Effect of antimicrobials on blood cultures in endocarditis

DIAGN MICROBIOLINFECTDIS 1987:8:165-172

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Effect of Antimicrobials on Blood Cultures in Endocarditis Robin McKenzie and Larry G. Reimer I

To study the effect of antimicrobials on bacterial growth in blood cultures, we used both simulated blood cultures and cultures obtained from rabbits with experimental endocarditis. Four strains of bacteria were incubated individually with six antimicrobials in nine blood culture media. Positivity rates varied with the ratio of the antimicrobial concentration to the MIC of the organism: 161 of 162 cultures (99%) were positive when the ratio was less than 1/10; 52 of 108 (48%) were positive when the ratio was between 1/10 and one; and none of 54 were positive when the ratio was greater than one. Endocarditis was produced in 28 rabbits with either E_. col__ii,Pp. aerul~inosa, S. aureus, or viridans streptococcus. Following a single dose of an antimicrobial, blood was taken for culture in eight media. Only for viridans streptococcus did recovery rates vary significantly in different media. Recovery rates for this organism in two supplemented peptone broths (78% and 89%) and in hypertonie supplemented peptone (78%) were each higher than in thioglycolate (22%), Columbia (22%), Bactec aerobic and anaerobic (11%), and trypticase soy broths (11%) (p < 0.05 for each pair). Growth of bacteria in blood cultures containing antimicrobials depended on the ratio of the antimierobial concentration to the MIC and, for viridans streptococcus, the blood culture medium.

INTRODUCTION Over 50% of patients with bacterial endocarditis may have received antimicrobials before h o s p i t a l i z a t i o n (Pazin et al., 1982). It is unclear, however, whether prior antimicrobial use increases the n u m b e r of negative blood cultures in endocarditis. A l t h o u g h several studies have shown an association of prior antimicrobial use and culture-negative e n d o c a r d i t i s (Pazin et al., 1982; Pesanti and Smith, 1979; Werner et al., 1967), one review failed to show this relationship (Pedersen and Petersen, 1976). Furthermore, blood cultures in staphylococcal endocarditis may remain positive for days after initiation of appropriate therapy (Reymann et al., 1978). Several aspects of blood cultures tend to decrease the effect of antimicrobials. Blood is u s u a l l y diluted in the media 1:5 to 1:10. Ingredients a d d e d to various media inactivate antimicrobials. S o d i u m polyanetholsulfonate (SPS) inhibits aminoglycosides and p o l y m y x i n s (Washington, 1975). Ten- to twenty-percent sucrose may inactivate p e n i c i l l i n (Simberkoff et al., 1970) or provide osmotic support to cell-walldefective bacterial variants i n d u c e d by beta-lactam antimicrobials (Eng and Maeland, 1982). Penicillinase destroys some beta-lactam drugs. In the following report, two methods were used to study the effect of antimicrobials

1Present address: Department of Pathology (113), VA Medical Center, Salt Lake City, UT 84148. From the Division of Infectious Diseases, Department of Medicine, West Virginia University. Morgantown, WV 26505. Address reprint requests to: Dr. Robin McKenzie, Building 10, Room 11S 242, NIAID, NIH, Bethesda, MD 20892. © 1987 Elsevier Science Publishing Co., Inc.

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R. McKenzie and L. G. Reimer

