A40926, a New Glycopeptide Antibiotic with Anti-Neisseria Activity

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Dec p /87/ $02.00/0 Copyright 1987, Americn Society for Microbiology Vol. 31, No. 12 A40926, New Glycopeptide Antibiotic with Anti-Neisseri Activity BETH P. GOLDSTEIN,* ENRICO SELVA, LUCIANO GASTALDO, MARISA BERTI, ROSA PALLANZA, FRANCA RIPAMONTI, PIETRO FERRARI, MAURIZIO DENARO, VITTORIO ARIOLI, AND GIOVANNI CASSANI Merrell-Dow Reserch Institute, Lepetit Reserch Center, Gerenzno (Vrese), Itly Received 4 June 1987/Accepted 22 September 1987 In the course of serch for glycopeptide ntibiotics hving novel biologicl properties, we isolted A Produced by n ctinomycete of the genus Actinomdur, A40926 is complex of four min fctors which contin ftty cid s prt of glycolipid ttched to the peptide bckbone. Its ctivity ws, in most respects, similr to tht of other glycopeptides, such s vncomycin nd teicoplnin. However, in ddition to inhibiting grm-positive bcteri, A40926 ws very ctive ginst Neisseri gonorrhoee. A40926 ws rpidly bctericidl for N. gonorrhoee clinicl isoltes t concentrtions equl to or slightly higher thn the MIC. In mice, levels in serum were higher nd more prolonged thn those of n equivlent subcutneous dose of teicoplnin. These properties suggest tht A40926 my hve potentil in the therpy of gonorrhe. The incresed frequency of isoltion of multiply ntibioticresistnt grm-positive bcteri hs led to wider use of ntibcteril gents which were originlly rrely considered for first-line therpy, such s the glycopeptide vncomycin (18). One of the dvntges of this ntibiotic is tht, thus fr, resistnce is rre nd restricted to certin cogulse-negtive stphylococci (25). The success of vncomycin hs stimulted the development of new glycopeptides, such s teicoplnin (5, 13, 32, 33). These ntibiotics inhibit cell wll biosynthesis by binding to the D-lnyl-D-lnine portion of nscent peptidoglycn (22). Specific binding to n ffinity resin contining cyl-d-lnyl-d-lnine groups (9) formed the bsis of our serch for new glycopeptides. Among the new glycopeptides identified, we focused our ttention on those hving novel biologicl properties. A40926 is the most interesting ntibiotic which hs emerged thus fr from our screening cmpign. In fermenttion broths, we found it s complex of four fctors (PA, PB, A, nd B). PA nd PB, described here for the first time, predominted in the broth; during purifiction, these two compounds were lrgely converted to A nd B, whose structures hve been described recently (31). One feture of ll four fctors, which reltes them to teicoplnin nd some other glycopeptides (ridicins nd kibdellins) (3, 27, 12) while distinguishing them from vncomycin, ristocetin, nd others, is the presence of ftty cid moiety ttched to one of the sugrs. In terms of biologicl ctivity, the most striking difference between the components of the A40926 complex nd other glycopeptide ntibiotics is their ctivity ginst Neisseri gonorrhoee. MATERIALS AND METHODS Isoltion of A We screened specificlly for glycopeptide ntibiotics by pssing fermenttion broths over the ffinity resin Sephrose-D-lnyl-D-lnine (9) nd eluting dsorbed glycopeptides with 1% queous mmoni. The strin producing A40926 ws found in soil smple collected in Indi. Btch dsorption to the sme ffinity resin described bove yielded nerly pure A40926 complex in one step. Quick neutrliztion of the mmoni elute yielded primrily the two fctors of the complex (PA nd PB) which * Corresponding uthor. predominted in the fermenttion broth. After prolonged incubtion t high ph, these components were grdully converted into fctors A nd B. The A40926 complex used in