JOURNAL OF CLINICAL MICROBIOLoGY, Feb. 1975, p. 119-123 Copyright 0 1975 Americn Society for Microbiology Vol. 1, No. 2 Printed in U.S.A. Effect of Sodium Polynethol Sulfonte in Blood Cultures JAN ENG The Microbiologicl Lbortory, Ullevdl Hospitl, Oslo 1, Norwy Received for publiction 7 October 1974 Fifteen-hundred hospitl blood cultures were mde in duplicte, with nd without 0.05% sodium polynethol sulfonte in the broth medium. A significntly higher rte nd speed of recovery of both grm-positive cocci nd grm-negtive bcilli ws ccomplished in sodium polynethol sulfonte broth. The effect ws independent of the content of 0.1% gr in the growth medium. In the cses of Neisseri meningitidis septicemi exmined, however, detrimentl result on recoveries ws observed. The ddition of sodium polynethol sulfonte lso resulted in n incresed frequency of recoveries of contminting orgnisms. Sodium polynethol sulfonte (SPS; Liquoid, Hoffmn-L Roche) is n nticogulnt nd surfce-ctive gent which is widely employed s n dditive to fluid blood culture medi. It is generlly considered to enhnce the rte nd speed of bcteril isoltions by counter-cting the bcteril inhibitors of humn blood. The ltter effect hs been clerly demonstrted in rtificil cultures (2, 3, 5, 6, 11). SPS is known to neutrlize the bctericidl ctivity of fresh humn serum (6) nd to inhibit phgocytosis (1). However, reltively few prospective clinicl trils using duplicte cultures with nd without SPS hve been published, nd some of their results re conflicting. Rosner reported tht the ddition of 0.05% SPS hd beneficil effect on the recovery of grm-positive cocci, wheres no effect ws demonstrted on the recovery of grm-negtive bcilli (8, 9, 10). Finegold et l. found fvorble effect of 0.5% SPS in smll study comprising ptients with both grm-positive nd grm-negtive bcteremi (4). Minkus nd Moffet, working with blood cultures from infnts nd children, concluded tht the ddition of 0.05% SPS did not increse the recovery rte of ny group of pthogenic bcteri nd tht it led to n incresed frequency of contmintions (7). In the present pper, the results of prospective clinicl study comprising 1,500 duplicte blood cultures re presented. A significnt fvorble effect of 0.05% SPS upon the rte nd speed of isoltions of both grm-positive nd grm-negtive pthogenic bcteri ws clerly demonstrted, with the exception of Neisseri meningitidis septicemis, in which n dverse effect ws observed. The ddition of SPS lso resulted in n incresed frequency of recoveries of typicl contminting orgnisms. MATERIALS AND METHODS Blood cultures were prepred in this hospitl by inoculting 5 ml of venous blood into 50 ml of broth medium contined in 100-ml screw-top bottles. The medium is nutrient broth with 0.1% dextrose produced in this lbortory. The broth bottles did not contin CO2 nd were not under vcuum. Broth with SPS ws mde by dding 0.05% SPS to the nutrient broth before utoclving. With view to ssessing the effect of SPS, Medicl Deprtments I nd VII nd Peditric Deprtment XVII of this hospitl used prllel tests in their blood culture routines during 1-yer period. Ten milliliters of venous blood were withdrwn in syringe, nd 5-ml smple ws immeditely inoculted into ech of two flsks, one with broth medium to which 0.05% SPS hd been dded nd one with exctly the sme medium but without SPS. (The two medi were inoculted in rndom order.) The bottles were kept t 37 C nd sent to the Microbiologicl Lbortory s soon s possible. The time which elpsed between inocultion of the bottles t the bedside nd the rrivl of the cultures t the lbortory vried from few hours to 2 dys. Immeditely upon the receipt in the lbortory, ll culture bottles were subcultured onto solid medi. The bottles were then incubted t 37 C nd inspected visully once dy. If both bottles remined cler, prt from the chrcteristic, fint clouding which regulrly ppered in the SPS bottles, they were subcultured fter 4 dys nd then discrded if negtive. Cultures from ptients with clinicl dignosis of endocrditis, however, were kept for 7 dys. If one of the bottles showed mcroscopic signs of bcteril growth, subcultures nd Grm stins were mde immeditely from both bottles in prllel. If the subculture from one bottle yielded colony formtion, wheres the other bottle ws negtive, the ltter ws subcultured dily until growth ppered or up to 7 dys, when the bottle ws discrded s negtive. All subcultures were mde onto chocolte gr pltes by mens of clibrted wire loop. The subculture pltes were incubted in ir t 37 C, except tht pltes from 119
