Correlation of Serum Bactericidal Activity with Antimicrobial Agent Level and Minimal Bactericidal Concentration

Transcription

1 THE JORNAL OF INFECTIOS DISEASES VOL. 145, NO.2. FEBRARY by the niversity of Chicago. All rights reserved /82/ $00.88 Correlation of Serum Bactericidal Activity with Antimicrobial Agent Level and Minimal Bactericidal Concentration Charles W. Stratton,* Melvin P. Weinstein.] and L. Barth Reller From the Division of Infectious Diseases and Clinical Microbiology, Department of Medicine, niversity of Colorado Health Sciences Center, Denver, Colorado Few data are available to show how serum dilution test results correlate with results of antimicrobial assays and determinations of minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs). Serum dilution tests were performed for 65 patients with bacteremia from serious infections. Assays for antimicrobial agent levels and determinations of MICs and MBCs against the infecting organisms were performed simultaneously. Mueller-Hinton broth and Mueller-Hinton broth supplemented with Ca'", Mg'", and pooled human serum were compared as diluents for the serum dilution test and as broths for the MIC and MBC determinations. Although the use of either medium yielded positive correlations, better correlations were found between measured and expected serum bactericidal activities when both the serum dilution test dilutions and the MIC and MBC determinations were done in the supplemented MHB-human serum solution. The serum dilution test is a direct method of measuring activity against a specific organism in a patient's serum during antimicrobial therapy [1, 2]. The serum dilution test has been used to assess the adequacy of antimicrobial therapy for infective endocarditis [2, 3] and has been recommended to guide antimicrobial therapy for other serious infections such as osteomyelitis [4] and septic arthritis and empyema [5], as well as infections in patients with cancer [6]. More recently, the test has been used to study the efficacy of combinations of antimicrobial agents and to detect synergism between antimicrobial agents in humans [7]. Although the serum dilution test is basically a simple variation of the broth dilution test and has been in use for over 30 years, there are few data available to show how this test correlates with results of antimicrobial assays and with determinations of MICs and MBCs. The objective of the present study was to determine whether serum dilution test results correlate with results that are Received for publication July 16, 1981, and in revised form September 2, Please address requests for reprints to Dr. L. Barth Reller, Box B-168, niversity of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, Colorado * Present address: Department of Pathology, Vanderbilt niversity Medical Center, Nashville, Tennessee. t Present address: Division of Infectious Diseases, Rutgers Medical School, Piscataway, New Jersey. calculated from antimicrobial agent levels and MBCs. Because human serum can alter the results of susceptibility tests, another objective was to compare Mueller-Hinton broth (MHB) and a solution containing pooled human serum and MHB supplemented with Ca" and Mg'" as the diluent in the serum dilution tests and as the broth in the MIC and MBC determinations. Patients and Methods Selection of patients. Blood culture results were monitored over an I8-month period at the niversity of Colorado and Veterans Administration Hospitals in Denver. Patients with blood cultures that were positive for bacterial infection were candidates for this study if they (1) had bacteremia from endocarditis or from another serious infection and (2) had begun therapy with a single antimicrobial agent within the first 48 hr of treatment. Patients being treated with more than one antimicrobial agent were candidates if they were diagnosed as having endocarditis. Study protocols. Once a patient was selected for inclusion in the study, the appropriate clinical data were recorded, blood samples were drawn, and the patient was followed during hospitalization. Test results of the individual patients were made available to the physician with primary responsibility for the care of a patient if the results revealed potentially harmful effects (for example, 160

