Aerobic Blood Culture Medium

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1981, p /81/ $02.00/0 Vol. 14, No. 3 Evaluation of Sucrose and Magnesium Sulfate as Additives in Aerobic Blood Culture Medium JAN ENG The Microbiological Laboratory, Ullevdl Hospital, Oslo 1, Norway Received 10 December 1980/Accepted 21 May 1981 Clinical blood cultures were made in duplicate in brain heart infusion broth with sodium polyanetholsulfonate and gelatin (P broth) and in the same medium with 20% sucrose added (S broth). In part of the study, 0.1% magnesium sulfate was also included in the medium with sucrose (SMg broth). The results from 1,287 positive blood cultures are reported. Significant differences among the rates and speeds of isolations from these media were found in Enterobacteriaceae and Staphylococcus aureus, which were isolated more frequently from S broth and SMg broth than from P broth; in addition, recoveries were accomplished earlier (1 or more days) from S broth and SMg broth than from P broth more often than the reverse growth patterns. An additional effect of magnesium sulfate upon recoveries could not be concluded. The possible mechanisms by which sucrose promotes recoveries from clinical blood cultures are discussed. Several authors, using different media and cultivation techniques, have reported increased rates of recovery from aerobic clinical blood cultures in broth media supplemented with 10 to 30% sucrose (2, 8, 10, 13; L. R. McCarthy and P. D. Ellner, Abstr. Annu. Meet. Am. Soc. Microbiol. 1973, M48, p. 81), but other investigators have been unable to reproduce the findings (4, 15). A possible explanation of these discrepancies may be that the effect of sucrose is medium dependent. We examined the effect of adding 20% sucrose (Saccharose; Merck & Co., Inc.) to brain heart infusion broth (Difco Laboratories) containing sodium polyanetholsulfonate (Hoffman-LaRoche, Inc.) and gelatin (Difco). In a part of the study, 0.1% magnesium sulfate was also included in the medium with sucrose. Magnesium confers stability to protoplast membranes (9), and one important hypothesis for the possible role of sucrose in blood culture media is that the sugar provides osmotic support to cell wall-defective bacterial variants. MATERLALS AND METHODS During a period of 3 years, from June 1977 to May 1980, all aerobic blood cultures in the Ulevil Hospital in Oslo, Norway, were made in duplicate in media with and without 20% sucrose. (From several patients, anaerobic blood cultures were also made. These cultures were made in bottles with prereduced medium not containing sucrose, and they are not a part of the present study.) During 22 months of the study period, the medium with sucrose also contained 0.1% magnesium sulfate. The compositions of the three media employed (plain broth [P], sucrose broth [S], and sucrose broth with magnesium sulfate [SMg]) are given in Table 1. The osmolalities of all batches of media were measured in an Advanced Digimatic osmometer (model 3D; Advanced Instruments, Inc.). The media were distributed in 50-ml amounts to 100-ml glass bottles fitted with rubber diaphragms and metal screw caps with central holes. The bottles were autoclaved at 120 C for 15 min, and then Penicillinase (Difco) was added aseptically. The bottles did not contain CO2. The blood cultures were made at the bedsides of the patients. Venous blood (10 ml) was withdrawn in a syringe, and a 5-ml sample was immediately inoculated into each of the two bottles employed (the bottles contained either P and S or P and SMg). The resulting bottle pair was considered as one blood culture. All cultures were incubated unvented at 37 C for 4 days and inspected visually once a day for macroscopic signs of bacterial growth. (Cultures from patients with clinical diagnoses of endocarditis or other symptoms which necessitated