Journal of Antimicrobial Chemotherapy (1982)9,141-148 Effects of antibiotics on the bactericidal activity of human serum Downloaded from https://academic.oup.com/jac/article/9/2/141/712569 by guest on 28 September 2021 Anna Fietta, Patrizia Mangiarotti and Giuliana Gialdroni Grassi Istituto Forlanini, University ofPavia, Italy The interaction between several antibiotics and either normal human serum or EGTA-chelated Mg-treated serum has been tested. Synergism has been observed with rifampicin, tetracycline and doxycycline, but not with minocycline, amino- glycosides (kanamycin, tobramycin, gentamicin, sisomicin and amikacin), chloramphenicol, fosfomycin and erythromycin. It has been demonstrated that Escherichia coli K12 strains bearing plasmids, conferring resistance to tetra- cycline, were killed in the presence of serum. Evidence has been presented that this synergistic action depends on com- plement and can be abolished by serum treatment with ethyleneglycol-tetraacetic acid. Introduction Interference of antimicrobial agents on several reactions involved in the host defence system has been demonstrated by many authors. It has been suggested that resistance to serum bactericidal activity can be an important factor in determining virulence of some Gram-negative bacteria (Durack & Beeson, 1977; Elgefors & Oiling, 1978; Howard & Glynn, 1971; McCabe et al., 1978; Medearis & Kenny, 1968; Oiling et al, 1973; Roantree & Rantz, 1960; Rowley, 1954), and serum bactericidal activity is an important mechanism of defence against bacterial invasion and spread (Agnella, 1978; McCabe et al, 1978; Roantree & Rantz, 1960; Johnston & Strand, 1977). Many Gram-negative Enterobacteriaceae causing bacteraemia are resistant to the bactericidal action of serum (Rowley & Wardlaw, 1958; Vosti & Randall, 1970). Conversion of serum-resistant Escherichia coli to serum-susceptible has been observed after treatment with diphenylamine (Feingold, 1969). Polymyxin B has been shown to be able to induce surface changes in some Gram-negative bacteria leading to serum susceptibility (Prull & Reynolds, 1972; Traub & Sherris, 1970) and subinhibitory concentrations of tetracycline, streptomycin and rifampicin have been shown to increase bacterial susceptibility to the killing activity by serum (Alexander, Gleen Cobbs & Curtiss, 1980; Dutcher et al, 1978). As a part of a study directed to determine the characteristics and the regulatory factors involved in the bactericidal activity of human serum and the possibilities of influencing this in some way, we have investigated the interaction between human 141 0305-7453/82/020141 +08 $01.00/0 © 1982 The British Society for Antimicrobial Chemotherapy 142 A. Fietta, P. Mangiarotti and G. G. Grassi serum and some antibiotics in the killing of rough strains of E. coli, under experimental conditions studying synergism among them.* Materials and methods Chemicals Penassay broth (Antibiotic Medium no. 3) and agar (Antibiotic Medium no. 1) were obtained from Difco; phosphate-buffered saline (PBS) (from Oxoid) and MgCl2 and ethyleneglycol tetraacetic acid (EGTA) (from C. Erba, Milan, Italy); tetracycline and Downloaded from https://academic.oup.com/jac/article/9/2/141/712569 by guest on 28 September 2021 doxycycline were a gift from Pfizer; chloramphenicol from Zambon Farmaceutici; rifampicin from Lepetit; sisomicin from Menarini; tobramycin from Lilly; erythromycin from Sigurta; fosfomycin from Italfarmaco; gentamicin from Essex; amikacin from Bristol; kanamycin was purchased commercially. Organisms The following strains were utilized: high level streptomycin-resistant (MIC > 1000 mg/1) mutant of E. coli K12 J6-2 pro,his,trp, lac; E. coli K12 J6-2 (R1066) and J6-2 (R27). R105 and R27 are R factors conferring transmissible resistance respectively to CTSSu and T. MICs of antibiotics for E. coli K12 were determined in Penassay broth with an inoculum size of 105 cells/ml. Serum The blood of ten healthy donors was allowed to clot for 1 h at room temperature. The sera were separated by centrifugation, pooled, divided into 02 ml samples and stored in liquid nitrogen. Bactericidal assay E. coli J6-2 was grown overnight in Penassay broth at 37°C with agitation and then diluted 1:40 in the same medium and grown to give 06 absorbance at 560 nm. The doubling time and the exponential nature of the growth were regularly checked spectrophotometrically. The bacteria were harvested by centrifugation, washed twice in PBS and resuspended at the concentration of lOVml in PBS. The bactericidal reactions were carried out in 1 ml volumes and incubated,at 37"C in a shaking water bath. Normal human serum at the final concentration of 1 /400 was used in combination with drug, since at this dilution it did not show any killing activity. Inactivation of serum complement was performed by heating at 56°C for 30 min (H56). In order to test the activity only through the activation of the alter- native pathway of complement EGTA and MgCl2 were added at final concentrations