Establishment of Infection in Mice

Transcription

1 INFECTION AND IMMUNITY, Jn. 1983, p /83/ $02.00/0 Copyright 1983, Americn Society for Microbiology Vol. 39, No. 1 Enhncement by Cndid lbicns of Stphylococcus ureus, Serrti mrcescens, nd Streptococcusfeclis in the Estblishment of Infection in Mice EUNICE CARLSON Deprtment of Biologicl Sciences, Michign Technologicl University, Houghton, Michign Received 16 August 1982/Accepted 8 October 1982 Cndid lbicns hs been shown to stimulte infection in mice by number of bcteri. Mice inoculted intrperitonelly with s little s 500 colony-forming units of Stphylococcus ureus long with nonlethl dose of 108 colony-forming units of C. lbicns (one-third the dose cusing 50% mortlity in 7 dys) developed widespred stphylococcl infection. S. ureus injected lone t tht or considerbly higher doses did not estblish infection. Phge typing methods demonstrted tht the stphylococcl infection ws due to the bcteril orgnisms originlly injected. A minimum dose of C. lbicns between 1.1 x 1 nd 1.1 x 10 colony-forming units ws necessry for the mplifiction of virulence of S. ureus to tke plce. Serrti mrcescens nd Streptococcus feclis inoculted intrperitonelly t smll nonlethl doses could lso be recovered from blood nd tissues 5 dys fter nimls were dully injected with C. lbicns but not from nimls which were inoculted with the sme mounts of these bcteri lone. A previous report described synergistic effect between Cndid lbicns nd toxic shock syndrome-ssocited Stphylococcus ureus on mortlity of mice when dully infected (4). The present study ws designed to exmine the nture of this synergistic effect between these two orgnisms. The following questions were ddressed. (i) Is stimultion mutul, or does one orgnism enhnce infection by the other? (ii) How much is the virulence of the stimulted orgnism incresed? (iii) Is the effect specific for C. lbicns nd S. ureus, or do other C. lbicns-bcteril combintions lso exhibit synergistic effect on niml mortlity or morbidity? MATERIALS AND METHODS Mice. Inbred mice of the CD-1 strin were obtined from Chrles River Breeding Lbortories, Inc., Wilmington, Mss. Mle mice weighing between 22 nd 25 g were used. Pthogens. S. ureus 2460 ws received from the Michign Deprtment of Public Helth, which originlly obtined it from n Ann Arbor hospitl t which it ws isolted from ptient with toxic shock syndrome. It hs the phge group I sensitivity pttern, nd the dose tht cuses 50% mortlity 7 dys fter intrperitonel (i.p.) injection of mice (LD50) is 5.3 x 109 (4). Serrti mrcescens (deep red pigment), Streptococcus feclis, nd C. lbicns (LD50, 2.9 x 108 [4]) were obtined from the microbiology lbortory stock culture collection t Michign Technologicl University, Houghton. Cultures used for injection were grown on 5% sheep blood gr (blood gr bse [BBL Microbiology Systems, Cockeysville, Md.] plus whole 193 blood) for 24 h t 37 C (S. ureus nd S. feclis), Sbourud dextrose gr (BBL) for 48 h (C. lbicns), or nutrient gr (Difco Lbortories, Detroit, Mich.) for 24 h (S. mrcescens). The cultures were eluted nd wshed with sterile physiologicl sline, mixed vigorously, nd used immeditely. Titers were estimted t the time of injection from clibrtion curve relting the opticl density t 660 nm (Colemn junior spectrophotometer) to colony-forming units (CFU). CFU were determined by plte counts on blood nd Sbourud gr medium. Injections. Orgnisms were injected i.p. with the desired dose of pthogen in 0.2 ml of sline. Injected nimls were housed singly in sterilized cges, nd gret cre ws tken to prevent pthogen exposure in ny wy other thn injection. Anhnl tissues. Five dys fter injection, nimls were scrificed (chloroform); fter deth, orgns were rinsed quickly in 70%o lcohol to remove ny surfce contmintion, rinsed in sterile