the microbiologicl studies consisted of 65% B nd 35% A. The structures of fctors PA nd PB were elucidted by fst tom bombrdment mss spectrometry, nd 1H nucler mgnetic resonnce spectroscopy t 250 MHz in dimethyl sulfoxide d-6 s described for fctors A nd B (31). Other glycopeptide ntibiotics. Actplnin, A35512B, A41030A, nd A47934 were obtined from Eli Lilly & Co.; voprcin ws from Lederle Lbortories. Ristocetin ws purchsed from Sigm Chemicl Co., nd ridicin (14) ws prepred by fermenttion of Kibdellosporngium ridum ATCC Preliminry ntibcteril ctivity of the fctors of the complex. The MICs for Clostridium di ffcile, Propionibcterium cnes, Bcteroides frgilis, nd Hemophilus influenze were determined by gr dilution (inocul, 104 CFU). MICs for other orgnisms were determined by broth mcrodilution (inocul, 104 to 105 CFU/ml) for Clostridium perfringens nd by broth microdilution for other bcteri. Incubtion times were 18 to 24 h, except for N. gonorrhoee, H. influenze, C. difficile, Propionibcterium cnes, nd B. frgilis (48 h). All orgnisms were incubted t 37 C. N. gonorrhoee nd H. influenze were incubted in 5% CO2 tmosphere; nerobic bcteri were incubted in n nerobic gs 1961 mixture. The medi used were Iso-Sensitest broth (Oxoid Ltd.) (stphylococci, Streptococcus feclis, Escherichi coli, Pseudomons eruginos, Proteus vulgris); Todd- Hewitt broth (Difco Lbortories) (other streptococci); GC bse broth (Difco) plus 1% IsoVitleX (BBL Microbiology Systems) (N. gonorrhoee); GC bse gr plus 1% IsoVitleX nd hemin (10,ug/ml) (H. influenze); AC medium (Difco) (C. perfringens); Wilkins-Chlgren gr (Oxoid) (other nerobic bcteri). MIC of A40926 complex for grm-positive bcteri nd neisserie. MIC studies were performed by gr dilution with inocul of 104 CFU. The medi used were Iso-Sensitest gr for stphylococci, Streptococcus feclis, nd Streptococcus fecium; Todd-Hewitt gr (Difco) for other streptococci; nd GC bse gr (Difco) plus 1% IsoVitleX nd hemin (10,ug/ml) for neisserie. Neisserie were incubted for 48 h in 5% C02; Streptococcus snguis, Streptococcus slivrius, nd Streptococcus mutns strins were incubted

2 1%2 GOLDSTEIN ET AL. for 48 h in n nerobic gs mixture; Streptococcus pneumonie ws incubted for 40 h nd other species were incubted for 24 h in norml tmosphere. Bctericidl ctivity. Inocul were t lest 106 CFU/ml. The time-kill experiment with Streptococcus feclis ws performed in Todd-Hewitt broth with log-phse inocul. At the indicted smpling times, duplicte 0.1-ml smples of suitble dilutions (t lest 10-fold in sline with peptone) were plted, by inclusion in 2.5 ml of Todd-Hewitt soft gr, on Todd-Hewitt gr pltes. For N. gonorrhoee MBC nd time-kill studies, contct with ntibiotics ws in GC bse broth with 1% IsoVitleX. Inocul were prepred from 24-h slnts; cultures were grown to log phse before ddition of ntibiotics. At the indicted smpling times, triplicte 0.01-ml smples were dripped cross the surfce of pltes contining GC bse gr plus 1% IsoVitleX nd hemin (10 pug/inl). The lowest concentrtion which killed t lest 99.9% of the initil inoculum ws considered the MBC. Preliminry studies in which ntibiotic ws dded immeditely before plting showed tht under our experimentl conditions, initil concentrtions of up to 16 times the MIC hd no effect on the plting efficiency of stphylococci, Streptococcus feclis, or N. gonorrhoee. Experimentl septicemi in mice. Ech control nd tretment group contined five mle nd five femle CD-1 mice (Chrles River Breeding Lbortories) weighing 18 to 22 g. They were infected intrperitonelly with 0.5 ml of bcteril suspension prepred by diluting n overnight culture with sterile sline- plus peptone (Streptococcus pyogenes nd Streptococcus pneumonie) or with 5% bcteriologicl mucin -(Difco) (Stphylococcus ureus). Inocul were djusted so tht untreted nimls died of septicemi within 48 h (2). Antibiotics were dministered subcutneously immeditely fter infection. The 50% effective dose (ED50) (dose t which 50% of nimls survived) ws clculted by the method of Spermn nd Krber (11) from the percentges of surviving nimls t ech dose on dy 7. Phrmcokinetics. For ech smpling time, five mle nd five femle mice were treted subcutneously with 10 mg of A40926 fctor B per kg. Blood (0.5 ml per niml) ws collected by crdic puncture; serum from pooled blood ws ssyed by the gr diffusion method on ntibiotic medium 3 (Difco) (ph 6) supplemented with 1% Noble gr (Difco), 6.65% NCl, 10% (vollvol) 1 M ph 4.2 phosphte buffer, nd 0.5% (vol/vol) bovine serum. The test orgnism ws S. ureus ATCC 6538; the ssy is sensitive to 1,ug of fctor B per ml. RESULTS Chrcteriztion nd txonomic identifiction of the producer strin. The substrte mycelium is composed of brnched hyphe. The eril hyphe form short nd compct closed spirls, some of them with hooklike structure, contining chins of 10 to 20 spores. The spores re irregulrly shped, roughly globulr-cylindricl, with n verge size of 0.8 by 1.2,um. The strin grows well on most medi used to study its culturl chrcteristics (26, 31) nd produces red-violet pigment on otmel gr. In the whole-cell hydrolyste (4, 21), mdurose ws the dignostic sugr. Anlysis of cell wll preprtions (17, 19, 29) showed the presence of meso-diminopimelic cid nd the bsence of glycine. These fetures identified the strin s n ctinomycete of cell wll type IIIB.(20). This informtion, together with the morphologicl chrcteristics of the strin nd the bsence of mycolic cids, led us to clssify it in the ANTIMICROB. AGENTS CHEMOTHER. genus Actinomdur. The strin hs been deposited with the Americn Type Culture Collection s ATCC Structure of the components of the complex. Figure 1 shows the structures of A40926 fctors PA, PB, A, nd B. All four contined two chlorine toms nd two sugrs, mnnose, nd N-cylminoglucuronic cid. The cyl groups were n- undecnoic cid in PA nd A nd isododecnoic cid in PB nd B. Among the glycopeptides, teicoplnin, ridicins, nd kibdellins lso contin ftty cid moieties (3, 12, 27). Another similrity between A40926 nd the ridicins ws the presence of two crboxylic cid functions which gve these molecules net negtive chrge t neutrl ph. From the moleculr weight differences nd the nucler mgnetic resonnce dt, we determined tht A nd B differed from PA nd PB in the loss of n cetyl group t position C-6 of mnnose, Preliminry chrcteriztion of ntibcteril ctivity. We compred the MICs of A40926 A+B complex nd of pure fctors A, B, PA, nd PB with those of teicoplnin for single strins o-f vrious bcteril species (Tble 1). Aginst the grm-positive bcteri, the MICs of the four A40926 fctors were similr to those of teicoplnin. The ctivity of A40926 ginst N. gonorrhoee stood out from spectrum otherwise similr to tht of teicoplnin. In common with other glycopeptides, A40926 ws not ctive ginst the other grm-negtive orgnisms tested (Tble 1) or ginst mycoplsms (dt not shown). As the nti-neisseri ctivity of glycopeptides hd not been previously described, we tested ll the vilble compounds of this clss under our own experimentl conditions. Activity might hve been missed if chocolte gr hd been used; we hve observed tht the dentured hemoglobin 04 O%C-R HN' Fctor R MW A (CHA)PH3 H 1716 PA (CH2)WH3 GOCH B (CH2)CH(CH3)2 H 1730 PB (CH 2WCH(CH3)2 CGOH FIG. 1. Structures of the four principl fctors of A40926 complex. MW, Moleculr weight.