2 Serum Dilution Test Correlates 161 toxic levels of aminoglycoside) or if the physician requested them. Serum samples were obtained according to the following protocol. (1) Serum was obtained 1 hr after the completion of injection or infusion of the antimicrobial agent(s) to determine the "peak" bactericidal titer, and serum was obtained 1 hr or less before the next dose to determine the "trough" bactericidal titer. (2) For patients receiving a single antimicrobial agent, serum specimens were obtained at least 24 hr but not more than 72 hr after treatment was begun. (3) If multiple antimicrobial agents had been administered before a single agent was finally chosen for therapy, serum was not obtained until the discontinued antimicrobial agents were no longer present in the patient's blood, as determined by pharmacodynamic tests and serum assays. If any possibility of interference by the discontinued antimicrobial agent existed, the patient was not included in the study. If the treatment regimen was changed in any way, the serum dilution studies were repeated, as long as only one antimicrobial agent remained in use. Media jor dilution tests. "Media were prepared and analyzed as described [8, 9]. Media used were MHB (Difco Laboratories, Detroit), trypticase soy broth (BBL Microbiology Systems, Cockeysville, Md.), and pooled human serum (Flow Laboratories, Inglewood, Calif.). A single batch of MHB (1ot no ), containing 9.8 mg of Ca" and 2.4 mg of Mg'" /liter, was used. MHB was supplemented with 50 mg of Ca" and 20 mg of Mg" /liter and then combined with pooled human serum in equal volumes to form supplemented MHB-human serum solution. All media were adjusted with 0.1 N NaOH or 0.1 N HCI to ph at 25 C. After adjustment of ph all sera were clarified by filtration and then passed through a filter (pore size, 0.45 urn; Millipore Corp., Bedford, Mass.). The pooled human serum was determined by radioimmunoassay to have no hepatitis B virus antigen. In addition, the pooled human serum was tested for the presence of {3-lactamase activity by the method of Thornsberry and Kirven [10]. Antimicrobial activity was screened by placing 20 I-tl of the pooled human serum on blank paper disks (BBL Microbiology Systems), placing the disks into antibiotic assay medium no. 5 (Difco) which had been seeded with a spore suspension of Bacillus subtilis (strain no. ATCC 6633), and looking for zones of inhibition. Another quality control measure used to evaluate the pooled human serum was the determination of MICs and MBCs of various antimicrobial agents for strains of bacteria in media supplemented with the serum. Reimer et al. recently published standard MICs and MBCs of most antimicrobial agents that were determined with and without human serum against strains of bacteria commonly used as quality controls in susceptibility tests [11]. Such a control strain was used each time MIC and MBC determinations were done. Preparation oj the inoculum. The inoculum was prepared according to the procedure of the National Committee for Clinical Laboratory Standards [12]. Organisms from each patient were grown in trypticase soy broth to a turbidity equal to a 0.5 McFarland standard. A 1:200 dilution was made with either MHB or with supplemented MHB depending on whether the final broth solution was to be MHB or supplemented MHB-human serum solution. This dilution yielded a final inoculum of viable bacteria/ml for both the broth and the serum dilution tests, as verified each time by the surface drop-count method of Miles and Misra [13]. Antimicrobial agents and assays jor antimicrobial agent levels. Solutions of antimicrobial agents were prepared with distilled water from standard powders of known potency. The antimicrobial agents and their sources were: methicillin, oxacillin, dicloxacillin, ampicillin, and kanamycin (Bristol Laboratories, Syracuse, N.Y.); penicillin, cephalothin, cefazolin, cephalexin, and vancomycin (Eli Lilly and Co., Indianapolis); c1indamycin (The pjohn Co., Kalamazoo, Mich.); gentamicin (Schering Corp., Bloomfield, N.J.); cefoxitin (Merck Sharp and Dohme, Rahway, N.J.); and chloramphenicol (Parke-Davis and Co., Detroit). All powders were obtained from single batches and were stored with a silica-gel desiccant at -70 C. Antimicrobial agent levels were determined by rapid bioassays [8, 9] with the exception of chloramphenicol levels, which were measured by a colorimetric method [14]. Rapid bioassay tests were done in quadruplicate, and zones of inhibition were averaged and plotted on semilogarithmic graph paper. The concentrations of antimicrobial agents in serum were determined from the graph based on zone sizes achieved with standard solutions of the agent prepared in serum. Broth dilution tests. The microtechnique used