sline, nd homogenized with mortr nd pestle; nutrient broth ws used to mke finl volume of 1 ml. The surfces of the peritonel cvity were wiped with moist Q-tip swb, which ws plced in 1 ml of broth. Blood ws drwn by crdic puncture, nd 0.1 ml ws trnsferred to 1 ml of nutrient broth. CFU determintions were mde on these 1-ml smples s mesurement of infection. The pthogen content of these initil 1-ml orgn smples ws determined by vigorous mixing nd dilution into nutrient broth nd ssyed between the dilutions of 1/102 through 1/105, depending on the CFU titers nticipted from pst experience. (Smples were refrigerted nd ressyed the next dy in those cses in which resulting colonies were too numerous to count.) Smples were plced in duplicte on selective medi. The numbers of CFU were determined by

2 194 CARLSON INFECT. IMMUN. TABLE 1. Effect of C. lbicns on infection by S. ureus inoculted i.p. into mice CFU of S. ureus nd C. lbicns recovered per orgn smple Animl group' Animl group (CoU) (CFU) Blood Pncres Kidney Spleen ~~~~~~~~~~~~~~Smll intestine Cecum S. ureus 7.1 X x 105b 1.8 x x x x x 104 C. lbicns 1.0 x X X x x x x 103 S. ureus 2.7 x x x 107b 3.0 x 104b 2.5 x x x 104 C. lbicns 1.0 X x x x x x x 105 S. ureus 5.0 x x x 106b < X 106 <104 C. lbicns 1.0 x x x x x x x 105 S. ureus 7.0 x x x 102 C. lbicns S. ureus 2.7 x x x 103 C. lbicns S. ureus 5.0 x x 103 <1o2 5.2 x 102 C. lbicns S. ureus 700c 2.2 x 102b C. lbicns 1.0 x x x 102d 2.3 x x X x 103 Animl groups consisted of two nimls, nd CFU numbers represent n verge. The kidney ws the only orgn in which considerble vrition ws observed. (In some cses, no CFU from one kidney were detected, wheres the other kidney in the sme niml yielded gret numbers.) Animls were inoculted with the dose indicted nd scrificed 5 dys lter. b S. ureus isolted from these orgns hd phge group I sensitivity pttern (29/52/80) which ws identicl to tht of the strin used in the injections. c S. ureus from this tissue ws not sensitive to ny phge used in testing. d This titer ws unusully low. CFU were from one niml only, s this orgn ws lost during processing of the second niml. using mnnitol slt gr medium (BBL) for S. ureus, Sbourud dextrose gr (BBL) contining 100 mg of chlormphenicl per liter (Sigm Chemicl Co., St. Louis, Mo.) for C. lbicns, McConkey gr medium (Difco) for S. mrcescens, nd 5% sheep blood gr with zide (zide blood gr bse [Difco] plus whole blood) for S. feclis. In ddition to growth on selective medi, typicl S. ureus nd S. feclis colonies were isolted nd sent to the Michign Stte Helth Lbortory (Houghton), where routine tests further confirmed identifiction. Colonies selected on Sbourud-chlormphenicl gr formed pseudohyphe nd chlmydospores on rice extrct gr (Difco). S. mrcescens ws identified by its growth on McConkey gr nd its deep red colony color. Bcteriophge typing. Bcteriophge typing of S. ureus isoltes ws done by the Michign Deprtment of Public Helth under the direction of R. Mrtin. The phges used included group 1 (29, 52, 52A, 79, 80); group 2 (3A, 3C, 55, 71); group 3 (6, 42A, 47, 53, 54, 76, 77, 83A, 84, 85); nd miscellneous (81, 94, 95, 96, 187). RESULTS Animls were inoculted dully with sublethl combintions of C. lbicns nd S. ureus. In the first set of experiments, C. lbicns t lmost one-third of its LD50 (108 CFU) ws combined with vrious low doses of S. ureus between 7.1 x 106 nd 5.0 x 102 CFU nd inoculted i.p. into mice. Five dys lter, nimls were scrificed, nd the titers of CFU of the two injected orgnisms in bdominl orgns were determined. Tble 1 shows tht the extent of bcteril infection in the presence of C. lbicns ws nerly the sme regrdless of the mount of bcteri initilly injected. In dully inoculted nimls, nerly ll bdominl orgns smpled yielded mixed infections, with S. ureus predominting over C. lbicns by CFU rtio of 10 to 100:1; the smll intestine nd pncres yielded the highest numbers. Tble 1 lso shows tht S. ureus inoculted lone t doses used in the combined injections developed no detectble infections, or infections in certin orgns only, with few CFU recovered; the level of cndidl infection ws the sme in the presence or bsence of the ccompnying S. ureus dose. A number of S. ureus colonies recovered from vrious orgns (Tble 1) were typed for phge sensitivity to determine whether