3 VOL. 31, 1987 NEW GLYCOPEPTIDE ANTIBIOTIC 1963 Orgnism TABLE 1. Teicoplnin Spectrum of ntibcteril ctivity of A40926 MIC (,ug/ml) A40926 A+ B complex Fctor A Fctor B Fctor PA Fctor PB Stphylococcus ureus Tour Streptococcus pyogenes C Streptococcus pneumonie UC Streptococcus feclis ATCC Clostridium perfringens ISS Clostridium difficile ATCC Propionibcterium cnes ATCC Bcteroides frgilis ATCC Neisseri gonorrhoee ISM 68/ Hemophilus influenze ATCC Escherichi coli SKF >128 >128 >128 >128 >128 >128 Proteus vulgris ATCC 881 >128 >128 >128 >128 >128 >128 Pseudomons eruginos ATCC >128 >128 >128 >128 >128 >128 The four fctors of the complex were isolted by reverse-phse high-pressure liquid chromtogrphy. present in this medium reduces the ctivity of glycopeptides A representtive experiment, in which Streptococcus (R. Pllnz nd R. Scotti, unpublished dt). The MICs of feclis strin ws exposed to ntibiotic concentrtions 8 these ntibiotics for test strins of vrious species re shown nd 16 times the MIC, is shown in Fig. 2. Vncomycin ws in Tble 2. The ctivities of most of these compounds ginst not bctericidl for this strin under these conditions. severl grm-positive species hve been compred previ- Anti-Neisseri ctivity of A Tble 4 summrizes our ously (14). Among the glycopeptides we tested, A40926 ws MIC dt for N. gonorrhoee clinicl isoltes, including the most ctive ginst our stndrd test strin of N. penicillinse-producing N. gonorrhoee, chromosomlly degonorrhoee. A47934, which is n glycone (7) (i.e., it termined penicillin-resistnt N. gonorrhoee, nd spectinocontins no sugrs), ws modertely ctive ginst N. gon- mycin-resistnt strins. The medin MIC of A40926 for the orrhoee, but less so thn A The other glycopeptide- 37 strins ws 1,ug/ml, compred with 16,ug/ml for glycone, A41030A (10), ws not ctive. teicoplnin nd 32,ug/ml for vncomycin. The ctivity of In vitro ctivity ginst stphylococci nd streptococci. We A40926 ginst N. gonorrhoee ws thus quite similr to its compred the MICs of A40926 A+B complex nd teicopl- ctivity ginst cogulse-negtive stphylococci (Tble 3). nin for totl of 132 clinicl isoltes (Tble 3). More thn The MBC, determined for seven strins of N. gonorrhoee, hlf of the Stphylococcus strins were methicillin resistnt ws equl to or twice the MIC even fter only 6 h of contct (MR). In our nlysis, we combined MR nd methicillin- (dt not presented). susceptible (MS) strins becuse previous studies with our The bctericidl ctivity of A40926 ginst N. gonorcollection filed to revel methicillin-relted differences in rhoee ws firly rpid. From 99.9 to 99.99% killing of four susceptibilities to glycopeptides (1). Nine of 10 Stphylococ- clinicl isoltes (including spectinomycin-resistnt nd cus hemolyticus (including two MS strins), three MR penicillinse-producing strin) occurred within 5 h of expo- Stphylococcus epidermidis, five of the six MR S. ureus, sure to concentrtions four to eight times the MIC (Fig. 3). nd two other MS cogulse-negtive stphylococci were A40926 ws somewht less ctive ginst Neisseri menresistnt to gentmicin. With one exception, the species or ingitidis. The MIC rnge for five stndrd strins from groups of species showed the sme medin MIC for A40926 serologicl groups A through D ws 4 to 16,ug/ml. nd teicoplnin; their medinr MICs differed (by fctor of 2) Activity of A40926 in experimentl septicemi in mice. We only for Streptococcus feclis. compred the ED50s of teicoplnin nd A40926 fctor B in The bctericidl ctivities of these two ntibiotics ginst nimls infected with vrious grm-positive species (Tble stphylococci nd Streptococcus feclis were lso similr. 5). A40926 ws slightly less ctive thn teicoplnin in these TABLE 2. Comprison of ntibcteril ctivity of vrious glycopeptides MIC (p.g/ml) for : Compound VNeisseri Streptococcus Streptococcus Stphylococcus Stphylococcus gonorrhoee pyogenes feclis ureus epidermidis A A Teicoplnin Vncomycin Ristocetin ,A35512B A41030A Aridicin Avoprcin Actplnin > Sme strins s in Tble 1, plus Stphylococcus epidermidis (ATCC 12228).