3 162 Stratton, Weinstein, and Reller in broth dilution tests was similar to that of MacLowry et al. [15], except that no holes were punched in the transport tape that sealed the polystyrene plates (Dynatech Laboratories, Alexandria, Va.). Each well contained 50 J.lI of inoculum and 50 J.lI of diluent. The final concentration of bacteria was organisms/rnl as recommended [16]. End-point determinations were made for bactericidal activity by transferring broth containing organisms onto antimicrobial agent-free Mueller-Hinton agar (plate diameter, 150 mm) with a multipoint inoculator and incubating the subcultures for 18 hr at 35 C [17]. By assaying the antimicrobial activity transferred from a standard solution, a volume of ml of broth (AJ1.5 X 10 3 organisms) was determined to be optimal for subcultures. The MBC has been defined as the lowest concentration of antimicrobial agent capable of killing 99.90/0 of the bacteria in the initial inoculum in both serum and broth dilution tests [18]. Thus, the bactericidal end point was defined here as the lowest concentration of antimicrobial agent capable of preventing growth (>99.9% killing) of bacteria on the agar surface during the given period of incubation. This multipoint inoculator technique for the determination of bactericidal end points is practical for microtiter systems. Reller and Stratton [9] previously compared this technique with the recommended technique for macrotube dilutions [18], in which O.OI-ml broth samples are removed and streaked onto blood agar plates, and found that both techniques produced identical end points. The original data of Reller and Stratton [9] were reviewed using rejection values for the macrotube dilution bactericidal end point that were determined by a method which takes into account pipetting error and the intrinsic sampling variability due to the Poisson distribution of sample responses [18]. Again, the techniques were found to produce identical end points. Broth dilution tests were performed in duplicate in both MHB and supplemented MHB-human serum solution. Serum dilution tests. The serum dilution tests were done by the same method as the broth dilution tests, except that samples of serum from the patients were used instead of the double-strength solution of antimicrobial agent in one well. Serum samples were diluted in twofold increments with antimicrobial agent-free human serum, and equal volumes of the inoculum prepared in supplemented MHB were added. In addition, serum samples were diluted in twofold increments with MHB, and equal volumes of the inoculum prepared in MHB were added. Each test was performed in duplicate. Theoretical results for serum bactericidal activity were calculated by dividing the assayed antimicrobial agent level by the MBC. These results were compared with the actual serum bactericidal titers by linear regression analysis with a programmable calculator (program STI-07A; model no. HP 97; Hewlett Packard Corp., Corvallis, Ore.). Statistical comparisons were performed with Student's t-test for paired data. P values that were <0.05 by the two-tailed test were considered significant. Results Patients. There were 65 patients (38 male and 27 female), who ranged in age from 10 to 84 years. One hundred thirty-six serum dilution tests were done. Although a wide spectrum of infections was present, a much narrower spectrum of organisms caused most of the infections. The patients were divided into four groups according to the organisms isolated from their blood. These groups were (1) patients with staphylococcal sepsis, (2) patients with streptococcal sepsis, (3) patients with sepsis due to Escherichia coli, and (4) patients with sepsis caused by other miscellaneous organisms. The groups were considered separately. Staphylococcal sepsis. Clinical information. There were 24 patients with staphylococcal sepsis, including nine with endocarditis and six with iv catheter-associated sepsis. Sepsis was considered to have been cured if the patient left the hospital or survived for at least one week after the termination of bacteremia. By these criteria there were 23 cures. Other staphylococcal infections included an infected hip joint, infection of a bovine dialysis shunt, pneumonia with empyema, two septic joints, and five abscesses associated with traumatic or postoperative wounds. The antimicrobial agents used to treat the patients with staphylococcal infections are shown in figure 1. No tolerant strains of staphylococci were noted among the 24 strains. (Tolerance was defined as an MBC:MIC ratio of 32.)