on blood cultures. First, bacteria and antimicrobials were inoculated directly into the media. Second, rabbits with endocarditis were given antimicrobials before blood was taken for culture. MATERIALS AND METHODS Microorganisms Isolates of microorganisms used were Escherichia coli ATCC 25922 (American Type Culture Collection, Rockville, MD), Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, and one isolate each of viridans streptococcus and Streptococcus pneumoniae from patients with bacteremia. After overnight growth, organisms were suspended in saline at a concentration of 10 ~ CFU/ml estimated by a 0.5 McFarland standard and confirmed by a modification of the Miles-Misra dropplate method (Postgate, 1969). Antimicrobials Penicillin G., cephalothin, and vancomycin were obtained from Eli Lilly, Indianapolis, IN; oxacillin and ticarcillin from Beecham, Bristol, TN; gentamicin from Elkins-Sinn, Cherry Hill, NJ; and tetracycline from Roerig Pfizer, NY, NY. Dilutions were made in saline. Minimum Inhibitory Concentrations The method for MIC determination was that approved by the National Committee for Clinical Laboratory Standards (1985). MICs for P. aeruginosa were 4.0 ~g/ml for gentamicin, 22 p.g/ml for ticarcillin, 83 p.g/ml for tetracycline, >512 p.g/ml for vancomycin, and >512 ~g/ml for cephalothin, oxacillin, and penicillin G. MICs for E. call were 1.4 ~.g/ml for gentamicin, 2.0 p.g/ml for tetracycline, 27 /~g/ml for cephalothin, 512 p.g/ml for oxacillin, 320/~g/ml for vancomycin, and 70 ~g/ml for penicillin G. For S. aureus, MICs were 0.6 p.g/ml for gentamicin, 1.2 p.g/ml for tetracycline, 0.3 p.g/ml for cephalothin, 0.7 p.g/ml for oxacillin, 1.0 p.g/ml for vancomycin, and 0.1 ~g/ ml for penicillin G. The MIC for penicillin against the strain of viridans streptococcus used was 0.12 ~g/ml. MICs for S. pneumoniae were not determined. Direct Inoculation of Blood Culture Media Nine commercial blood culture media were studied: supplemented peptone broth, hypertonic supplemented peptone broth, supplemented peptone broth If, Columbia broth, trypticase soy broth, thioglycollate broth (Becton Dickinson, Rutherford, NJ); Bactec aerobic (6B) broth, Bactec anaerobic (7C) broth (Johnson Laboratories, Townson, MD); and thio] broth with SPS (Difco, Detroit, MI). Each of these media was inoculated with 1 ml of 0.9% saline containing 102 CFU of E. coli, P. aeruginosa, S. aureus, or S. penumoniae. Antimicrobials were also added in a volume of 0.1 ml to produce final concentrations equal to 10% of expected peak serum levels following common doses given orally (penicillin--.09 p.g/ml, tetracycline--.3 p.g/ml, and oxacillin--l.0 p.g/ml) or parenterally (cephalothin--l.5 p.g/ml and gentamicin--.8 p.g/ ml) (Wise, 1978). The vancomycin level (.4 p.g/ml) was 5% of the expected peak of 8 p.g/ml following a 500-mg dose given intravenously. These levels similate the concentrations of antimicrobials present in blood cultures when blood containing peak or 1/2-peak levels of antimicrobials are diluted in blood culture media 1 part in 10. Duplicate cultures were inoculated with human blood producing the 1:10

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dilution of blood recommended for many blood culture media. Cultures were incubated at 37°C and subcultured after 1, 2, and 7 days.

Production of Endocarditis Experimental endocarditis was produced by a modification of the procedure previously described (Garrison and Freedman, 1970). On day 1, New Zealand white rabbits weighing 2-4 kg were anesthetized with 9 mg/kg of xylazine (Haver-Lockhart, Shawnie, KA) and 40 mg/kg of ketamine hydrochloride (Park-Davis, Morris Plains, NJ). Sterile polyethylene tubing (Intramedic PE-90, I.D. 0.034, O.D. 0.05 in., Clay-Adams, Parsippany, NJ) was passed through the right carotid artery into the left ventricle and secured. On the fourth day, 1 ml of saline containing 108 CFU of bacteria was injected into a lateral ear vein. Seven hours later, 5 ml of blood were taken from the median ear artery and cultured to ensure that bacteremia was present before administering antimicrobials.

Antimicrobial Administration On the fifth day, 1 ml of antimicrobia] was injected into a lateral ear vein of the rabbits. Six rabbits infected with S. aureus received oxacillin 30-70 p.g/kg; eight rabbits infected with viridans streptococcus were given penicillin G 60,000-180,000 U/kg; eight infected with E. coli received gentamicin 1.7 ~g/kg; and six infected with P. aeruginosa were given gentamicin 1.7 p.g/kg. Two of these last six also received ticarcillin 50 p.g/kg. Four control rabbits infected with one of each of the four organisms received no antimicrobials.

Blood Cultures Between 20 min 0nd 3 hr after administration of a single dose of antimicrobial, rabbits were anesthetized and exsanguinated by cardiac puncture. Five ml-aliquots of blood were inoculated into bottles of eight of the nine commercial blood culture media listed above (thiol broth was omitted.) For each media, 5 ml of blood is the recommended inoculum. The resultant dilution of blood was 1:10 in all media except the Bactec bottles, which gave a dilution of 1:6. Eight to 17 blood culture bottles were inoculated for each rabbit. Cultures were incubated at 37°C and subcultured onto agar plates at 24 hr, 48 hr, 1 wk, and 2 wk. Subculture plates of viridans streptococci were incubated in a CO2 incubator.