3 VOL. 39, 1983 FUNGAL ENHANCEMENT OF BACTERIAL INFECTION 195 TABLE 2. Effect of dose of C. lbicns on infection by S. ureus inoculted i.p. into mice CFU of C. lbicns nd S. ureus recovered per orgn smple Animl group' (CiU) DoseSmllLg g os Blood Pncres Kidney Spleen intestme Cecum nrtestie Peritoneum S. ureus 1.0 X X x x x x x x x 105 C. lbicns 1.1 X x x x x x x x x 103 S. ureus 1.0 X X x 103 C. lbicns 1.1 X 105 S. ureus C. lbicns 1.1 X X x X x x x 104 S. ureus C. lbicns 1.1 x 105 S. ureus 1.0 X X X 103 C. lbicns Animl groups consisted of two nimls, with the CFU numbers representing n verge. they mtched the strin originlly used in the dully injected nimls. In ll cses, the phge type mtched tht of the S. ureus originlly inoculted. In n niml injected only with C. lbicns, the pncres nd smll intestine were found to hrbor low-level stphylococcl infections. In the cse of the smll intestine, the phge type mtched tht used in the experiment, wheres S. ureus from the pncres ws not sensitive to ny phge used in testing. In nother set of experiments, S. ureus ws exmined to determine whether it ffected infection by C. lbicns in dully injected nimls. S. ureus t lmost one-third of its LD50 (1.7 x 109 CFU) ws dded in combintion with vrious low doses of C. lbicns (6 x 105 nd 6 x 104 CFU) nd inoculted i.p. into mice. It ws found tht C. lbicns did not infect bdominl orgns when injected t these doses either lone or in combintion with S. ureus. To determine the minimum mount of C. lbicns necessry to ffect S. ureus infection, vrious doses of C. lbicns nd S. ureus were combined nd injected i.p.; orgn titers were determined 5 dys lter. As seen in Tble 2, when S. ureus t dose of 108 CFU ws combined with C. lbicns t dose of 1.1 x 107 CFU, S. ureus ws recovered from bdominl orgns 5 dys lter, wheres when 1.1 x 105 CFU of C. lbicns ws injected together with S. ureus, neither pthogen could be recovered. The lst two sets of experiments were designed to determine whether C. lbicns ffected the virulence of other bcteri. Tble 3 shows tht S. mrcescens inoculted i.p. t vrious concentrtions with C. lbicns could be recovered 5 dys lter in vrious bdominl orgns. Tble 4 shows similr effect with combintions of C. lbicns nd S. feclis. CFU of S. mrcescens nd S. feclis when inoculted lone could not be recovered from bdominl orgns. DISCUSSION Animls chllenged simultneously with C. lbicns nd Mycobcterium tuberculosis (14) or C. lbicns nd Escherichi coli (7) hve been reported to show lower resistnce to these fungl-bcteril combintions thn to the fungus or bcteri lone. Additionl studies on niml mortlity employed combintions of extrcts of C. lbicns nd vrious bcteri (16, 20) nd produced conflicting results. These studies were done more thn 20 yers go, nd this line of reserch hs not been ctively pursued. In recent yers, number of new serious diseses hve been reported involving orgnisms which re frequently crried without clinicl symptoms. Reserch reports on the illnesses hve emphsized need for studies on interctions between commonly encountered, usully nonvirulent, orgnisms (6, 12). In response to this need, some new studies hve recently emerged. One such study showed cytomeglovirus to lower resistnce of nimls to infection by vrious bcteri nd C. lbicns (9) İn our lbortory, study hs been conducted in which synergistic effect on mouse mortlity ws demonstrted in combined infection of mice with C. lbicns nd n S. ureus strin isolted from ptient with toxic shock syndrome. Dul