prolonged incubation, however, were incubated for 1 or more weeks.) Subcultures onto solid medium were made from all bottles upon receipt of the cultures in the laboratory and again after 3 days of incubation or when macroscopic signs of bacterial growth appeared. If one of the bottles in a pair showed macroscopic signs of bacterial growth, both bottles were subcultured in parallel. If one of the bottles in a pair yielded colony formation on subculture and the other bottle was negative, the latter was reincubated and subcultured when macroscopic signs of growth developed or after 6 days. Five different growth patterns were thus recorded: simultaneous growth in the two media, earlier growth (1 or more days) from P broth or S (or SMg) broth, respectively, and growth from P broth or S (or SMg) broth only. The subcultures made upon receipt of the blood 247

2 248 ENG J. CLIN. MICROBIOL. TABLE 1. Composition of three aerobic blood culture media under investigation Composition Broth Brain Sodium poly- MgSO,- Osmolality heart odium fo- Gelatin Penicillinase Sucrose 0, 7H0, (avg., ph infusion anetholsulfo- (%) (IU/ml) wt/vol) wtvl mosmol/kg) (g/liter) nate (%) wt/vol) mosmol/kg) SMg , S , p cultures in the laboratory were made by means of a pipette yielding an inoculum size of approximately 0.07 ml, whereas all subsequent subcultures were made with a wire loop delivering approximately 0.01 ml. All subcultures were made onto chocolate agar plates, which were incubated at 37 C overnight in air containing 5% CO2. The bacterial strains isolated were identified by standard methods (3). The statistical procedure used was that of binomial testing of equal distribution in the two groups compared. RESULTS During the 3-year period under study, 1,287 monomicrobial blood cultures were received. Some of these (573) were made in parallel in P broth and S broth, and 714 were made in P broth and SMg broth. The growth patterns of the various groups of bacteria isolated from these media are shown in Tables 2 and 3. Significant differences in the rates of isolations were found with Enterobacteriaceae and Staphylococcus aureus. The cultures yielding growth of these two groups of bacteria in S broth only or in SMg broth only significantly outnumbered the cultures showing growth in P broth only (Tables 2 and 3). Also, the number of cultures in which growth appeared earlier (1 or more days) in S broth or SMg broth than in P broth was significantly higher than the number of cultures showing the reverse growth pattern, except for the difference noted in footnote c of Table 2, which was not statistically significant but showed a strong tendency in the same direction. The cultures with nonfermentative gram-negative rods, as well as those with streptococci, including Streptococcus pneumoniae, did not show significant growth differences in the broth media under comparison. An insufficient number of strains of the other bacterial groups was isolated for a statistically significant analysis. Twenty-nine cultures made in P and S broths and 44 cultures made in P and SMg broths yielded growth of two or more bacterial species. These cultures could not be included in the present examination, as they were unsuitable for a comparison of the growth of each separate TABLE 2. Growth patterns from P broth and S broth in 573 monomicrobial clinical blood cultures made in duplicate from the same venipuncture No. of cultures with growth pattern: Total no. of Total no. of Organism PP+ S- P+ earlier, P+, S+ (simul- P+, S+ ear- p- S+ cultures patients P s+ P,+ taneous) lier Enterobacteriaceae 22a 7b b Nonfermentative gramnegative rods Staphylococcus aureus ' Streptococci, including S. pneumoniae Neisseria meningitidis Haemophilus influenzae Streptobacillus moniliformis Aerobic gram-positive rods, not identified Yeasts Total 40 8b b P< < C p > 0.10.

