver chloride~ Elution was performed with water:aceto~
nitrile (6:4) at 15 ml./min., collecting 1 1,. fractions.
Fractions were analyzed for activity against B. subtilis.
The active fractions were combined, concentrated under reduced pressure, and lyop~ilized to give 2.85 9. of material.
A 0.5 9. portion of this material was dissolved in 10 ml. of a solvent consisting of water:acetoni~
trile:dibutylamine (80:20:0.03M, which solvent had been adjusted to pH 7.S with phosphoric acid) by addition of dibutylamine until solution had been accomplished (final pH 8.2~ The solution was applied to a 2.~ x 59 cm.
Michel-Miller ~PLPLC glass column packed with 25-40 micron LiChroprep RP 8 ~from MC/B Manufacturing Chemist, Inc., Cincinnati, OH).
Using an FMI pump, the column was eluted at 4 ml./min. with the same solvent combination used for sample dissolution. The eluate was monitored at 254 nm using an ISCO Model UA-5 W detector. Selected 10 ml. fractions were analyzed for the presence of factor G by the analy-tical HPLC procedure described in preceding Examples.
Fractions 54-74, inclusive, rich in factor G, were combined with fractions from two similar purif i-cations and applied at 10 ml~/min. to 100 mlO of ~iaion HP-20 resin in a colum~ (2~8 x 22 cm)~ previously equilibrated with water. The column was washed with r _95_ water (300 ml.) adjusted to p~I 2.5 with formic acid, until no chloride was detected in the wash by precipitation as silver chlorideb Elution was performed with 0. 75 L. of water:acetonitrile t6:4). The eluate was concentrated 5 under reduced pressure and lyophilized to give 960 mg. of factor G.

Example 10 Sample Preparation for Biological Assay and Quantitative Analysis of A41030 Factor A in Dried Whole Broth One liter of whole broth was concentrated to a volume of 200 ml. and lyophilized to give 31.5 9. of dried whole broth. A 400 mg. sample of the dried whole hroth was extracted 3 times with 10 ml. portions of water at pH
8.5. The extracts were combined, concentrated to a volume of 10 ml., and portions of this concentrate used for bio-logical assay. The turbidimetric assay was conducted on a semiautomated system (Autoturb~ microbiological assay system, Elanco) described by N.R. Kuzel and F.W. Kavanaugh in J. Pharmaceut. .Sci. 60(5~, 764 and 767 ~1971). In testing the A41030 complex, the following test parameters were used: Staphylococcus aureus ATCC 9144 in a nutrient broth medium (pH 7), incubated for Eour hours at 37C~
Test samples and standard were dissolved in methanol:water (l l)o The standard, A41030 factor A, was presented to the Autoturb~ carrousel at concentrations of 0.4, 0,6, 0.9, 1.2, and 1.5 mcg./ml~

One milliliter of the above concentrate was purified by the following procedure to be used for analysis by HPLC.
(a) One C-18 SEP-PA ~ cartridge (silica gel cartridge J Waters Associates, Inc., Milford, Mass.) was washed with 10 ml. of methanoll using a 10 ml. syringe with a Luer fitting, as known to the art.
(b) Wash the same cartridge with 10 ml. of water.
(c~ Apply 1 ml. of the concentrate from above to the cartridge at the rate of approximately 1 ml./min.
(d) Wash the cartridge with 1 ml. of water and blow the cartridge dry.
(e) Elute the cartridge ~ith 1 ml. of a solution of tetrahydrofuran:water (1:1) at about 0.5 ml./min.
(f) Remove the tetrahydrofuran from the eluate in vacuo, or alternativelyr under a nitrogen stream, and reconstitute the eluate to a volume of 1 ml. with water.
(g~ Analyze the solution by ~PLC procedure as described hereinbefore~

The results of the assay for biological activity and the HPLC analysis of the whole broth are recorded in Table 25, which follows.

U~ l U-w Ta33le 2 5 13iological Activity and HPLC Analysis of A41030A in Whole Broth Concentr. Total. Wt. Total Wt.
o~ + of o~ 9~ o~
S~Lmpl~ ~7O. W~. A41030A Activity* A41030A A41~30A
106.8 k~ 4,d~ mg~g 49~ g 472 ~ 96.1 14~ . 5 kg 10. 8 mg/g 1685 g 1582 g 93 . 9 .

ot~l }Jiolo-3ical activi.iy comprised of A41030 factors At B, C, D, E, F and G~
~,s deit~L mined ~3y ~PLC .

Claims (19)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows-

1. A process for preparing A41030 complex, or Factor A, B, C, D, E, F, or G, or a pharmaceutically-acceptable salt thereof, which comprises cultivating Streptomyces virginiae NRRL 15156, in a culture medium containing assimilable sources of carbon, nitrogen, and inorganic salts under submerged aerobic fermentation conditions.

2. A method according to claim 1 followed by separation of the complex from the culture medium.

3. A method according to claim 2, in which the factors A, B, C, D, E, F or G are isolated from the complex.

4. A process according go claim 3 for preparing A41030 factor A of structure:

or a pharmaceutically-acceptable salt thereof.