4 Serum Dilution Test Correlates ' PENICILLIN, METHICILLIN.' CEPHALOTHIN :0' CEFAZOLIN...J cl 32 e cr W I b cl CD ::> cr w l/) CI W cr ::> l/) cl W. 0 0 o CALCLATED RECIPROCAL SERM BACTERICIDAL TITER ( antimicrobial level) MBC II'CEFOXITIN.' CLINDAMYCIN 6' VANCOMYCIN.' OXACILLIN O'NAFCILLIN.'OICLOXACILLIN Figure 1. Correlation of titers of bactericidal activity determined in serum dilution tests with calculated values based on MBCs and antimicrobial agent levels for 24 patients undergoing treatment for staphylococcal bacteremia. Left, results obtained using Mueller-Hinton broth supplemented with Ca'", Mg'", and pooled human serum as the diluent for serum dilution tests and MBC determinations; right, results obtained using unsupplemented Mueller Hinton broth. Serum dilution tests. Sixty-two serum dilution tests were done for patients with staphylococcal sepsis. All except one patient (with the infected bovine dialysis shunt) had peak serum bactericidal titers of>1:8. Antimicrobial agents that are highly. protein-bound, such as oxacillin or nafcillin, had serum inhibitory titers and serum bactericidal titers that were four- to eightfold higher in MHB than in supplemented MHB-human serum solution. Similarly, the results of MIC and MBC determinations for these antimicrobial agents were four- to eightfold lower in MHB than in supplemented MHB-human serum solution. The geometric means and ranges of all serum bactericidal titers against staphylococci are shown in table 1. The serum bactericidal titers obtained using supplemented MHB-human serum solution as the diluent were compared with theoretical results obtained by dividing the observed antimicrobial agent level by the MBC determined in supplemented MHB-human serum solution. The results are plotted in figure 1, left. A statistically significant relationship (r = 0.67, P < 0.001) was demonstrated between observed serum bactericidal titers and calculated serum bactericidal values. Serum bactericidal titers that were determined with unsupplemented MHB as diluent were generally lower than values calculated from MBCs that were determined in unsupplemented MHB (figure 1, right). Linear regression analysis showed a correlation (r = 0.45, P< 0.001) that was not as high as the correlation between calculated and observed results obtained with supplemented MHB-human serum solution. Streptococcal sepsis. Clinical information. "There were 16 patients with streptococcal sepsis, including two with endocarditis, who received four weeks of parenteral therapy. All patients were cured. Ten patients had pneumonia (three of these also had empyema). One patient had an infected bovine dialysis shunt and three patients had soft-tissue infections. The antimicrobial agents used to treat the patients with streptococcal infections are shown in figure 2. Serum dilution tests. Thirty-two serum dilution tests were done for patients with streptococcal sepsis. Serum bactericidal titers were high, as they were for patients with staphylococcal sepsis. All 16 patients had peak levels of>1:8, and only two patients had trough levels of <1 :8. The geometric mean titers and ranges in titers are shown in table 1. Linear regression analysis showed a significant relationship (r = 0.95, P < 0.001) between observed serum bactericidal titers and calculated

5 164 Stratton, Weinstein, and Reller Table t. Serum bactericidal titers in patients undergoing therapy with antimicrobial agents for bacteremia due to staphylococci, streptococci, Escherichia coli, or other miscellaneous organisms. Geometric mean serum bactericidal titer (range) with Infecting organism (no. of samples tested) Staphylococci (62) Streptococci (32) E. coli (34) Miscellaneous organisms (14) Peak 1: (1:8-1:2,048) 1:512 (1:32-1:4,096) 1:16 (1:2-1:) 1:16 (1:2-1:2,048) MHB Trough 1:16 (1:2-1 :256) 1: (1:2-1:2,048) 1:4 (1:2-1:16) 1:8 (1:2-1:) Supplemented MHB-pooled human serum solution Peak 1:32 (1:2-1:512) 1:256 (1:8-1 :2,048) 1:16 (1:2-1:) 1:16 (1:2-1:2,048) Trough 1:8 (1:2-1:) 1:64 (1:2-1 :2,048) 1:4 (1:2-1:16) 1:8 (1:2-1:) NOTE. Serum bactericidal titers were determined by serum dilution tests. Peak bactericidal titers were determined in sera obtained from the patients 1 hr after the completion of injection or infusion of the antimicrobial agent(s); trough bactericidal titers were determined in sera obtained I hr or less before the next dose. For a description of the serum dilution test and the diluents (Mueller-Hinton broth [MHB] and supplemented MHB-pooled human serum solution), see Patients and Methods; for a description of miscellaneous organisms, see Results. serum bactericidal values when supplemented MHB-human serum solution was used (figure 2, left). The correlation was equally high (r = 0.95, P< 0.001) between observed and calculated serum bactericidal titers obtained with unsupplemented MHB (figure 2, right). E. coli sepsis. Clinical information. There were 15 patients with E. coli sepsis; none had endocarditis. The only patient not cured was afebrile by day 6 of treatment, when he died of massive gastrointestinal bleeding. Six patients had acute pyelonephritis, three had pneumonia, and two had acute cholecystitis. Others had diverticulitis, peritonitis, sepsis secondary to a transrectal prostate biopsy, and sepsis (in an immunosuppressed patient) from an unknown source. The antimicrobial agents used to treat the patients with E. coli sepsis are shown in figure 3. Serum dilution tests. Thirty-four serum dilution tests were done for patients with E. coli sepsis. Serum bactericidal titers were lower for these patients than for those with streptococcal or staphylococcal sepsis. Although most patients had peak serum bactericidal titers of 1:8, four patients with lower levels were cured. Twelve of the 15 patients in this group had trough serum bactericidal titers of <1 :8. The geometric mean titers and ranges in titers are shown in table 1. Once again, a statistically significant relationship was demonstrated (r = 0.56, P < 0.01) between observed and calculated serum bactericidal values obtained with supplemented MHB-human serum solution (figure 3, left). The correlation between observed and calculated values obtained with unsupplemented MHB was similar, but not as high (r = 0.47, P < 0.01) (figure 3, right). Sepsis with miscellaneous organisms. Clinical information. There were 10 patients with sepsis caused by a variety of organisms, all of whom were cured. Two patients had endocarditis, one caused by Brucella melitensis and the other by Fusobacterium necrophorum. Two immunosuppressed patients had infections due to Listeria monocytogenes. Two patients had iv catheter-associated sepsis with gram-negative bacilli (Enterobacter cloacae and Klebsiella pneumoniae). Other patients had pyelonephritis caused by Pseudomonas aeruginosa or pneumonia with empyema caused by Haemophilus influenzae, and two had abscesses with anaerobic organisms (Bacteroides fragilis for one patient and anaerobic diphtheroids for the other) isolated from the blood and from the abscesses. The antimicrobial agents used to treat these patients are shown in figure 4. Serum dilution tests. Only 14 serum dilution tests were done for the patients with miscellaneous infections. Serum bactericidal titers were low, but all patients with peak levels of <1:8, except those with endocarditis, were cured. The geometric mean titers and ranges in titers are shown in table 1. Linear regression analysis showed a statistically significant relationship (r = 0.78, P < 0.01) between observed and calculated serum bactericidal values obtained using supplemented MHB-human serum solution (figure 4, left). When unsupplemented MHB was used, the correlation was

6 Serum Dilution Test Correlates 165 1I s 12 S12 a: J '::......J 0 eo a: J ld :e ::l a: J Vl 0 J a: ::l Vl J :e o 0 C] 0 ant imicrobial level) CALCLATED RECIPROCAL SERM BACTERICIDAL TITER ( o MBC o ' PENICjLLI, AMPICILLIN.' CEFAZOLIN.00 'VANCOMYCIN Figure 2. Correlation of titers of bactericidal activity determined in serum dilution tests with calculated values based on MBCs and antimicrobial agent levels for 16 patients undergoing treatment for streptococcal bacteremia. Left, results obtained using Mueller-Hinton broth supplemented with Ca'", Mg'", and pooled human serum as the diluent for serum dilution tests and MBC determinations; right, results obtained using unsupplemented Mueller Hinton broth. similar but not as high (r = 0.66, P< 0.02) (figure 4, right). Discussion Although the serum dilution test has been in use for over 30 years, its clinical utility has not been clearly defined. Several factors have contributed to this problem. One has been the lack of standardization for the test [19]. Other factors are the choice of diluent for the test [8, 20] and