4 1964 GOLDSTEIN ET AL. ANTIMICROB. AGENTS CHEMOTHER. TABLE 3. MICs of A40926 for stphylococci nd streptococci MIC (jig/ml), Orgnism (no. of isoltes) Teicoplnin A40926 A + B complex Rnge 50%o 90%o Rnge 50%o 90%o Stphylococcus ureus (6/6) Stphylococcus epidermidis (7/5) Stqphylococcus hdemolyticus (9/3) Other stphylococcic (9/7) Streptococcus pyogenes (11) Streptococcus pneumonie (10) Streptococcus feclis (10) Streptococcusfecium (10) Streptococcus glctie (10) Streptococcus mutns (13) Other streptococcid (16) For stphylococci, shown s number MR/number MS. b 50% nd 90o, MIC for 50 nd 90% of isoltes, respectively. S. simulns, S. cpitis, S. wrneri, S. cohnii (two strins ech); S. xylosus (one strin) S. hominis (three strins), nd S. sprophyticus (four strins). d 5. slivrius (seven strins), S. snguis (six strins), nd S. bovis (three strins). g model systems. In Streptococcus pneumonie infections, A40926 complex nd fctors A, PA, nd PB ll hd ctivity t*0similr to tht of fctor B (dt not presented). Phrmcokinetics of A40926 in mice. We compred the levels of A40926 fctor B nd teicoplnin in serum fter single subcutneous dministrtion of 10 mg/kg (Fig. 4). At this dose, the pek level in serum of A40926 ws much higher thn tht of teicoplnin, nd significnt concentrtions of A40926 were mintined for longer period of time. 7- DISCUSSION By using n ffinity resin to screen fermenttion broths, we were ble to identify strins which produced glycopeptides even when other types of ntibiotics were produced by 6- the sme strin. This pproch ws therefore more efficient E \ thn methods bsed on the reversl of ntibcteril ctivity i~ 2 t when ntibiotic preprtions re mixed with cyl-d-lnylo \\\ D-lnine (23, 28). The ltter method my miss glycopeptides when other nonsuppressed ntibiotic ctivities re present. O \,\o A40926 complex emerged from this screening cmpign. Although structurlly very similr to some other previously described glycopeptide ntibiotics, in prticulr the ridicins 4-\ (27), A40926 ws unique in its ctivity ginst N. gonorrhoee. In ll other respects, its in vitro ntibcteril ctivity \\&. ppers, thus fr, to be similr to tht of teicoplnin nd 3- vncomycin. Another novel feture of A40926 ws its high levels in serum nd long hlf-life in the mouse compred with teicoplnin nd vncomycin (5, 30). 0 A40926 ws somewht less ctive thn teicoplnin in the tretment of grm-positive septicemi in mice, even though 2_ the infecting orgnisms hd the sme in vitro susceptibilities h FIG. 2. Bctericidl effect of A40926 ginst Streptococcus feclis ATCC Concentrtions of ntibiotics used were 8 nd 16 times the MIC determined under the sme culture conditions used in this experiment: A40926 A+B complex, 8 (0) nd 16 (A) Fg/ml; teicoplnin, 8 (-) nd 16 (A),ug/ml; vncomycin, 16 (l) nd 32 (*),ug/ml; *, untreted control. TABLE 4. MICs of A40926, teicoplnin, nd vncomycin for 37 clinicl isoltes of N. gonorrhoee MIC (i±g/ml)b Antibiotic Rnge 509o 90%o A Teicoplnin Vncomycin 8-> The isoltes included penicillin-resistnt nd spectinomycin-resistnt strins. b 5 nd 90%, MIC for 50 nd 90o of isoltes, respectively.

5 VOL. 31, 1987 NEW GLYCOPEPTIDE ANTIBIOTIC 1965 Strin L 1001 MIC 1 L L L 1601 E 40\ 30\ h FIG. 3. Bctericidl effect of A40926 ginst four clinicl isoltes of N. gonorrhoee. Concentrtions of A40926 A+B complex were four nd eight times the MIC for ech strin, determined under the sme culture conditions used in this experiment. Symbols: 0, untreted control; 0, 4 jig/ml; O, 8,ug/ml; A, 16,ug/ml. Strin L1596 is spectinomycin resistnt, nd L1601 is penicillin resistnt. to both ntibiotics. One feture of our infection model is rpid dissemintion of bcteri to vrious orgns (2). In our experience, differences in ED50 s low s two- to threefold re of little significnce in this system. The phrmcokinetics of A40926 nd teicoplnin in the mouse hve thus fr been compred t the single dose of 10 mg/kg. The pek levels of the two ntibiotics might be more similr t the lower doses used to