120 ENG cultures suspected of contining N. meningitidis were incubted in ir with n incresed content of crbon dioxide. Anerobic pltings were performed from turbid culture bottles yielding negtive erobic subcultures. The dy when growth first ppered in the subcultures ws noted, s well s the pproximte number of colonies. The bcteril strins isolted were identified by stndrd methods. RESULTS During 1-yer period, 1,500 blood cultures sets were exmined. Two-hundred nd sixtyfive sets of cultures were positive, i.e., orgnisms were recovered from one or the other, or both, bottles in the pir. Of these, 151 cultures obtined from 66 ptients yielded growth of potentilly pthogenic orgnisms. From the remining 114 positive cultures were isolted Stphylococcus epidermidis, diphtheroid bcilli, Bcillus sp., or yests. Cses in which rel S. epidermidis bcteremi could be suspected on clinicl nd bcteriologicl grounds were not encountered during the study period. The two groups of positive duplicte cultures will be treted seprtely in the following. No nerobic orgnisms were recovered. During the lst one-third of the study, 0.1% gr ws dded to ll broth medium bottles for blood cultures in this hospitl, wheres during the first two-thirds of the period only broth medium without gr ws used. Growth of potentilly pthogenic orgnisms from 151 duplicte blood cultures. One hundred of these cultures were mde in broth medium without dded gr; their growth ptterns in grm-positive nd grm-negtive bcteremis re shown in Tble 1. Tble 2 shows the corresponding findings in the remining 51 culture pirs, positive in broth medium with 0.1% gr dded. The centrl columns of both tbles show the number of culture pirs from which n pproximtely equl number of colonies were formed from both bottles t the sme time. The neighboring columns show the number of pirs from which colony formtion rose from both bottles in simultneous pltings, but TABLE 1. J. CLIN. MICROBIOL. with higher colony count from one bottle thn from the other. In the second columns from the left nd right re given the number of pirs of bottles in which growth in the subcultures differed by 1 or more dys between the two bottles in the pir. The outside columns show the number of pirs in which only one bottle yielded growth, the other bottle in the pir remining negtive throughout 7 dys of incubtion. In both tbles n symmetricl distribution is evident, in tht the number of culture pirs showing erlier growth from SPS broth, or growth exclusively from SPS broth (i.e., the totls of the figures in the two outermost left columns), clerly outweighs the number of pired cultures giving the reverse growth ptterns. The effect is present in both grm-positive nd grm-negtive bcteremis, nd in broth medium with, s well s without, 0.1% gr dded. In N. meningitidis septicemis, however, n dverse effect ws observed. Eleven pirs of cultures from seven ptients were positive for this orgnism; 10 of these pirs yielded better growth, erlier growth, or growth exclusively from the broth medium without SPS. The two subgroups of the dt which re given in Tble 1 nd 2 re combined in Tble 3, which lso shows the vrious orgnisms isolted. Out of totl of 88 pired cultures yielding growth of grm-negtive bcilli, erlier growth or growth exclusively from SPS broth ws found in 46 (52.3%), wheres the sme growth ptterns were observed in 17 out of 52 pired cultures yielding growth of grm-positive orgnisms (32.7%). In the extreme right column of Tble 3 is given the number of ptients in whom the orgnism listed ws isolted from one pired culture only. Of the three cses of single isoltions of Diplococcus pneumonie, two ptients suffered from pneumoni nd the third one from meningitis; in the ltter cse, D. pneumonie ws isolted from cerebrospinl fluid s well. Hemophilus influenze ws isolted from one pired blood culture only in four cses, ll of Effect of 0.05% SPS on recovery ofgrm -positive nd grm -negtive orgnisms from 100 pired blood cultures in broth medium without gr Orgnisms +, - Erlier Hevier +1 + +.,+ + + _-_.- _I!_ (equl) nevier + erlie?r + Grm-positive orgnisms 5 1 4 8 2 1 Grm-negtive bcilli 21 17 1 20 1 4 N. meningitidis 3 4
VOL. 1, 1975 EFFECT OF SPS IN BLOOD CULTURES 121 TABLE 2. Effect of 0.05%,7 SPS on recovery of grm-positive nd grm-negtive orgnisms from 51 pired blood cultures in broth medium with 0.1% gr dded Orgnisms Erlier Hevier +,± h. + + ±,+ (equl) hevier + erlier + Grm-positive orgnisms 3 8 5 9 1 2 Grm-negtive bcilli 6 2 9 2 2 N. meningitidis 1 1 TABLE 3. Effect of 0.05% SPS on recovery of vrious orgnisms isolted from 151 pired blood cultures Orgnisms Erlier Hevier +, + +, +, Single +,+ + (equl) hevier + erlier +-. isoltions Grm-positive orgnisms Stphylococcus ureus 3 6 4 3 1 2 Diplococcus pneumonie 1 1 2 1 3,-Hemolytic streptococci 1 1 2 1 0 -Hemolytic streptococci 2 3 3 3 2 1 1 3 Enterococci 7 1 3 Listeri monocytogenes 1 1 0 Grm-negtive bcilli Escherichi coli 12 4 1 12 1 4 2 3 Klebsiell sp. 3 5 1 4 Proteus mirbilis 2 5 1 1 1 Slmonell sp.5 3 9 6 1 1 Alcligenes feclis 1 1 Acinetobcter sp. 2 3 1 Hemophilus influenze 1 2 1 4 No genus determined 4 1 3 Neisseri meningitidis 1 4 4 2 5 'S. prtyphi A, two ptients; S. prtyphi B, twco ptients; S. heidelberg, one ptient. whom were meningitis ptients in whom the sme orgnism ws cultivted from cerebrospinl fluid. Of the seven ptients whose blood cultures yielded growth of N. meningitidis, five suffered from meningitis; in four of them, N. meningitidis ws isolted from cerebrospinl fluid nd in one cse ttempts t spinl puncture were unsuccessful. Two ptients suffered from septicemi with n exnthem, but meningitis ws not present. In the pired cultures showing time difference in the growth from individul bottles, difference of 1 dy ws observed most frequently (Tble 4). Of the cultures yielding erlier growth from SPS broth, however, n equl number differed by 2 or more dys, 5 dys being the highest difference observed. In five ptients, different orgnisms were isolted from different duplicte cultures. In one bottle pir, two orgnisms (Escherichi coli nd Proteus mirbilis) were isolted from both bottles in the pir. In the remining 61 ptients, only one species ws isolted. In the ptient mteril under study elderly people predominted; thus, hlf of the ptients were 60 yers or more. Growth of S. epidermidis, diphtheroid bcilli, Bcillus sp., or yests from 114 duplicte cultures. An enhncing effect of 0.05% SPS on the recovery of the bove-mentioned orgnisms is evident from Tble 5. Sixty-two pired cultures yielded erlier growth or growth exclusively from SPS broth, wheres only 37 bottle pirs exhibited the reverse growth ptterns. In only 23 of the 114 duplicte cultures (20.2%), the orgnisms listed were isolted from both bottles in the pir. This is in shrp contrst to the corresponding finding in the
122 ENG group of isoltions of potentilly pthogenic orgnisms, in which recovery ws ccomplished from both bottles in 104 (68.9%) of 151 bottle pirs (Tble 3). DISCUSSION The group of recoveries of potentilly pthogenic orgnisms my contin some cses in which the orgnisms recovered were introduced s contminnts during inocultion of ptient's blood into the culture bottles, or during the subsequent processing of the cultures in the lbortory. As n id in the evlution of the occurrence of possible contmintions, the number of single isoltions is given in Tble 3 for ech orgnism recovered. All single isoltions of D. pneumonie, H. influenze, nd N. meningitidis were proven to be rel bcteremis ccording to the clinicl dt given bove. If the number of cses of possible contmintion is sought, in ccordnce with generlly ccepted rules, mong the remining single isoltions, the frequency of contmintion will probbly be low, most likely below 10% of the 151 positive duplicte cultures. Hence, it is inferred tht the conclusions which cn be drwn from the mteril with regrd to the effect of SPS re vlid for true bcteremis. These conclusions re: (i) significntly higher rte nd speed of recovery of both grm-positive cocci nd grm-negtive bcilli were ccomplished in broth medium TABLE 4. Time difference between growth from individul bottles in 37 pired blood cultures Difference (dys) Erlier +, +, erlier + 1 14 7 2 5 1 3 4 4 4 1 5 1 TABLE 5. J. CLIN. MICROBIOL. with 0.05% SPS, s compred with control broth medium without SPS; (ii) lthough present in both min groups of bcteri, the effect ws more pronounced on grm-negtive rods thn on grm-positive cocci; (iii) the effect ws independent of the content of 0.1% gr in the broth medium; nd (iv) N. meningitidis septicemis constituted n exception to the generl fvorble effect of 0.05% SPS, in tht the cultures showed better growth, erlier growth, or growth exclusively from the broth medium without SPS. In ccordnce with the findings of Minkus nd Moffet (7), the inclusion of 0.05% SPS in the broth medium resulted in n incresed recovery rte of typicl contminting orgnisms from blood cultures (Tble 5). The effect must be explined by the ntgonizing ctivity of SPS ginst the nonspecific bcteril inhibitors of fresh humn blood. I do not consider tht the incresed recovery rte of contminting orgnisms interferes seriously with the usefulness of SPS in prcticl work. The repressed growth of N. meningitidis from