the choice of broth for the MIC and MBC determinations [21]. Another is the lack of data showing that the serum dilution test correlates well with results of assays for antimicrobial agent levels and MIC and MBC determinations [21]. A previously developed standardized serum dilution test [9] and physiologic diluent [8] were used in the present study. Supplemented MHB-human serum solution was used for the MIC and MBC determinations and for the serum dilution tests because of its similarity to human serum with respect to protein binding, ph, concentration of calcium, magnesium, and phosphate ions, and performance in previous tests [8, 9, 22]. The serum bactericidal activity and the MICs and MBCs were influenced by the choice of MHB or supplemented MHB-human serum solution as the diluent for the serum dilution test and as the broth for the MIC and MBC determinations; the addition of human serum increased the MICs and MBCs and decreased the serum bactericidal activity. The decreasing of serum bactericidal activity by human serum was somewhat unexpected. Human serum was expected to potentiate the in vitro activity of antimicrobial agents because of its intrinsic antibacterial defense mechanisms [23-31]. However, heat inactivation of the pooled human serum appears to have inactivated most of these mechanisms. Similar inactivation has been noted by Jordan and Kawachi [21]. Serum bactericidal titers have been compared with values calculated from antimicrobial agent levels and MBCs [21]. Although a positive correlation was found (r = 0.43, P < 0.05), the investigators did not think that the magnitude of the titer could be accurately estimated [21]. The use of human serum in the present study resulted in a better correlation of calculated and observed activities (figures 1, 3, and 4). Binding to proteins appears to decrease the in vitro activity of some antimicrobial agents. The serum bactericidal activity of antimicrobial agents that are highly protein-bound was overestimated

7 166 Stratton, Weinstein, and Reller I :: o 64 '"-..2.-/ J <l: o a:: 1J.J... <l: CD : APICILLI a: CEPHALOTHIN.' CEFAZOLIN ;0' CEPHALEXIN 'K ANAhlYC1N.' GENTAMICIN 6.' CHLORAMPHENICO antimicrobial levelj CALCLATED RECIPROCAL SERM BACTERICIDAL TITER MBC J ( Figure 3. Correlation of titers of bactericidal activity determined in serum dilution tests with calculated values based on MBCs and antimicrobial agent levels for 15 patients undergoing treatment for bacteremia due to Escherichia coli. Left, results obtained using Mueller-Hinton broth supplemented with Ca", Mg", and pooled human serum as the diluent for serum dilution tests and MBC determinations; right, results obtained using unsupplemented Mueller Hinton broth. when tests were done with unsupplemented MHB, as seen in figure 1, right. Previous studies have shown decreases in the in vitro activities of certain antimicrobial agents in the presence of human serum [8,9, 11, 19-21]. Human serum clearly affects antimicrobial activity. The fact that antimicrobial activity in the presence ofhuman serum is different from activity in broth media is not surprising because human serum is a complex mixture with little in common with most broth media. Human serum is the milieu in which antimicrobial agents are expected to function, and so a physiologic broth is more likely to correlate with in vivo activity. Over an 18-month period, a total of 65 patients with bacteremia was studied, most of whom had staphylococcal, streptococcal, or E. coli infections. Recent surveys show that staphylococcal, streptococcal, and, E. coli infections continue to be among the most common causes of bacteremia [32-34]. Thus, our group of patients constitutes a representative clinical sample. Most of our patients were cured of bacteremia. This may be attributed as much to host factors as to the effects of antimicrobial agents. The functional state of host defense mechanisms is one of the most important determinants of the effectiveness of antimicrobial agents [23-25]. Few patients with severe host defense problems were included in the present study because such patients generally receive more than one antimicrobial agent at a time during therapy. Serum bactericidal titers were much higher for the patients with bacteremia due to gram-positive bacteria than for those with bacteremia due to gram-negative bacteria. This result may reflect