Other Laboratory Procedures At the time of exsanguination, blood was obtained for antimicrobial levels and serum bactericidal titers. Oxacillin levels were determined by bioassay using Bacillus subtilis as the test organism (Bennet et al., 1966). Gentamicin levels were measured by radioimmunoassay. Due to a technical error, penicillin levels were not obtained. Serum bactericidal titers (SBT) were determined by a previously described method (Stratton and Relier, 1977) using an inoculum size of 105 CFU/ml. To examine the anesthetic agents xylazine and ketamine hydrochloride for possible inhibition of growth of the organisms used in this study, 20 p.1 of a 1/100 dilution of each drug was absorbed onto a 6-mm blank-paper disc, which was placed on a blood agar plate streaked with each organism. No inhibition of growth was shown. Necropsy of each rabbit confirmed the presence of vegetations.

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TABLE 1. N u m b e r of Positive Cultures Following Direct Innoculation of Bacteria and A n t i m i c r o b i a l s into Nine Blood Culture Media ° No. of positive blood cultures Antimicrobial Gentamicin Tetracycline Cephalothin Oxacillin Vancomycin Penicillin Total

E. coli

P. aeruginasa

S. aureus

S. pneumoniae

17 14 18 18 18 18 103

5 17 18 18 18 18 94

0 0 0 0 2 14 16

10 3 0 0 4 6 23

°Each bacterium-antimicrobial combination was set up in duplicate in each blood culture medium giving 18 cultures for each combination.

Calculation of Results Blood from each rabbit was inoculated into eight different blood culture media. Only c o m p l e t e sets of eight bottles were included in the analysis of the data. Twenty-eight rabbits p r o v i d e d thirty-two complete sets of blood cultures. Comparison of positivity rates of blood cultures was made using the Fischer's exact test and chi-square test.

RESULTS

Direct Inoculation of Blood Culture Media Duplicate cultures were set up for four isolates of bacteria incubated i n d i v i d u a l l y with six antimicrobials in nine blood culture media (432 cultures). Human blood was a d d e d at 1:10 dilution to one-half of the cultures. Of cultures containing blood, 116 of 216 (54%) were positive. Of those without blood, 119 of 216 (55%) were positive. Since the a d d i t i o n of blood had little effect on the positivity rate of cultures, results with and without blood were combined. Table 1 shows recovery rates for the four strains of bacteria according to the antimicrobial used. Recovery rates varied according to the ratio of antimicrobial level to MIC. When the antimicrobial level was less than 1/10 of the MIC, 161 of 162 (99%) of cultures were positive; when the level was between 1/10 of the MIC and equal to the MIC, 52 of 108 (48%) were positive; w h e n the level exceeded the MIC, none of 54 cultures were positive. Overall positivity rates were 95% for E. coil, 87% for P. aeruginosa, 15% for S. aureus, and 21% for S. pneumoniae. Whereas all six antimicrobials used were active against the 2 gm-positive organisms, only two of the drugs had significant gin-negative activity. Table 2 presents the recovery rates according to the culture m e d i u m used. Fifty percent to 63% of cultures were positive in each medium. Recovery was highest in the three s u p p l e m e n t e d p e p t o n e broths, but differences were not statistically significant.

Experimental Endocarditis Four control rabbits infected with one each of the four organisms--S, aureus, viridans streptococcus, E. coli, and P. aeruginosa--but not given antimicrobials gave positive blood cultures in each of the eight different blood culture media. Endocarditis was p r o d u c e d in another 28 rabbits that were subsequently given a single antimicrobial dose. Two h u n d r e d and fifty-six blood cultures from these animals were analyzed.

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169

TABLE 2. N u m b e r of Positive Cultures Following Direct Innoculation of Bacteria and A n t i m i c r o b i a l s into Nine Blood Culture Media ° No. of positive cultures Blood culture media

E. coli

P. aeruginosa

S. aureus

S. pneumoniae

Total

Supplemented peptone Supplemented peptone II Hypertonic supplemented peptone Thioglycollate Trypticase soy Columbia Thiol Bactec aerobic Bactec anaerobic

12 11

12 11

2 2

2 4

28 28

12 11 11 11 12 12 11

11 10 10 10 10 10 10

3 1 3 2 1 1 1

4 2 3 2 2 2 2

30 24 27 25 25 25 24

°Each bacterial strain was cultured with six antimicrobials in duplicate in each blood culture medium giving a total 12 cultures for each bacterium~lood culture medium combination.

