0031-3998/86/ 2009-0909$02.00/0 PEDIATRIC RESEARCH Vol. 20, No.9, 1986 Copyright© 1986 International Pediatric Research Foundation, Inc. Printed in U.S.A.
Cellular Uptake and Cell-Associated Activity of Third Generation Cephalosporins
RICHARD F. JACOBS, JOSEPH W. THOMPSON, DEBORAH P. KIEL, AND DIANNE JOHNSON University ofArkan sas for Medical Sciences, and Arkansas Children 's Hospital, Little Rock, Arkansas 72202
ABSTRACT. The ability of the third generation cephalo ogens that have been demonstrated to be "cell-associated" in sporins to penetrate human polymorphonuclear leukocytes human disease and experimental infections may also be clinically (PMNs) and their antibacterial activity against cell-asso significant ( 1-4). If these pathogens are capable of intracellular ciated Staphylococcus aureus (SA) and Haemophilus injlu survival, they may evade killing by antibiotics that do not enter enzae, type b (Hib) were studied. Utilizing radioactive phagocytic cells (5-9). uptake experiments, the cellular to extracellular concen The newer {J-lactam antibiotics have been shown to have tration ratios were determined to be less than one for all marked activity against Hib and good (although variable) activity cephalosporins at 10 and 120 min: cefotaxime (0.08 ± against may strains of SA (10). We evaluated the ability of these 0.02, 0.34 ± 0.08), ceftizoxime (0.21 ± 0.11, 0.52 ± 0.18), antibiotics to penetrate PMNs and kill cell-associated SA and ceftriaxone (0.12 ± 0.04, 0.38 ± 0.23), and N-formimidoyl Hi b. thienamycin (0.18 ± 0.09, 0.33 ± 0.14). Third generation cephalosporins were similar to penicillin in their exclusion from PMNs. The killing of cell-associated SA and Hib METHODS were evaluated in a preopsonized cell-associated bacterial assay with radiolabelled SA/Hib (cfu/cpm) comparing ac PMN isolation. PMNs were obtained from whole blood of tivity of PMNs + antibiotics to the PMN cell control (no adult donors free of infection and antibiotics. Heparinized (20 antibiotics) at 0.5, 2, and 4 h. PMNs alone killed ::50.5 log U/ml) blood was added to dextran (Macrodex, 6% w/v, in saline, SA/Hib over 4 h. Clindamycin killed significantly more Pharmacia Laboratories, Piscataway, NJ) at a ratio of 5 ml blood: SA (p < 0.01) than all other antibiotics; nafcillin killed 1 ml dextran and was allowed to sediment in an upright position significantly fewer SA (p < 0.05) than all other antibiotics. until the erythrocytes settled to 50% of the total volume. The Although each third generation cephalosporin showed good harvested leukocyte-rich-plasma supernate was washed in HBSS activity against cell-associated Hib, chloramphenicol had without Ca++ and Mg++ (Grand Island Biological Co, Gibco; a significantly greater effect (p < 0.05). N-formimidoyl Grand Island, NY) by centrifugation at 300 x g for 7 min. The thienamycin demonstrated good activity only after the con cell button was incubated in 0.84% NH4CI at 37" C x 5 min to centration was increased in vitro to 8 Although lyse retained erythrocytes and washed in HBSS at 300 x g for 7 cellular penetration of antibiotics may be important in the min. The PMN fraction was isolated by centrifugation of the eradication of cell-associated pathogens, the overall cellu leukocyte button resuspended in HBSS at 500 x g for 30 min lar activity would appear to be multifactorial. (Pediatr Res over Ficoii-Hypaque, specific gravity 1.077 (Sigma Chemical Co., 20:909-912, 1986) St. Louis, MO). The PMNs were washed twice in HBSS at 300 x g for 7 min, resuspended in RPMI 1640 (Gibco) and counted Abbreviations in a hemacytometer. Viability as determined by trypan blue dye exclusion was consistently >97%. SA, Staphylococcus aureus Uptake ofantibiotics by PMNs. PMN penetration by cefotax Hib, Haemophilus injluenzae, type b ime, ceftizoxime, ceftriaxone, and N-formimidoyl thienamycin cfu, colony forming unit was assayed as previously described (5, 6, 11). Each reaction tube cpm, counts per minute 6 HBSS, Hanks' balanced salt solution contained I ml of 5 x 10 PMN in RPMI 1640 with 10% fetal calf serum (Gibco) and 100 of one ofthe following in RPMI GBSS, Geys balanced salt solution 3 PMN, polymorphon-uClear leukocytes 1640 yielding a final concentration as listed: 8 [ H] H20 (SpAc-100 New England Nuclear, Boston, MA), 44 MIC, minimum inhibitory concentration 35 Na2 S04 (SpAc-500 New England Nuclear), MBC, minimum bactericidal