4 196 CARLSON INFECT. IMMUN. TABLE 3. Effect of C. lbicns on infection by S. mrcescens inoculted i.p. into mice. CFU of C. lbicns nd S. mrcescens recovered per orgn smple Animl group Dose (CFU) Blood Pncres Kidney Spleen S. mrcescens 6.0 x x x x x 104 C. lbicns 9.0 x X x x x 105 S. mrcescens 6.0 x x x X 103 C. lbicns 9.0 x X x 102 S. mrcescens 6.0 x x X x x 103 C. lbicns 9.0 X 107 < x x x 104 S. mrcescens 6.0 x 106b 1.5 x x 102 C. lbicns S. mrcescens C. lbicns 9.0 x X x x 104 Animl groups consisted of two nimls, with CFU numbers representing n verge. b Animls were lso infected with lower doses of S. mrcescens (6.1 x 104 nd 6.0 x 102), without subsequent recovery, indicting less thn 100 CFU per orgn smple. infection with the two orgnisms t doses which seprtely cused no niml deths resulted in 100% mortlity (4). In the reserch reported here, sublethl mounts of C. lbicns inoculted i.p. into mice gretly mplified the virulence of ll three of the bcteril species tested; minimum dose of C. lbicns between 105 nd 107 CFU is pprently necessry for this effect to tke plce. Sublethl doses of S. ureus did not stimulte infection by smll doses of C. lbicns, nd it ws therefore concluded tht the synergism observed with the fungl-bcteril combintions ws not mutul. The experimentl pproch ws in response to the observtion (unpublished) tht nimls inoculted with C. lbicns lone were unexpectedly highly sensitive to ccidentl secondry bcteril infection by pthogens used in our lbortory, norml flor, or environmentl orgnisms. Should this mplifiction lso tke plce in humns who hrbor cndidl infections, the medicl implictions re gret. Although these studies show tht C. lbicns hs strong mplifying effect on the virulence of other orgnisms, how this is chieved is mystery. One possibility is tht the cndidl infection process cuses physicl dmge to orgn wlls which mkes them "leky," llowing other microbes or chemicls (perhps toxins) or both to penetrte more esily; it is lso possible tht C. lbicns directly stimultes the growth of S. ureus. Studies re in progress to revel the nture of this C. lbicns virulence mplifying effect. Preliminry (unpublished) findings indicte tht viruses nd lrge molecules, s well s bcteri, penetrte orgns more esily in the presence of C. lbicns, which supports the "leky" hypothesis. Cndidosis is well known for its occurrence with vriety of diseses; one study (8) lists 39 such conditions. The literture throughout the century includes stedy flow of reports ssocited Cndid species with bronchitis, sthm, pneumoni, nd llergy (10, 11, 15, 17, 19). The cuse-effect role of Cndid species in these mny conditions hs been uncler nd controversil. C. lbicns hs not been directly implicted in the disese process; the medicl dogm hs developed tht these re conditions which predispose to secondry fungl infection. It hs been noted by other uthors tht the lck of cuse-effect reltionships my result minly from the lck of investigtion of cndidosis experimentlly (13, 18). It is hoped tht these findings will estblish the experimentl groundwork which eventully my provide informtion necessry for repprisl of the role of C. lbicns in humn disese. The possibility of n s yet unidentified involvement of C. lbicns in diseses is further strengthened by the elusiveness of this infecting orgnism. For exmprle, C. lbicns involvement in denture stomtitis hs only been elucidted in studies since 1963 (5), lthough the condition ws known for mny decdes (3). It is now believed to be very common nd is estimted in one report (2) to occur in 60% of ll denture werers. Biopsies of inflmed res consistently filed to demonstrte tissue invsion (1). One cn speculte tht n equivlent infection of the smll intestine ( trget orgn in the experimen-