5. A process according to claim 3 for preparing A41030 factor B of structure:

or a pharmaceutically-acceptable salt thereof.

6. A process according to claim 3 for preparing A41030 factor C of structure:

or a pharmaceutically-acceptable salt thereof.

7. A process according to claim 3 for preparing antibiotic A41030 factor D, which is a white, amorphous solid, having:
(a) an approximate elemental analysis of 54.46%
carbon, 4.35% hydrogen, 7.58% nitrogen, 4.27 chlorine, and by difference, 29.34% oxygen;
(b) an observed molecular weight of about 1326, as determined by fast atom bombardment mass spectrometry;

(c) an infrared absorption spectrum having the following distinguishable maxima: 3448-3226 (strong, broad), 2959 (weak), 1661 (strong), 1592 (strong), 1511 (strong), 1429 (weak), 1290 (weak), 1227 (weak), 1212 (medium), 1163 (weak), 1143 (weak), 1053 (medium), and 1010 (strong) cm l;
(d) ultraviolet adsorption spectra with an absorption maximum, in acidic or neutral methanol:water (1:1), at 278 nm (.epsilon. 10,600) and, in basic methanol:water (1:1), at 298 nm (.epsilon.
19,900);
(e) two titratable groups in 66% aqueous di-methylformamide with pKa values of about 5 . 5 and 7.6;
(f) is soluble in alcohol-water mixtures, in dimethylsulfoxide, in dimethylformamide, in dimethylsulfoxide-water mixtures, in di-methylfoxmamide-water mixtures, in dilute aqueous acid, or in dilute aqueous base; and (g) the pharmaceutically-acceptable, non-toxic salts of said A41030 factor D.

8. A process according to claim 3 for preparing A41030 factor E of structure:

or a pharmaceutically-acceptable salt thereof.

9. A process according to claim 3 for preparing A41030 factor F of structure:

or a pharmaceutically-acceptable salt thereof.

10. A process according to claim 3 for preparing antibiotic A41030 factor G, which is a white solid having:
(a) an approximate elemental analysis of 50.02%
carbon, 4.61% hydrogen, 4.74% chlorine, 6.11%
nitrogen, and 30.70% oxygen;
(b) an observed molecular weight of about 1684, as determined by fast atom bombardment mass spectrometry;

(c) an infrared absorption spectrum having the following distinguishable absorption maxima:
3320 (very broad, strong), 2975 sharp, weak), 2920 (sharp, weak), 1659 (normal, strong), 1594 (broad, strong), 1512 (sharp, strong), 1492 (shoulder) 1430 (sharp, weak), 1386 (broad) weak), 1337 (broad, weak), 1308 (sharp, weak), 1264 (sharp, weak), 1230 (broad, medium), 1145 (broad, medium), 1077 (sharp, medium), 1062 (sharp, medium), 1014 (sharp, medium), and 846 (broad, medium) cm-1;
(d) ultraviolet absorption spectra with an absorption maximum, in acidic or neutral methanol:water (1:1), at 278 nm (.epsilon. 15,000) and, in basic methanol:water (1:1), at 298 nm (.epsilon.
18,000);
(e) two titratable groups in 66% aqueous di-methylformamide with pKa values of about 5.4 and 7.0;
(f) is soluble in alcohol-water mixtures, in dimethylsulfoxide, in dimethylformamide, in dimethylsulfoxide-water mixtures, in dimethylformamide-water mixtures, in dilute aqueous acid, or in dilute aqueous base; and (g) the pharmaceutically-acceptable, non-toxic salts of said A41030 factor G.

11. A41030 antibiotic complex or a pharmaceutically acceptable salt thereof, whenever prepared by a process according to claim 2, or by an obvious chemical or biological equivalent thereof.

12. A41030 antibiotic factor A or a pharmaceutically acceptable salt thereof, whenever prepared by a process according to claim 4, or by an obvious chemical or biological equivalent thereof.

13. A41030 antibiotic factor B or a pharmaceutically acceptable salt thereof, whenever prepared by a process according to claim 5, or by an obvious chemical or biological equivalent thereof.

14. A41030 antibiotic factor C or a pharmaceutically acceptable salt thereof, whenever prepared by a process according to claim 6, or by an obvious chemical or biological equivalent thereof.

15. A41030 antibiotic factor D or a pharmaceutically acceptable salt thereof, whenever prepared by a process according to claim 7, or by an obvious chemical or biological equivalent thereof.

16. A41030 antibiotic factor E or a pharmaceutically acceptable salt thereof, whenever prepared by a process according to claim 8, or by an obvious chemical or biological equivalent thereof.

17. A41030 antibiotic factor F or a pharmaceutically acceptable salt thereof r whenever prepared by a process according to claim 9, or by an obvious chemical or biological equivalent thereof.

18. A41030 antibiotic factor G or a pharmaceutically acceptable salt thereof, whenever prepared by a process according to claim 10, or by an obvious chemical or biological equivalent thereof.

19. The axenic culture of the microorganism Streptomyces virqiniae NRRL 15156.