3 VOL. 14, 1981 ADDITIVES IN AEROBIC BLOOD CULTURE MEDIUM 249 TABLE 3. Growth patterns from P broth and SMg broth in 714 monomicrobial clinical blood cultures made in duplicate from the same venipuncture No. of cultures with growth pattern: Total no. Total no. Organism P+ earlier, P+, SMG+ (si- P+o SMg+ cfcul- of pa- P+, SMg- SMg+mutnos(i + ealr P-, SMg+ tures tients SMg+ multaneous) earlier Enterobacteriaceae 23a 5a a 82a Nonfermentative gram-negative rods Staphylococcus aureus... 5a a a 21a Streptococci, including S. pneumoniae Neisseria meningitidis Neisseria gonorrhoeae Haemophilus influenzae Listeria monocytogenes Clostridium sp Lactobacillus sp Yeasts Total. 66a 12a a 144a ap < species in the cultures from the two media under examination. For the purpose of this study, cultures showing growth of Bacillus, corynebacteria, micrococci, or coagulase-negative staphylococci were not recorded, as the vast majority of these reflected contaminations. Eight cases judged to represent true bacteremias caused by micrococci or coagulase-negative staphylococci were encountered during the study period. DISCUSSION The composition of the P broth employed in the present investigation was based upon earlier findings on the growth-promoting capacity of 0.05% sodium polyanetholsulfonate in blood culture media (1), as well as on the ability of 1.2% gelatin to neutralize the unwanted, inhibitory effects which sodium polyanetholsulfonate exerts upon the growth of certain bacteria (5, 7, 16). Brain heart infusion broth was chosen as one among several choices of well-recommended broth bases (1). The amounts of sucrose used in S broth and SMg broth (20%) were chosen from the range of sucrose concentrations employed by earlier workers in this field (2, 4, 8, 10, 13, 15; McCarthy and Ellner, Abstr. Annu. Meet. Am. Soc. Microbiol. 1973, M48, p. 81). Several authors have demonstrated that the bacterial counts in bacteremia may be quite low; thus, in the work of Finegold et al. (6), over half of the positive cultures with gram-negative rods had colony counts of only cl/ml of blood. It seems probable, therefore, that in a proportion of the cultures in the present study, only one of the two 5-ml inocula harbored viable bacteria. This would result in a random and equal distribution of cultures yielding growth in one of the bottles only upon the two media under comparison. Enterobacteriaceae and Staphylococcus aureus were isolated significantly more frequently from S broth and SMg broth than from P broth. In addition, recoveries were accomplished earlier from S broth and SMg broth than from P broth more often than the reverse growth patterns. It is concluded that the inclusion of 20% sucrose in brain heart infusion broth with sodium polyanetholsulfonate and gelatin improved the recoveries of these two groups of bacteria from clinical blood cultures, and this result was reproduced when 0.1% magnesium sulfate was included in the broth medium in addition to 20% sucrose. These effects are held to be of considerable practical importance, since both groups of bacteria are very common causes of septicemia. In the present study, they constituted 70.2% of all isolates. Magnesium sulfate was added to sucrose broth in a part of the study to elucidate whether the addition would add to the growth-promoting capacity of sucrose alone. A somewhat higher proportion of cultures yielded growth of Enterobacteriaceae in SMg broth than in S broth (Tables 2 and 3). The significance of this finding is uncertain, as different series of blood cultures were examined in S broth and SMg broth, and the occurrence of the (unknown) factors in the blood of bacteremic patients which caused some cultures to grow better in sucrose broth may vary from time to time. The growth-promoting

4 250 ENG effects observed in SMg broth, therefore, may well have been due to sucrose alone. The mechanism by which sucrose promotes bacterial growth in blood cultures is not clarified. A commonly cited explanation is that sucrose offers osmotic support to cell wall-defective bacterial variants, which may have been induced in vivo by cell wall-active antibiotics or other factors (enzymes or immune factors), thus furthering their multiplication and reversion to classical bacteria. This hypothesis has, however, not been definitely proved, and alternative explanations are being discussed. Simberkoff et al. (14) demonstrated that penicillins are rapidly inactivated by 10% sucrose at an alkaline ph. Provided this inactivation phenomenon is operative in blood cultures, better growth of blood cultures from patients on penicillin therapy may be expected in the bottles containing sucrose. Theoretically, two possible effects of sucrose in blood cultures made during penicillin therapy may thus be conceived. In the present