5 VOL. 39, 1983 FUNGAL ENHANCEMENT OF BACTERIAL INFECTION 197 TABLE 4. Effect of C. lbicns on infection by S. feclis inoculted i.p. into mice. CFU of S. feclis nd C. lbicns per orgn smple Animl group Dose (CFU) Blood Pncres Kidney Spleen Peritoneum S. feclis 1.0 X x x x x x 102 C. lbicns 1.0 x x x x x 102 S. feclis 1.0 x 105 b _ 2.0 x x 103 C. lbicns 1.0 x x x 104 S. feclis 1.0 X x x 103 C. lbicns 1.0 x x x x x 103 S. feclis C. lbicns 1.0 X 107c S. feclis C. lbicns 1.0 x x x x 103 Animl groups consisted of two nimls, with CFU numbers representing n verge. b, Smple not processed to completion. c Animls were lso infected with lower doses of S. feclis (1.0 x 105 nd 1.0 x 103 CFU) without subsequent recovery, indicting less thn 100 CFU per orgn smple. tl system described here) would be virtully undetectble. It must be noted tht physicins hve reported therpeutic cures for vriety of diverse disese conditions by nticndidl drugs (15, 19). It now ppers possible tht this fungus my ply key role in mny disese conditions, not by its own toxic or invsive growth, but rther by enhncing secondry infection. ACKNOWLEDGMENTS I thnk F. Erbisch for invluble discussions nd dvice nd Sue Rydeski for excellent technicl ssistnce. In ddition, N. Stonemn of the Michign Deprtment of Public Helth (Houghton) is grtefully cknowledged for help nd confirmtory testing. LITERATURE CITED 1. Budtz-Jorgensen, E Denture stmtitis. 3. Histopthology of trum- nd Cndid-induced inflmmtory lesions of the pltl mucos. Act Odontol. Scnd. 28: Budtz-Jorgensen, E., A. Stenderup, nd M. Grbowski An epidemiologic study of yests in elderly denture werers. Community Dent. Orl Epidemiol. 3: Chn, L. R The denture sore mouth. Ann. Dent. 3: Crlson, E Synergistic effect of Cndid lbicns nd Stphylococcus ureus on mouse mortlity. Infect. Immun. 38: Cwson, R. A Denture sore mouth nd ngulr cheilitis. Orl cndidisis in dults. Br. Dent. J. 115: Durck, D. T Opportunistic infections nd Kposi's srcom in homosexul men. N. Engl. J. Med. 305: Gle, D., nd B. Sndovl Response of mice to the inocultion of both Cndid lbicns nd Escherichi coli. J. Bcteriol. 73: Gentles, J. C., nd C. J. Le Touche Yests s humn nd niml pthogens, p In A. H. Rose nd J. S. Hrrison (ed.), The yests, vol. 1. Acdemic Press, Inc., London. 9. Hmilton, J. R., J. C. Overll, nd L. Glsgow Synergistic effect on mortlity in mice with murine cytomeglovirus nd Pseudomons eruginos, Stphylococcus ureus, or Cndid lbicns infections. Infect. Immun. 14: Holti, G Cndid llergy, p In H. L. Winner nd R. Hurley (ed.), Symposium on Cndid infection. Livingstone, Edinburgh. 11. Keeney, E. L Cndid sthm. Ann. Intern. Med. 34: Lrsen, B., nd R. P. Glsk Vginl microbil flor: composition nd influences of host physiology. Ann. Intern. Med. 96: Mibch, H. I., nd A. M. Kligmn The biology of experimentl humn cutneous monilisis (Cndid lbicns). Arch. Dermtol. 85: Mnkiewicz, E., nd M. Lilvk Effect of Cndid lbicns on the evolution of experimentl tuberculosis. Nture (London) 187: Oliver, H. G Asthm due to n infection: clinicl study. Med. Off. 12: Rzucidlo, L., A. Stghow, E. Rudzki, I. Mnkiewicz, nd S. Sobolews Influence of zymosn from yest nd yest-like fungi on the virulence of Slmonell typhi (901-0) nd Stphylococcus ureus in mice. Bull. Acd. Pol. Sci. Cl. 2 5: Steinfield, E A study of the yests found in the sputum of ptients with sthm nd chronic bronchitis. J. Lb. Clin. Med. 8: Stovll, W. D., nd S. B. Pessin. Clssifiction nd pthogenicity of certin monilis. Am. J. Clin. Pthol. 3: Truss, C The role of Cndid lbicns in humn illness. J. Orthomol. Psychitry 10: Ymbyshi, H A zymosn-like substnce extrcted from Cndid lbicns. Med. J. Osk Univ. 9:11-21.