concentration 14 5 [ C] cefotaxime (SpAc-100 Hoechst, Frank C/E, cellular to extracellular concentration ratio fort, Germany), 5 [14C] ceftizoxime (SpAc-17.2 mCij SpAc, specific activity mmol, Smith, Kline, and French, Philadelphia, PA), 5 4 [I C] ceftriaxone (SpAc-100 HotTman-LaRoche, Nu tley, NJ), 5 N-formimidoyl thienamycin (SpAc-0.4 mCi/29.4 mg, Merck, Sharpe, and Dohme, Rahway, NJ), 5 Bacterial pathogens that are capable of survival and replication ml PH] clindamycin (SpAc-50 UpJohn Co, Kalama 14 intracellularly are a significant cause of human infections; path- zoo, MI), or 5 [ C] penicillin (SpAc-25 New England Nuclear). The reaction mixtures were incubated in a Received September 17, 1985; accepted May 7, 1986. 37" C shaking water bath at 200 rpm. At 10 and 120 min a 500- Correspondence and reprint requests to Dr. Richard F. Jacobs, Department of aliquot of each was removed, layered over I ml inert silicone Pediatrics, Arkansas Children's Hospital, 804 Wolfe Street, Little Rock, AR 72202. Supported in part by the Chancellor's Grant-University of Arkansas for oil (Versilube, General Electric Co, Schenectady, NY), and cen Medical Sciences and a grant from the UpJohn Company, Kalamazoo, MI. R.F.J. trifuged at 10,000 x g for 2 min in a microcentrifuge (Eppendorf, is an EL Trudeau Scholar of the American Lung Association. Brinkman Electronics, Westburg, NY). The radioactivity of the 909 910 JACOBS ET AL. PMN pellet and a 100-,ul aliquot of the supernate was measured The cfu/cpm ratios of cell-associated SA/Hib were compared in 10 ml Aquasol-2 (New England Nuclear) using a scintillation between reaction mixtures containing antibiotics and parallel spectrometer (Beckman Instruments, Fullerton, CA). The total mixtures containing PMNs without antibiotics (cell control). The and extracellular water content of the PMN pellet was deter results were calculated as a fraction survival (percent) relative to · mined by dividing the cpm in the PMN pellet by the cpm in the this antibiotic-free cell control at each time point (5, 6, 14). known volume of supernate for PH] H20 and Nal5So4, respec Statistics. Data are expressed as the mean ± SD. The signifi tively. The extracellular water content was consistently <10% of cance of differences between means was determined by the the total water content. The cellular water content of the PMN Student's t test ( 15). pellet was determined by subtracting the extracellular water content from the total water content of the PMN pellet. The C/ RESULTS E of the antibiotics was determined as previously described (5). MIC/MBC determination. The MIC/MBC values for the an Uptake of antibiotics by PMNs. The mean ± 1 SD for the tibiotics against an ATCC 25923 strain of SA (Difco Laborato cellular to extracellular concentration ratios with PMNs are ries, Detroit, Ml) and a clinical isolate of Hib were determined shown in Table I. Penicillin was excluded from PMNs with a C/ by the tube dilution method using a standard inoculum (12). E ratio of less than one (0.29 ± 0.05 at 10 min, 0.58 ± 0. I 6 at Effects of antibiotics on cell-associated SA and Hib. The anti 120 min). In contrast, clindamycin was concentrated within microbial activity of each antibiotic against cell-associated SA PMNs with C/E ratios of 2.95 ± 0.64 and 3.39 ± 1.04 at 10 and and Hib was assayed as previously described (5, 6, 11). To prepare 120 min, respectively. The {J-lactam antibiotics, cefotaxime, cef radioactive bacterial cultures, 10 ml of RPMI 1640 without tizoxime, ceftriaxone, and N-formimidoyl thienamycin were leucine (Gibco) plus 2.5 J.LCi [14C] leucine (SpAc-344 mCij similar to penicillin with C/E ratios always less than one. The mmol, New England Nuclear) was inoculated with 100 J.Ll of a clindamycin C/E ratio was significantly greater than the other fresh SA stock culture (tryptic soy broth) and placed in a shaking antibiotics at both time points (p < 0.00 I). Although ceftizoxime 37" C water bath at 200 rpm X 21 h. Radioactive Hib was grown seemed to have better penetration than other newer {J-lactams, from four, 1-day-old, chocolate agar colonies placed in 20 ml no significant differences were found. The C/E ratios were con Catlin's media without leucine (13) and 12 J.LCi [ 14C] leucine in sistent and no effect on PMNs was seen at the completion of the a 5% C02 incubator X 24 h. Both the SA and Hib cultures were assay due to: I) PMN clumping-phase contrast microscopy, 2) washed twice in cold (4• C), GBSS (Gibco) by centrifugation at PMN viability-trypan blue dye exclusion, or 3) a change in the 1900 x g for 15 min. The concentration of SA was adjusted to extracellular or total water content of the PMN pellet on intraas an OD of 0.15 at 490 nm to yield 2 x 107 cfu/ml. The Hib say analysis (p > 0.05). washed button was resuspended in 12.5 ml RPMI 1640 to yield MIC/MBC determination. The MIC/MBC values ofthe study an inoculum of 2 X 109 cfu/ml. bacteria used in the cell-associated killing assays are shown in Reaction tubes for each antibiotic studied and an antibiotic Table 2. A marked difference in the susceptibility of the strain free cell control (PMNs + bacteria without antibiotics) contained of SA against N-formimidoyl thienamycin was found when 1.4 ml bacteria (SA: 2 x 107 cfu/ml or Hib: 2 x 109 cfu/ml), I compared to the other {J-lactam antibiotics. This is in contrast ml of 18 x 106/ml PMN, and 0.6 ml serum. The reaction to an increased MIC/MBC of N-formimidoyl thienamycin for mixtures were incubated in a 37" C shaking water bath at 200 the strain of Hib ({J-lactamase negative). rpm x 30 min. Following this incubation of bacteria and PMNs, extracellular organisms were removed by the addition of I 0 U I I. Cellular/extracellular concentration ratios versus time mllysostaphin (Sigma) X 10 min to the reaction tubes contained Table SA and by differential centrifugation of the reaction tubes con inPMNs taining Hib (5). The PMN-bacteria reaction mixtures were re C/E* suspended in 3 ml RPMI I 640 + I 0% fetal calf serum with aliquots containing the described final concentrations of anti Drug (n = 6) !Omin 120min biotics or media+ serum alone (cell control). The final concen Penicillin 0.29 ± 0.05 0.58 ± 0.16 trations of antibiotics used in the reaction mixtures were equal Clindamycin 2.95 ± 0.64t 3.39 ± l.04t to or exceeded the bacterial MIC/MBC. The concentrations were: Cefotaxime 0.08 ± 0.02 0.34 ± 0.08 SA-4 J.Lg/ml, nafcillin; 2 J.Lg/ml, clindamycin; I J.Lg/ml, genta Ceftizoxime 0.21 ± 0.11 0.52 ± 0.18 micin; 2 J.Lg/ml, cefotaxime; 2 J.Lg/ml, ceftizoxime; 2 J.Lg/ml, Ceftriaxone 0.12 ± 0.04 0.38 ± 0.23 ceftriaxone; and I ,ug/ml, N-formimidoyl thienamycin; Hib-8 N-formimidoyl thienamycin 0.18 ± 0.09 0.33 ± 0.14 J.Lg/ml, ampicillin; 2 and 4 J.Lg/ml, chloramphenicol; 0.5 J.Lg/ml, * C/E results expressed as mean ± SD. cefotaxime; 0.5 J.Lg/ml, ceftizoxime; 0.5 J.Lg/ml, ceftriaxone; 2 t p < 0.00 I, compared to all other variables. and 8 J.Lg/ml, N-formimidoyl thienamycin. The reaction mixture was incubated in a 37" C shaking water bath at 200 rpm. At 30 min, 2 h, and 4 h, 1-ml aliquots were removed and centrifuged Table 2. Minimum inhibitory/bactericidal concentrations of at 300 x g for 7 min. A growth control (organisms without cells investigative isolates or antibiotics) was assayed in parallel to the above variables. SA ATCC During the phagocytic incubation, the growth control contained Hib I ml RPMI 1640, I ml bacterial inoculum, and 0.6 ml serum. 25923 The growth control was centrifuged at 1900 x g for 2 min and Drug* MIC MBC MIC MBC resuspended in 3 ml RPMI 1640 + 10% fetal calf serum for the remainder of the experiment. To induce cell lysis, I ml of 0.2% Ampicillin 0.1 0.3 Triton X-100 (Sigma) was added to all reaction tubes containing Nafcillin 2.0 4.0 PMNs, and serial dilutions were made in GBSS. Cell lysates Clindamycin 0.5 1.0 containing SA were spread on tryptic soy agar and incubated Gentamicin 0.5 1.0 overnight at 37" C. Cell lysates containing Hib were spread on Chloramphenicol 1.0 2.0 Cefotaxime 1.0 2.0 0.01 0.03 chocolate agar and incubated overnight in a 37• C, 5% C02 incubator. A 0.4-ml aliquot of the lysed cell suspensions and Ceftizoxime 1.0 2.0 0.01 0.03 growth control was counted in 10 ml Aquasol-2 on the scintil Ceftriaxone 1.0 2.0 0.01 0.03 lation spectrometer. The bacterial cfu and cpm were enumerated N-formimidoyl thienamycin 0.1 0.2 1.0 2.0 the following day. * Drug concentrations in !Lg/ml. CELLULAR ACTIVITY OF NEW CEPHALOSPORINS 911
o Ampicillin 18 /19 /mll Cell-associated killing of SA and Hib by antibiotics. After the 100 # 8 Chfor•mphenicolj2,g/mll initial 30-min incubation, cell control (PMN) mixtures were • Chloc•mphenlcoi 14 fiQ /mll treated with lysostaphin (SA) or differentially washed (Hib) and Cl Cefotaxime tO .S ,gl mll