study, all media contained Penicillinase in amounts considered sufficient to inactivate relatively high levels of Penicillinase-sensitive penicillins. The possibility cannot be excluded, however, that some penicillin activity may have existed in some of the cultures owing to very high dosages or a possible deterioration of the enzyme. Studies to determine the correlation between the growth patterns observed in the blood culture media described here and the presence or absence of antimicrobial therapy are currently being performed. Muschel and Larsen (12) reported that sucrose inhibits bactericidal reactions through an anticomplementary effect. All of the media compared here, however, contained 0.05% sodium polyanetholsulfonate, which inactivates complement (11). Henrichsen and Bruun (10) found that media containing 10% sucrose yielded growth of Enterobacteriaceae and Staphylococcus aureus significantly more frequently than media without sucrose. In that study, blood was transported to the laboratory in a glass tube containing sodium polyanetholsulfonate and thereafter distributed to different growth media, and it has been questioned whether the sucrose effect demonstrated might be attributable, at least in part, to the specimen handling (15). In the present study, however, blood was inoculated into the media at the bedsides of the patients; even so, our results are similar to those of Henrichsen and Bruun (10). Incidentally, the latter authors found no J. CLIN. MICROBIOL. correlation between the effect of sucrose on the isolation of Escherichia coli and the presence or absence of the ability of the strains to ferment sucrose. We observed, as did other investigators (15), that most blood cultures made in sucrose broth developed spontaneous hemolysis after incubation for about 3 days, and this interfered with the macroscopic reading of the cultures. For that reason, and also to obtain more information on the effect of 20% sucrose upon the growth of the various groups of bacteria which were too scarcely represented in the present study, it seems advisable to use plain broth and sucrose broth in parallel as a routine method. ACKNOWLEDGMENT I gratefully acknowledge the assistance of Ingar Holme, of the Life Insurance Companies' Institute for Medical Statistics at the Oslo City Hospitals, who performed the statistical analysis of the data. LITERATURE CITED 1. Bartlett, R. C., P. D. Ellner, and J. A. Washington II Cumitech 1, Blood cultures. Coordinating ed., J. C. Sherris. American Society for Microbiology, Washington, D.C. 2. Coleman, R. M., W. W. Laslie, and D. W. Lambe, Jr Clinical comparison of aerobic, hypertonic, and anaerobic culture media for the radiometric detection of bacteremia. J. Clin. Microbiol. 3: Cowan, S. T Cowan and Steel's manual for the identification of medical bacteria, 2nd ed. Cambridge University Press, London, United Kingdom. 4. Ellner, P. D., T. E. Kiehn, J. L. Beebe, and L. R. McCarthy Critical analysis of hypertonic medium and agitation in detection of bacteremia. J. Clin. Microbiol. 4: Eng, J., and E. Holten Gelatin neutralization of the inhibitory effect of sodium polyanethol sulfonate on Neisseria meningitidis in blood culture media. J. Clin. Microbiol. 6: Finegold, S. M., M. L. White, I. Ziment, and W. R. Winn Rapid diagnosis of bacteremia. Appl. Microbiol. 18: Graves, M. H., J. A. Morello, and F. E. Kocka Sodium polyanethol sulfonate sensitivity of anaerobic cocci. Appl. Microbiol. 27: Gross, P. A., R. Fryda, and K. Reilly Comparative evaluation of different types of blood culture media for isolation of aerobes. J. Clin. Microbiol. 6: Guze, L. B. (ed.) Microbial protoplasts, spheroplasts and L-forms. The Williams & Wilkins Co., Baltimore. 10. Henrichsen, J., and B. Bruun An evaluation of the effects of a high concentration of sucrose in blood culture media. Acta Pathol. Microbiol. Scand. Sect. B 81: Kocka, F. E., T. Magoc, and R. L. Searcy Action of sulfated polyanions used in blood culture on lysozyme, complement and antibiotics. Ann. Clin. Lab. Sci. 2: Muschel, L. H., and L. J. Larsen Effect of hypertonic sucrose upon the immune bactericidal reaction. Infect. Immun. 1:51-55.

5 VOL. 14, Rosner, R A quantitative evaluation of three blood culture systems. Am. J. Clin. Pathol. 57: Simberkoff, M. S., L Thomas, D. McGregor, L. Shenkein, and B. B. Levine Inactivation of penicillis by carbohydrate solutions at alkaline ph. N. Engl. J. Med. 283: Washington, J. A., II, M. M. Hall, and E. Warren. ADDITIVES IN AEROBIC BLOOD CULTURE MEDIUM Evaluation of blood culture media supplemented with sucrose or with cysteine. J. Clin. Microbiol. 1: Wilkins, T. D., and S. E. H. West Medium-dependent inhibition of Peptostreptococcus anaerobius by sodium polyanetholsulfonate in blood culture media. J. Clin. Microbiol. 3: