Enhanced Phagocytosis of Encapsulated Escherichia coli Strains

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May 16, 1989 - The influence of five antibiotics (netilmicin, ceftriaxone, cefepime, fleroxacin, and ciprofloxacin) on capsular polysaccharide distribution and on ...
Vol. 34, No. 2

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1990, p. 332-336 0066-4804/90/020332-05$02.00/0 Copyright ©) 1990, American Society for Microbiology

Enhanced Phagocytosis of Encapsulated Escherichia coli Strains after Exposure to Sub-MICs of Antibiotics Is Correlated to Changes of the Bacterial Cell Surface GIAMMARCO RAPONI,1t* NATHAN KELLER,"2 BERRY P. OVERBEEK,1 MAJA ROZENBERG-ARSKA,"' KOK P. M. VAN KESSEL,1 AND JAN VERHOEF"2 Department of Clinical Microbiology and Laboratory for Infectious Diseases, University Hospital of Utrecht, De Uithof, 3584 CX,1 and U-GENE Research, 3584 CS,2 Utrecht, The Netherlands Received 16 May 1989/Accepted 2 November 1989

The influence of five antibiotics (netilmicin, ceftriaxone, cefepime, fleroxacin, and ciprofloxacin) on capsular polysaccharide distribution and on opsonophagocytosis by human polymorphonuclear leukocytes of unencapsulated and encapsulated Escherichia coli strains was studied. Unencapsulated E. coli strains were readily opsonized in serum and easily ingested by polymorphonuclear leukocytes, and antibiotics did not further enhance the phagocytosis rates. In contrast, encapsulated bacteria were poorly opsonized in human serum, and phagocytosis was enhanced after overnight exposure to O.5x the MICs of the antibiotics, with the exception of cefepime. Incubation of unencapsulated as well as encapsulated bacteria in complement-inactivated serum markedly reduced the bacterial uptake by polymorphonuclear leukocytes regardless of the presence of antibiotics. Slide agglutination assays, performed either with a monoclonal antibody for capsular polysaccharide or with an antiserum raised against the stable unencapsulated mutant E. coli 07:K-, showed reduction but not lack of the capsular polysaccharide of encapsulated E. coli 07:K1, and better exposure of subcapsular epitopes, after incubation with 0.5 x the MICs of antibiotics. Flow cytometric analysis of encapsulated E. coli exposed to netilmicin, ciprofloxacin, and fleroxacin revealed that the reduction in capsular material was homogeneous among the bacterial population. Treatment with cefepime and ceftriaxone induced two populations of bacteria that differed in the amount of K antigen present. These results indicate that sub-MICs of netilmicin, ceftriaxone, fleroxacin, and ciprofloxacin influenced complement-mediated opsonization, probably due to changes in the capsular polysaccharide structure. one, cefepime, fleroxacin, and ciprofloxacin on opsonophagocytosis of encapsulated and unencapsulated strains of E. coli. Data are provided that show that enhanced opsonophagocytosis induced by certain antibiotics is due to diminished production of capsular antigen by bacteria.

The outer structures of bacteria play an important role in the interaction between bacteria and host defenses during infections. Encapsulated strains of Escherichia coli have been shown to be resistant to the lytic and opsonic activities of serum (6, 7) and to phagocytosis by human polymorphonuclear leukocytes (PMN) (8). The invasive capacity of these bacteria is correlated to their ability to resist killing by normal human serum (9, 14). Especially Kl capsule appears to be responsible for serum resistance of strains that are otherwise exquisitely sensitive to the action of serum (2) and that are easily ingested and degraded by PMN (21). However, during infection bacteria are exposed not only to the defense mechanisms of the host but also to the action of antimicrobial agents. It is well known that antibiotics exert part of their action in the body fluids, at concentrations below the MICs. Sub-MICs of certain antibiotics have been shown to modify the morphological and ultrastructural characteristics of bacteria, influencing the interactions with host defense mechanisms, i.e., opsonophagocytosis (5, 15). In previous studies we reported that preexposure of encapsulated E. coli strains to sub-MICs of an aminoglycoside (netilmicin) and of a cephalosporin (ceftriaxone) enhanced the phagocytosis rate by PMN compared with that of unexposed bacteria (18). We speculated that this effect was due to alterations of the K antigens by the exposure to antibiotics. In the present study we extended our previous work and investigated the effect of sub-MICs of netilmicin, ceftriax-

MATERIALS AND METHODS Bacteria. E. coli 01:K1, E. coli 07:K1, and the stable unencapsulated mutant strain E. coli 07:K-, kindly provided by P. A. M. Guinee (National Institute of Public Health and Environmental Hygiene, Bilthoven, The Netherlands) and by A. M. J. J. Verwey-van Vught (Free University, Amsterdam, The Netherlands), were used. The bacteria were kept in 15% glycerol at -70°C until use. Strains were grown overnight in Mueller-Hinton broth (MHB; Difco Laboratories, Detroit, Mich.) at 37°C in the presence or absence of 0.5x the MICs of antibiotics. For the phagocytosis assay, bacteria were radioactively labeled by the addition of 2 mCi of [methyl-3H]thymidine (specific activity, 5.0 Ci/mmol; Radiochemical Centre, Amersham, United Kingdom) per ml to MHB. After incubation for 18 h, the bacteria were washed three times in phosphate-buffered saline and adjusted spectrophotometrically to yield 2.5 x 108 CFU/ml. Antibiotics and MIC determination. Netilmicin (Essex Labo, Heist op den Berg, Belgium), ceftriaxone and fleroxacin (Hoffmann-La Roche, Mijdrecht, The Netherlands), cefepime (Bristol Myers, Brussels, Belgium), and ciprofloxacin (Bayer, Mijdrecht, The Netherlands) were used at 0.5 x the MIC. MICs were determined by a broth dilution method by using serial twofold dilutions of the antibiotics in MHB and adding an inoculum of 105 CFU/ml adjusted spectropho-

Corresponding author. t Present address: Raponi-GIPAC Laboratories, Via Oderisi da

*

Gubbio 10, 00146 Rome, Italy.

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tometrically. The results were read after incubation for 18 h at 370C.

Sera and leukocytes. Human serum was pooled from healthy donors (HPS) and stored in small volumes at -70°C until use. For the opsonophagocytosis assay, HPS was

diluted in Hanks balanced salt solution (pH 7.4) and used unheated and heated for 30 min at 56°C to inactivate complement factors. Heparinized venous blood samples (10 U of heparin per ml of blood) were obtained from healthy volunteers. PMN were isolated by dextran (molecular weight, 70,000; Pharmacia Fine Chemicals, Uppsala, Sweden) sedimentation and Ficoll-Paque (Pharmacia) centrifugation as previously described (22). Cells were suspended in Hanks balanced salt solution containing 0.1% gelatin to a final concentration of 5 x 106 cells per ml. Viability was assayed by trypan blue exclusion and was greater than 95%. Opsonophagocytosis assay. In polypropylene vials (Biovials; Beckmann Instruments, Mervue, Galway, Ireland), 0.2 ml of radiolabeled bacteria (2.5 x 10' CFU/ml) was added to 0.8 ml of diluted HPS for different incubation times, at 37°C, in a shaking water bath (150 rpm). Opsonization was stopped by the addition of 2.5 ml of ice-cold phosphatebuffered saline, and serum was removed by centrifugation for 15 min at 1,600 x g at 4°C. Subsequently, bacteria were suspended in 1 ml of Hanks balanced salt solution to a final concentration of 5 x 107 CFU/ml. The uptake of opsonized bacteria by PMN was then measured as described by Verhoef et al. (24). In brief, equal volumes of opsonized E. coli (5 x 107 CFU/ml) and PMN (5 x 106 cells per ml) were incubated in a shaking water bath (150 rpm) at 37°C. After 6 min, phagocytosis was stopped by the addition of ice-cold phosphate-buffered saline. Radioactivity not associated with phagocytes was removed from PMN by three cycles of differential centrifugation (160 x g for 5 min at 4°C). The pellets were suspended in 2.5 ml of scintillation liquid (Aqua Luma Plus; Lumac/3M, Schaesberg, The Netherlands). Radioactivity was determined by a liquid scintillation counter (Philips, Eindhoven, The Netherlands). The percentage of bacteria taken up by PMN after 6 min was calculated from the uptake of radioactivity by the phagocytes, and the total added radioactivity was determined in a separate vial. Each experiment was done in triplicate. Results, expressed as the mean value and the standard deviation of at least three experiments, were statistically analyzed according to the Student t test. P values of