determine the ED50. (It is not possible to ssy blood levels t these doses.) Furthermore, the potentil dvntges of long-lsting ntibiotic my fil to emerge in such n cute infection. Another fctor which my ffect the ED50 is serum binding. Preliminry dt, bsed only on the MIC in the presence of vrious concentrtions of bovine serum, indicte tht A40926 my be bound somewht more strongly thn teicoplnin. Any or ll of these fctors might ccount for the modest differences in ED50- Although we cnnot yet fully ccount for the unusul ctivity of A40926 ginst N. gonorrhoee, the ntibiotic hs h fter dministrton FIG. 4. Levels of A40926 fctor B nd teicoplnin in mouse serum. Groups of five mle nd five femle mice were treted once with ntibiotic t 10 mg/kg. Serum smples from pooled blood smples were ssyed. Symbols: 0, A40926 fctor B; 0, teicoplnin. two structurl fetures which correlte with its longer hlflife of elimintion (14). One is its net negtive chrge t neutrl ph; in this, A40926 resembles the ridicins (27). The second feture is the lipophilicity conferred by the ftty cid moiety ttched to the N-minoglucuronic cid. Along with teicoplnin, ridicins, nd kibdellins, A40926 might be better described s lipoglycopeptide. N. gonorrhoee is n orgnism in which resistnce to two of the most widely used clsses of therpeutic drugs, penicillins nd tetrcyclines, hs been spreding worldwide (6, 15). Some penicillin-resistnt strins re lso reltively resistnt to cephlosporins nd erythromycin (24). Spectinomycin is n effective ntigonorrhel gent which, like penicillin, hs the dvntge of single-dose therpy. Spordic occurrences of primry resistnce, s well s of resistnce secondry to therpy, re, however, cuse for concern (8, 16). A totlly new clss of ntigonorrhel drug could thus be of gret interest. In the field of grm-positive infections, in which vncomycin hs been used for number of yers, resistnce to glycopeptide ntibiotics is rre mong normlly sensitive bcteril species. The phrmcokinetics of A40926 nd its ctivity ginst N. gonorrhoee suggest tht this new glycopeptide my hve potentil in single-shot therpy of gonorrhe. TABLE 5. Orgnism Activity of A40926 in experimentl septicemi in mice' Teicoplnin ED50 (mg/kg) A40926 fctor B Stphylococcus ureus Tour Streptococcus pyogenes C Streptococcus pneumonie UC Single subcutneous dministrtion of ntibiotic immeditely fter intrperitonel infection. The ED50 ws clculted t dy 7 from the percentges of nimls surviving t ech tretment level (11). LITERATURE CITED 1. Arioli, V., nd R. Pllnz Teicoplnin-resistnt cogulse-negtive stphylococci. Lncet i: Arioli, V., R. Pllnz, S. Furesz, nd G. Crniti Rifmpicin: new rifmycin. I. Bcteriologicl studies. Arzneim. Forsch. 17: Brn, J. C. J., D. H. Willims, D. J. M. Stone, T.-W. C. Leung, nd D. M. Doddrell Structure elucidtion of teicoplnin ntibiotics. J. Am. Chem. Soc. 106: Becker, B., M. P. Lechevlier, R. E. Gordon, nd H. A. Lechevlier Rpid differentition between Nocrdi nd Streptomyces by pper chromtogrphy of whole-cell hydroly-

6 1966 GOLDSTEIN ET AL. stes. Appl. Microbiol. 12: Berti, M., R. Pllnz, B. P. Goldstein, nd V. Arioli Teichomycin A2, new ntibiotic from Actinoplnes: ctivity in vitro nd in vivo, p In P. Periti nd G. Gildroni Grssi (ed.), Current chemotherpy nd immunotherpy, vol. 1. Americn Society for Microbiology, Wshington, D.C. 6. Biddle, J. W., Y. Jenlouis, nd M. S. McGlohn Detection of cliniclly significnt ntimicrobil resistnce in gonococci by gr-disk diffusion, p In G. K. Schoolnik, G. F. Brooks, S. L. Flkow, C. E. Frsch, J. S. Knpp, J. A. McCutchn, nd S. A. Morse (ed.), The pthogenic neisserie. Americn Society for Microbiology, Wshington, D.C. 7. Boeck, L. D., nd F. P. Mertz A47934, novel glycopeptide-glycone ntibiotic produced by strin of Streptomyces toyocensis. Txonomy nd fermenttion studies. J. Antibiot. 39: Boslego, J. W., E. C. Trmont, E. T. Tkfuji, B. M. Dinieg, B. S. Mitchell, J. W. Smll, W. N. Khn, nd D. C. 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