both higher MBCs for gram-negative bacilli and the greater safety in administering high doses of penicillins and cephalosporins rather than aminoglycosides. Previous reports have recommended achieving serum bactericidal activity in titers ranging from 1:2 to 1:32 during antimicrobial therapy for infective endocarditis and other serious infections [1-3, 5-7,20,35]. However, in only three reports were the dilutions made in serum [5, 6, 20]. Peak serum bactericidal titers of 1:8 were good indicators of successful antimicrobial therapy, but the only failures that occurred in the treatment of patients with lower peak serum bactericidal titers were those involving patients with infective endocarditis. Because of the low numbers of patients in homogeneous groups (for example, there were only nine patients with endocarditis due to Staphylo-

8 Serum Dilution Test Correlates 167 I 2 Cl:.....J et C Cl:... et 1Il 6, Il' At,lP ICILL IN e CEF"AZOLIN 0: KANAMYCIN iii' GENTAt,lILIN.' CHLOR AMPHENICOL :::) Cl: IF) C Cl: :::) IF) et antimicrobial level \ CALCLATED RECIPROCAL SERM BACTERICIDAL TITER ( MBC ) e 256 Figure 4. Correlation of titers of bactericidal activity determined in serum dilution tests with calculated values based on MBCs and antimicrobial agent levels for 10 patients undergoing treatment for bacteremia caused by miscellaneous organisms. Left, results obtained using Mueller-Hinton broth supplemented with Ca'", Mg", and pooled human serum as the diluent for serum dilution tests and MBC determinations; right, results obtained using unsupplemented Mueller-Hinton broth. For a description of the miscellaneous organisms, see Results. coccus aureus), no valid conclusions regarding the level of serum bactericidal activity needed to cure specific bacteremias can be made. Nevertheless, cogent arguments have been made for the use of the serum dilution test to guide antimicrobial therapy for infective endocarditis and infections in granulocytopenic patients [4-7, 21, 36, 37]. The standardized test used in the present study can be useful for clinical microbiology laboratories that are unable to perform MIC and MBC determinations or assays for antimicrobial agent levels. When the serum dilution test is used to guide therapy for systemic infections, we recommend that the test be performed with a physiologic diluent. We also recommend that physiologic medium be used for MIC and MBC determinations. However, when human serum is used in susceptibility testing, stiringent quality controls measures must be taken to ensure uniformity. References 1. Schlichter, J. G., MacLean, H. A method of determining the effective therapeutic level in the treatment of subacute bacterial endocarditis with penicillin: a preliminary report. Am. Heart J. 34: , Schlichter, J. G., MacLean, H., Milzer, A. Effective penicillin therapy in subacute bacterial endocarditis and other chronic infections. Am. J. Med. Sci. 217: , Mandell, G. L., Sande, M. A. Some newer aspects of infective endocarditis. Geriatrics 30:97-102, Prober, C. G., Yeager, A. S. se of the serum bactericidal titer to assess the adequacy of oral antibiotic therapy in the treatment of acute hematogenous osteomyelitis. J. Pediatr. 95: , Jawetz, E. Combined antibiotic therapy in serious infections caused by drug-resistant staphylococci ( ). Arch. Intern. Med. 103: , Klastersky, J., Daneau, D., Swings, G., Weerts, D. Antibacterial activity in serum and urine as a therapeutic guide in bacterial infections. J. Infect. Dis. 129: , Klastersky, J., Meunier-Carpentier, F., Provost, J. M., Staquet, M. Synergism between amikacin and cefazolin against Klebsiella: in vitro studies and effect on the bactericidal activity of serum. J. Infect. Dis. 134: , Stratton, C. W., Reller, L. B. Serum dilution test for bactericidal activity. I. Selection of a physiologic diluent. J. Infect. Dis. 136: , Reller, L. B., Stratton, C. W. Serum dilution test for bactericidal activity. II. Standardization and correlation with antimicrobial assays and susceptibility tests. J. Infect. Dis. 136: , Thornsberry, c., Kirven, L. A. Ampicillin resistance in Haemophilis influenzae as determined by a rapid test for beta-iactamase production. Antimicrob. Agents Chemother. 6: , Reimer, L. G., Stratton, C. W., Reller, L. B. Minimal in-