clinicl blood cultures in the presence of 0.05% SPS observed in the present study (Tble 3) provided the impetus for n investigtion in the present lbortory of the sensitivity of number of N. meningitidis strins to 0.05% SPS. The study ws performed using broth titrtion technique; it showed tht severl of the strins were mrkedly inhibited. Also, pper disk method ws evolved for screening the sensitivity of strins of vrious species to SPS; the highest formtion of zones of inhibition ws seen in N. meningitidis nd N. gonorrhoee. When viewing the results of these experiments, which will be published seprtely, together with those of the present study, it is considered likely tht the ddition of 0.05% SPS to blood culture broth my exert toxic effect on N. meningitidis outweighing the fvorble effect on the bctericidl components of ptients' blood, thus yielding detrimentl totl result on the rte nd speed of recovery. Recovery of Stphylococcus epidermidis, diphtheroid bcilli, Bcillus sp., nd yests from 114 pired blood cultures Orgnisms +_Q Erlier Hevier +, + +, + +, + (equl) hevier + erlier + S. epidermidis 46 7 7 7 1 31 Diphtheroid bcilli 3 1 1 Bcillus sp. 6 1 Yests 3
VOL. 1, 1975 EFFECT OF SPS IN BLOOD CULTURES The beneficil overll effect of 0.05% SPS on recovery from clinicl blood cultures, including the striking effect in grm-negtive rod bcteremis on the one hnd, nd the strong indictions of detrimetitl effect in N. meningitidis bcteremis on the other, nturlly led to the conclusion tht duplicte blood cultures with nd without SPS should be used routinely. The possibility tht lowering of the SPS concentrtion below 0.05% could eliminte toxic effects with preservtion of the growthpromoting cpcity might lso be considered. Lowrnce nd Trub, however, exmined the effect of vrying SPS concentrtions below 0.05% on the killing of serum-sensitive strins of E. coli in fresh humn serum nd concluded tht t lest 0.025% SPS should be dded to broth medium for blood cultures to improve the chnces of isolting pthogens present in smll numbers (6). Where routine duplicte blood cultures with nd without SPS re not considered prcticble nd the use of one blood culture medium only is preferred, it is considered tht this medium should contin 0.05% SPS, whose beneficil effect in most clinicl bcteremis hs been clerly demonstrted. It should be recognized, however, tht this prctice involves dnger of delying or repressing the isoltion of the custive gent in N. meningitidis bcteremis. N. meningitidis septicemis re often recognizble or suspected on clinicl grounds. When blood cultures re mde from such ptients to verify the dignosis bcteriologiclly, broth medium without SPS should be used. 123 ACKNOWLEDGMENTS I m indebted to the stffs of Deprtments I, VII, nd XVII of UllevAl Hospitl for their redy coopertion, nd to Inger Bjerke for skillful work in the lbortory. LITERATURE CITED 1. Allgower, M. 1947. Ueber die Wirkung von Heprin, polynetholsulfosurem Ntrium (Liquoid Roche) und tribsischem Ntriumcitrt uf menschliche Leukozyten in vitro. Schweiz. Med. Wochenschr. 77:40-43. 2. Ellner, P. D., nd C. J. Stoessel. 1966. The role of temperture nd nticogulnt on the in vitro survivl of bcteri in blood. J. Infect. Dis. 116:238-242. 3. Evns, G. L., T. Cekoric, R. L. Sercy, nd L. R. Hines. 1966. Effects of nticogulnts on ntibcteril ction of blood. Clin. Res. 14:484. 4. Finegold, S. M., I. Ziment, M. L. White, W. R. Winn, nd W. T. Crter. 1968. Evlution of polynethol sulfonte (Liquoid) in blood cultures, p. 692-696. Antimicrob. Agents Chemother. 1967. 5. Grrod, P. R. 1966. The growth of Streptococcus viridns in sodium polynethol sulphonte ("Liquoid"). J. Pthol. Bcteriol. 91:621-623. 6. Lowrnce, B. L., nd W. H. Trub. 1969. Inctivtion of the bctericidl ctivity of humn serum by Liquoid (sodium polynetholsulfonte). Appl. Microbiol. 17:839-842. 7. Minkus, R., nd H. L. Moffet. 1971. Detection of bcteremi in children with sodium polynethol sulfonte: prospective clinicl study. Appl. Microbiol. 22:805-808. 8. Rosner, R. 1968. Effect of vrious nticogulnts nd no nticogulnt on bility to isolte bcteri directly from prllel clinicl blood specimens. Am. J. Clin. Pthol. 49:216-219. 9. Rosner, R. 1970. Comprison of blood culture system contining Liquoid nd sucrose with systems contining either regent lone. Appl. Microbiol. 19:281-282. 10. Rosner, R. 1972. A quntittive evlution of three blood culture systems. Am. J. Clin. Pthol. 57:220-227. 11. Von Hebler, T., nd A. A. Miles. 1938. The ction of sodium polynethol sulphonte ("Liquoid") on blood cultures. J. Pthol. Bcteriol. 46:245-252.