of 9 and 4 mM, respectively. In this case a serum dilution of 1:200 was used. The reaction was stopped after 60 min by putting the tubes on ice. Viable counts were obtained by plating suitable dilutions in PBS on Penassay agar plates. The results were expressed as percentage survival, that is as Ns/No x 100, where Ns and No were respectively the number of surviving cells at 60 min and that of cells at zero time. *A preliminary report on some of these findings has been presented by Fietta A., Mangiarotti P., Sacchi F. and G. Gialdroni Grassi Ilth Mediterranean Congress of Chemotherapy, 1980-Nice, October. Antibiotics and bactericidal activity of human serum 143 100 Downloaded from https://academic.oup.com/jac/article/9/2/141/712569 by guest on 28 September 2021 I/I00 1/2001/300 Serum dilution Figure 1. Efficiency of normal human serum (NHS) and EGTA-chelated-Mg2+ treated serum (CHS) bactericidal activity. *, Normal human serum (NHS); •, EGTA-chelated-Mg2+ treated serum (CHS) Samples were incubated for 60 min at 37'C. However, in order more clearly to present the phenomena observed, the results in Figure 1 were expressed as percentage killing, defined as 100—Ns/No x 100. Results Bactericidal activity of normal and EG TA-chelated-Mg2*-treated human serum Incubation of rough strains with human serum results in rapid killing of the cells, that involves either the classical or the alternative pathway of complement activation. In fact, the bactericidal activity is expressed to a different extent by normal human serum (NHS) and by EGTA-chelated-Mg2+ treated serum (CHS). NHS reaction is more efficient than CHS. If serum is sufficiently diluted (1/400 for NHS; 1 /200 for CHS) there is no observable killing (survival = 96 ± 6%) (Figure 1). Table I. Effect of some antibiotics on bactericidal activity of normal human serum (NHS)* Antibiotic alone* Antibioticf + NHSf (1/400) Antibiotic 05 10 2-5 50 05 10 2-5 50 (mg/1) (mg/1) Kanamycin 103 ±5 100±10 95±8 50 100±12 90±6 86±9 56 Tobramycin 95±8 90±ll 77±9 — 75± 7 70+10 68±5 — Gentamicin 102±6 35±4 — — 98±5 39±3 — — Sisomicin 102±12 85±7 88±9 71 ±7 Amikacin 79±9 63±3 36±9 66±10 49±6 30+10 Chloramphenicol 110±10 110±ll 100±9 91 rt 12 106±5 103 ±2 102 + 6 94 + 6 Fosfomycin 100±3 89±8 81 ±8 3O±5 102 + 8 87±8 76+9 29±8 Erythromycin 99±5 96±4 92±6 92±2 98±6 84±6 83 + 5 80+3 •Results are expressed as % of-survival and they represent the mean values of at least three determi- nation. The standard deviations are also reported. tThe concentrations (mg/1) of antibiotic adopted for this test range from 1/16 to 1/2 of MIC. f NHS, Normal human serum diluted 1 /400 (survival % = 96 ± 6). 144 A. Fietta, P. Mangiarotti and G. G. Grassi 100 Downloaded from https://academic.oup.com/jac/article/9/2/141/712569 by guest on 28 September 2021 mg/l mg/l Figure 2. Effects of tetracycline derivatives and rifampicin on NHS bactericidal activity, (a) Tetracycline alone (*); tetracycline plus NHS (•); (b) doxycycline alone (*);doxycycline plus NHS (•); (c) minocycline alone (*); minocycline plus NHS (•); (d) rifampicin alone (*); rifampicin plus NHS (•). Results represent the means of at least four experiments; vertical lines represent the standard deviations from means. Bactericidal activity of human serum plus antibiotics No significant killing was observed when cells were incubated with antibiotics at concentrations ranging from 1/16 to 1/2 of the MICs. MICs of antibiotics for E. coli K12, were the following: aminoglycosides (4-8 mg/l), rifampicin (5 mg/l), tetra- cyclines (1-1-5 mg/l), chloramphenicol (2*5 mg/l), fosfomycin (8 mg/l), erythro- mycin (32 mg/l). When aminoglycosides (tobramycin, sisomicin, amikacin, gentamicin and kanamycin), chloramphenicol, fosfomycin and erythromycin were added to diluted NHS no significant variation of the bactericidal activity was observed after 60 min of incubation (Table I). The same results were obtained with CHS (data not shown). A marked decrease in percentage survival of the cells was shown with rifampicin as well as with tetracycline and doxycycline+ NHS. Minocycline however, showed a lesser effect (Figure 2). At all tested doses the per cent survival with tetracycline, Antibiotics and bactericidal activity of human serum 145 Table II. Bactericidal activity of serum plus tetracycline against E. coli J6-2 (R1066) and J6-2 (R27) Survival (%)t Reagents* J6-2(R1066) J6-2R27 NHS 97 98 H56 100 100 Tetracycline 87 90 Tetracycline + H56 88 99 Downloaded from https://academic.oup.com/jac/article/9/2/141/712569 by guest on 28 September 2021 Tetracycline + NHS 38 28 *NHS and H56 are diluted 1:400; tetracycline is at 50 mg/1, final concentration. tResults are the means of two experiments. IOO 90 Time (min) Figure 3. Time course of bactericidal effect of NHS plus tetracycline or rifampicin (a) • NHS (1/400); • tetracycline alone (25 mg/1; • tetracycline (25 mg/1) plus NHS (1/400) Vertical lines represent the standard deviation from means, (b) • NHS; D rifampicin (1 mg/1); • rifampicin (1 mg/1) plus NHS.