9 168 Stratton, Weinstein, and Reller hibitory and bactericidal concentrations of 44 antimicrobial agents against three standard control strains in broth with and without human serum. Antimicrob. Agents Chemother. 19: , National Committee for Clinical Laboratory Standards. Performance standards for antimicrobic disc susceptibility tests. 2nd ed. National Committee for Clinical Laboratory Standards, Villanova, Pa., 1979, p Miles, A. A., Misra, S. S. The estimation of the bactericidal power of the blood. J. Hyg. (Lond.) 38: , O'Brien, D., Ibbott, F. A., Rodgerson, D. O. Determination of whole blood chloramphenicol (colorimetric). In Laboratory manual of pediatric microbiochemical techniques. 4th ed. Harper and Row, New York, 1968, p MacLowry, J. D., Jaqua, M. J., Selepak, S. T. Detailed methodology and implementation of a semi-automated serial dilution microtechnique for antimicrobial susceptibility testing. Applied Microbiology 20:46-53, Ericsson, H. M., Sherris, J. C. Antibiotic sensitivity testing: report of an international collaborative study. Acta Pathol. Microbiol. Scand. [B) 217(Suppl.):1-90, Steers, E., Foltz, E. L., Graves, B. S. An inocula replicating apparatus for routine testing of bacterial susceptibility to antibiotics. Antibiot. Chemother. 9: , Pearson, R. D., Steigbigel, R. T., Davis, H. T., Chapman, S. W. Method for reliable determination of minimal lethal antibiotic concentrations. Antimicrob. Agents Chemother. 18: , Pien, F. D., Vosti, K. L. Variation in performance of the serum bactericidal test. Antimicrob. Agents Chemother. 6: , Pien, F. D., Williams, R. D., Vosti, K. L. Comparison of broth and human serum as the diluent in the serum bactericidal test. Antimicrob. Agents Chemother. 7: , Jordan, G. W., Kawachi, M. M. Analysis of serum bactericidal activity in endocarditis, osteomyelitis, and other bacterial infections. Medicine (Baltimore) 60: 49-61, Kunin, C. M. Blood level measurements and antimicrobial agents. Clin. Pharmacol. Ther. 16: , Weinstein, L., Dalton, A. C. Host determinants of response to antimicrobial agents [in three parts]. N. Engl. J. Med. 279: , , , Jackson, G. G., Riff, L. J. Pseudomonas bacteremia: pharmacologic and other bases for failure of treatment with gentamicin. J. Infect. Dis. 124: (Suppl):SI85-S191, Adam, D. Interaction of antibiotics with host factors. In W. Braun and J. S. nger [ed.]. "Non-specific" factors influencing host resistance, a re-examination. Kriger, New York, 1973, p Feingold, D. S., Goldman, J. N., Kuritz, H. M. Locus of the action of serum and the role of lysozyme in the serum bactericidal reaction. J. Bacteriol. 96: , Feingold, D. S., Goldman, J. N., Kuritz, H. M. Locus of the lethal event in the serum bactericidal reaction. J. Bacteriol. 96: , Schreiber, R. D., Morrison, D. c.. Podack, E. R., Muller Eberhard, H. J. Bactericidal activity of the alternative complement pathway generated from 11 isolated plasma proteins. J. Exp. Med. 149: , Traub, W. H., Sherris, J. C. Studies on the interaction between serum bactericidal activity and antibiotics in vitro. Chemotherapy 15:70-83, Traub, W. H., Kleber, I. Studies on the additive effect of polymyxin B and bactericidal activity of human serum against Serratia marcescens. Chemotherapy21: , Dutcher, B. S., Reynard, A. M., Beck, M. E., Cunningham, R. K. Potentiation of antibiotic bactericidal activity by normal human serum. Antimicrob. Agents Chemother. 13: , Nolan, C. M., Beaty, H. N. Staphylococcus aureus bacteremia-current clinical patterns. Am. J. Med. 60: , Duma, R. J., Weinberg, A. N., Medrek, T. F., Kunz, L. J. Streptococcal infections: a bacteriologic and clinical study of streptococcal bacteremia. Medicine (Baltimore) 48:87-127, DuPont, H. L., Spink, W. W. Infections due to gramnegative organisms: an analysis of 860 patients with bacteremia at the niversity of Minnesota Medical Center, Medicine (Baltimore) 48: , Anhalt, J. P., Sabath, L. D., Barry, A. L. Special tests: bactericidal activity, activity of antimicrobics in combination and detection of f3-lactamase production. In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and J. P. Truant [ed.]. Manual of clinical microbiology. 3rd ed. American Society for Microbiology, Washington, D.C., 1980, p Sande, M. A., Scheld, W. M. Combination antibiotic therapy of bacterial endocarditis. Ann. Intern. Med. 92: , Parker, R. H., Fossieck, B. E., Jr. Intravenous followed by oral antimicrobial therapy for staphylococcal endocarditis. Ann. Intern. Med. 93: , 1980.