The n u m b e r of positive blood cultures, antimicrobial levels, and serum bactericidal titers for each rabbit are shown in Table 3. Overall for the four organisms, 69% of blood cultures were positive. Fifty-six percent of cultures of blood with serum bactericidal titers of 1:8 or greater were positive c o m p a r e d to 80% of cultures with serum bactericidal titers less than 1:8 (p < 0.001, chi-square test). The percent positive blood cultures for the four organisms in the eight different m e d i a are s h o w n in Table 4. The percent of positive cultures was significantly lower for the 2 gm-positive cocci (43%) than for the 2 gm-negative rods (90%) (p < 0.001, chi-square test). Overall, s u p p l e m e n t e d peptone broth II gave a higher recovery rate (88%) than Bactec, trypticase soy, or thioglycollate broths (each 63%) (p < 0.05, chisquare test). For v i r i d a n s streptococcus, the highest recovery rates were in supplem e n t e d p e p t o n e II (89%) and s u p p l e m e n t e d peptone and hypertonic s u p p l e m e n t e d p e p t o n e broths (each 78%). These two rates were higher than the 11% recovery rate in both Bactec and trypticase soy broths (p = 0.002 and p = 0.007, respectively, Fischer's exact test) and the 22% recovery rate in both thioglycollate and Columbia broths (p = 0.007 and p = 0.027, respectively, Fischer's exact test). Seventy-one percent of positive blood cultures from rabbits with endocarditis showed growth w i t h i n 24 hr. Ninety-three percent were positive in 48 hr. There was little difference in rates of growth in different media.

DISCUSSION W h e n a n t i m i c r o b i a l s were a d d e d directly to blood culture media to simulate cultures of blood containing peak or ~-peak concentrations of antimicrobials, 55% of cultures were positive. W h e n rabbits with endocarditis were given antimicrobials and blood was taken containing SBTs of 0 to 1:128, 69% of blood cultures were positive. Recovery of gm-negative organisms was higher than that of gm-positive organisms. Higher MICs and lower serum bactericidal levels for the gin-negative organisms probably account for this difference. For S. oureus with increasing time following antibiotic injection, serum bactericidal activity and antibiotic levels t e n d e d to fall and a higher percentage of blood cultures became positive. For viridans streptococcus with increasing time, serum bactericidal activity tended to decrease but no increase in the n u m b e r s of positive blood cultures occurred. This organism may be more vulnerable to longer exposure to antimicrobials.

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T A B L E 3. Data O b t a i n e d from I n d i v i d u a l Rabbits Infected w i t h O n e of F o u r Organisms and Treated with Antibiotics

Organism S. aureus

Viridans streptococcus

E. coil

P. aeruginosa

Time ° (min)

SBT~'

Antibiotic ~ level ~g/ml

Blood cultures no. positive/ no. obtained (%)

30 30 30 55 75 180

128 16 16 16 8 0

44 10 4 14 9 <2

0/8 4/8 4/8 5/8 5/8 8/8

(0) (50) (50) (63) (63) (100]

30 30 30 40 60 70 80 120 20 30 30 30 40 40 90 180 30 d 30 30 30 30 40 ~

128 32 8 8 16 2 16 0 8 8 8 4 4 NA 4 1 4 2 2 1 <2 2

NA NA NA NA NA NA NA NA 8 9 9 7 NA 6 2 0 5 8 6 4 3 4

6/8 2/8 4/8 4/8 6/16 2/8 3/8 1/8 8/8 8/8 8/8 6/8 8/8 8/8 16/16 8/8 8/8 0/8 16/16 8/8 16/16 4/8

(75) (25) (50) (50) (38) (25) (38) (13) (100) {I00) (100) (75) (100) (100) (100) (100) (100} (0) (100) (100) (100) (50)

°Time since antibiotic given. bReciprocal of serum bactericidal titer. ~Oxacillin for S, aureus, gentamicin for E. coli and P. aeruginosa. dTicarcillin also given. NA, not available. W h e n d i f f e r e n t m e d i a w e r e c o m p a r e d using the e n d o c a r d i t i s model, growth of v i r i d a n s s t r e p t o c o c c u s in e a c h of the three s u p p l e m e n t e d p e p t o n e m e d i a was sign i f i c a n t l y h i g h e r t h a n in e i t h e r the Bactec system, trypticase soy broth, C o l u m b i a broth, or t h i o g l y c o l l a t e broth. P e n i c i l l i n a s e in the s u p p l e m e n t e d p e p t o n e broths m a y h a v e i n c r e a s e d the r e c o v e r y rate of streptococci in the p r e s e n c e of p e n i c i l l i n . Hyp e r t o n i c b r o t h has b e e n s h o w n to increase the sensitivity of blood cultures in patients w i t h S. a u r e u s b a c t e r e m i a treated w i t h beta-lactam a n t i m i c r o b i a l s (Eng and M a e l a n d , 1982). In the t w o m o d e l s used, h o w e v e r , no i n c r e a s e d r e c o v e r y was seen w h e n h y p e r t o n i c s u p p l e m e n t e d p e p t o n e broth was c o m p a r e d to the o t h e r two s u p p l e m e n t e d p e p t o n e broths. S e v e r a l t e c h n i c a l factors m a y h a v e i n f l u e n c e d our results. D i l u t i o n of b l o o d from the rabbits was 1:6 in the Bactec bottles and 1:10 in the other bottles as r e c o m m e n d e d by the m a n u f a c t u r e r s . T h e resultant l o w e r d i l u t i o n of a n t i m i c r o b i a l s in the Bactec bottles m a y h a v e led to l o w e r r e c o v e r y rates in this system. W e r n e r et ah (1967) f o u n d a m e d i a n c o l o n y c o u n t b e t w e e n 2 1 - 3 0 C F U / m l in the

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171

TABLE 4. Percent Positive Blood Cultures in Different Blood Culture Media for Rabbits Infected with One of Four Organisms and Treated with Antimicrobials Blood culture media ~ SP SPII HSP Tg TS C B aer Ban B aer&an Total

No. of positive cultures/no, obtained (%] P. E. coli

8/9 9/9 9/9 9/9 9/9 9/9 8/9 9/9 9/9 70/72

(89) (100) (100) (100) (100] (100) (89} (100) (100) (97)

aeruginosa

6/8 (75) 7/8 (88) 7/8 (88) 6/8 (75) 6/8 (75) 6/8 (75) 7/8 (88} 7/8 (88) 7/8 (88) 52/64 (81)

S. aureus

3/6 4/6 4/6 3/6 4/6 4/6 2/6 2/6 3/6 26/48

(50) (67) (67} (50] (67} (67) (33) (33} {50) (54}

V strep ~ 7/9 8/9 7/9 2/9 I/9 2/9 0/9 1/9 1/9 28/72

(78} (89) (78) (22) (11) (22) (0) (11) (11) (39}

Total 24/32 28/32 27/32 20/32 20/32 21/32 17/32 19/32 20/32 176/256

(75) (88} (84) (63) (63) (66) {53) (59) (63) (69)

°SP. supplemented peptone; SPII.supplemented peptone 11;HSP. hypertonic supplemented peptone; TR. thioglycollate; TS, trypticase soy; C. Columbia; B aer. Bactec aerobic; B an, Bactec anaerobic. ~Viridans streptococcus. blood of 415 patients with streptococcal endocarditis and 11-20 CFU/ml in 17 patients with staphylococcal endocarditis. These figures probably overestimate the magnitude of bacteremia since they do not include specimens with positive broth cultures (in w h i c h 10 ml of blood was cultured) but negative pour plates (in w h i c h 1 ml of blood was cultured). For our experiments, twenty CFU/ml of blood was taken as an a p p r o x i m a t i o n of the m e d i a n colony found in h u m a n endocarditis. In the simulated blood cultures, 100 CFU of bacteria were a d d e d to blood culture media approximating the n u m b e r of organisms obtained in 5 ml of blood from patients with endocarditis. Blood obtained from rabbits with endocarditis probably contained more than 20 CFU/ml. Quantitative cultures have been done in our laboratory in rabbits infected with the same isolate of viridans streptococcus. Colony counts in the blood of 32 rabbits with positive blood cultures after the carotid catheter was removed ranged from 0 - 9 6 8 0 CFU/ml with a mean of 961 CFU/ml (Robin McKenzie and Debra Pederson, u n p u b l i s h e d results). In the current study, the catheter was left in place, a factor w h i c h might p r o d u c e higher counts. The 5 ml-inoculum of rabbit blood, therefore, probably contained a mean of more than 5,000 CFU of bacteria. This higher i n o c u l u m may explain w h y cultures from the rabbits were more often positive than simulated cultures. In several instances, blood culture positivity correlated poorly with the SBT (Table 3). These discrepancies are probably due to the wide range of colony counts in the blood of i n d i v i d u a l rabbits. In a study measuring antibacterial activity by bioassay in 2,010 blood cultures (Rodriguez and Lorian, 1985), organisms were isolated less frequently from blood cultures with than those without antibacterial activity. Organisms sensitive and resistant to the antimicrobial used were recovered from nine blood cultures with low or m e d i u m antibacterial activity. Only resistant organisms were recovered from five cultures with high antibacterial activity. MICs for the organisms and drug levels were not given. This data, however, supports ours in suggesting that the level of antimicrobial activity and the sensitivity of the organism are important factors. Our results indicate that blood culture media are frequently capable of supporting the growth of organisms in the presence of antimicrobials. The recovery rate appears to d e p e n d on the relative concentration of the antimicrobial to the MIC of the organism. Selection of m e d i a may be important for viridans streptococcus. For patients who have taken prior antimicrobials, blood cultures should be obtained after anti-

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m i c r o b i a l s h a v e c l e a r e d from the b l o o d s t r e a m or are present at low levels. To o p t i m i z e c o n d i t i o n s for r e c o v e r y of v i r i d a n s streptococcus, a s u p p l e m e n t e d p e p t o n e m e d i u m s h o u l d be used. This work was presented in part at the American Society for Microbiology meeting held in St. Louis, MO, March 1984. This study was supported by a Biomedical Research Grant from West Virginia University. We thank Barbara Roberts for her valuable technical work and Gretchen Winstein for excellent secretarial assistance.

REFERENCES Bennet JV, Brodie JL, Benner El, Kriby WMM (1986) Simplified accurate antibiotic assay of clinical specimens. Appl Microbiol 14:170. Eng J, Maeland A (1982) Correlation of growth of aerobic blood cultures in hypertonic broth with antibiotic therapy. J Clin Microbiol 16:890. Garrison PK, Freedman LR (1970) Experimental endocarditis. 1. Staphyloccal endocarditis in rabbits resulting from placement of a polyethylene catheter in the right side of the heart. Yale J Biol Med 42'.394. National Committee for Clinical Laboratory Standards (1985) Reference methods for the determination of minimum inhibitory concentration (MIC) of antimicrobial agents with bacteria that grow aerobically; broth microdilution, broth macrodilution, and agar dilution methods. Approved standard M7-A. National Committee for Clinical Laboratory Standards, Villanova, PA. Pazin GJ, Saul S, Thompson ME (1982) Blood culture positivity--suppression by outpatient antibiotic therapy in patients with bacterial endocarditis. Arch Intern Med 142:263. Pedersen FK, Petersen EA (1976) Bacterial endocarditis in Blegdamshospitalet in Copenhagen 1944-1973. Scand J Infect Dis 8:99. Pesanti EL, Smith IM (1979) Infective endocarditis with negative blood cultures: An analysis of 52 cases. Am I Med 66:43. Postgate JR (1969) Viable counts and viability. In Methods in Microbiology, Vol 1. Eds., JR Norris and DW Ribbons. London and New York: Academic Press, pp. 611-628. Reyman MT, Holley HP Jr, Cobbs CG (1978) Persistent bacteremia in staphylococcal endocarditis. Am J Med 65:729. Rodriguez F, Lorian V (1985) Antibacterial activity in blood cultures. J Clin Microbiol 21:262. Simberkoff MS, Thomas L, McGregor D, Shenkein I, Levine BB (1970) Inactivation of penicillins by carbohydrate solutions at alkaline pH. New Engl J Med 283:116. Stratton CW, Relier LB (1977) Serum dilution test for bactericidal activity. I. Selection of a physiologic diluent. J Infect Dis 136:187. Washington JAII (1975) Blood cultures: Principles and techniques. Mayo Clinic Proc 50:91. Werner AS, Cobbs CG, Kaye D, Hook EW (1967) Studies on the bacteremia of bacterial endocarditis, lAMA 202:127. Wise R (1978) Table of expected concentrations of antibiotic. In Laboratory Methods in Antimicrobial Chemotherapy. Eds., DS Reeves, I Phillips, JD Williams, and R Wise. Edinburgh: Churchill Livingstone, pp. 151-156.