Effect of Subinhibitory Concentrations of Amikacin and Ciprofloxacin on the K1 Antigen Expression and Phagocytosis of Escherichia coli Strains

original papers Adv Clin Exp Med 2010, 19, 4, 429 436 ISSN 1230-025X Copyright by Wroclaw Medical University Dorota Wojnicz, Dorota Tichaczek-Goska, Agnieszka Cisowska Effect of Subinhibitory Concentrations of Amikacin and Ciprofloxacin on the K1 Antigen Expression and Phagocytosis of Escherichia coli Strains Wpływ stężeń subinhibicyjnych amikacyny i ciprofloksacyny na ekspresję antygenu K1 oraz fagocytozę szczepów Escherichia coli Department of Biology and Medical Parasitology, Wroclaw Medical University Abstract Background. Escherichia coli strains with capsular Kl antigen are the major cause of neonatal meningitis and are also responsible for a large number of cases of bacteraemia and urinary tract infections in children and adults. The polysaccharide K1 antigen of E. coli is an important virulence factor that increases resistance to phagocytosis. Although the subinhibitory concentrations (sub-mics) of antibiotics do not kill microorganisms, they are able to modulate their characteristics, e.g., the morphology of cells, the bacterial surface components. As a result a reduction of bacterial pathogenicity is observed. This may also be associated with changes in the susceptibility of pathogens to the host immune response. Objectives. The aim of this study was to evaluate the influence of sub-mics of amikacin and ciprofloxacin on the presence of cell-surface K1 antigen of uropathogenic E. coli strains and their phagocytosis by neutrophils. Material and Methods. Fifteen E. coli strains expressing K1 antigen were selected from among the one hundred and twenty samples isolated from the urine of children with urinary tract infections. The bacteriophage K1A was used to detect the presence of the capsular antigen K1. The MICs of amikacin and ciprofloxacin for each strain incubated in Mueller-Hinton broth were obtained by microdilution method. The phagocytic index was determined by microscopic examination of slides prepared with May-Grünwald stain. Results. Exposure of E. coli K1 strains to 1/2, 1/4 and 1/8 of MICs of amikacin and ciprofloxacin reduced the number of clones sensitive to K1A phage, however the effect of amikacin was minute. Moreover, changes in the susceptibility to phagocytosis of E. coli strains incubated in the presence of antibiotics were observed. Conclusions. The treatment of encapsulated bacteria with sub-mics of amikacin or ciprofloxacin inhibits the K1 capsule expression and enhances phagocytosis by human neutrophils (Adv Clin Exp Med 2010, 19, 4, 429 436). Key words: Escherichia coli, K1 antigen, phagocytosis, subinhibitory concentrations, antibiotics. Streszczenie Wprowadzenie. Szczepy Escherichia coli z otoczkowym antygenem K1 są częstą przyczyną zapalenia opon mózgowo-rdzeniowych u noworodków, przypadków bakteriemii oraz zakażeń układu moczowego u dzieci i dorosłych. Polisacharydowy antygen K1 pałeczek E. coli jest ważnym czynnikiem wirulencji, który zwiększa oporność na fagocytozę. Mimo, że stężenia subinhibicyjne antybiotyków (sub-mics) nie zabijają bakterii, mogą zmieniać ich cechy fenotypowe, np. morfologię komórek, czy struktury powierzchniowe. W wyniku tego obniża się patogenność mikroorganizmów oraz zmienia się ich podatność na odpowiedź immunologiczną gospodarza. Cel pracy. Określenie wpływu sub-mics amikacyny i ciprofloksacyny na zdolność syntezy powierzchniowego antygenu K1 przez uropatogenne pałeczki E. coli oraz ich fagocytozę przez neutrofile. Materiał i metody. W badaniach wykorzystano piętnaście szczepów E. coli K1 spośród stu dwudziestu izolatów otrzymanych z moczu dzieci z zakażeniem dróg moczowych. Obecność antygenu K1 na powierzchni komórek bakterii oznaczono za pomocą faga K1A. Wartości MIC amikacyny i ciprofloksacyny wobec badanych pałeczek oznaczono metodą mikrorozcieńczeń w podłożu Mueller-Hinton. Na podstawie mikroskopowej oceny rozmazów krwi barwionych odczynnikiem May-Grünwalda określono indeks fagocytarny. Wyniki. Liczba klonów szczepów E. coli K1 wrażliwych na faga K1A poddanych działaniu 1/2, 1/4 oraz 1/8 MIC amikacyny lub ciprofloksacyny zmniejszyła się, w przypadku amikacyny obserwowane zmiany nie były jednak

430 D. Wojnicz, D. Tichaczek-Goska, A. Cisowska znaczące. Ponadto zaobserwowano zwiększoną podatność na fagocytozę bakterii inkubowanych w obecności antybiotyków. Wnioski. Działanie sub-mics amikacyny i ciprofloksacyny hamuje zdolność syntezy antygenu K1 przez pałeczki E. coli oraz zwiększa ich fagocytozę (Adv Clin Exp Med 2010, 19, 4, 429 436). Słowa kluczowe: Escherichia coli, antygen K1, fagocytoza, stężenia subinhibicyjne, antybiotyki. Escherichia coli strains with capsular Kl antigen are the major cause of neonatal meningitis and are also responsible for a large number of cases of bacteraemia and urinary tract infections in children and adults [1]. The cell surface antigen K1, such as the capsule of group B meningococci, is a linear homopolymer of α-2-8-linked N-acetylneuraminic acid (NeuNAc) and is poorly immunogenic, because NeuNAc residues are also found in gangliosides and many mammalian serum and cell membrane glycoproteins [2]. K antigens of E. coli have been described as virulence factors because of their higher incidence in strains causing invasive infections. The greater virulence of bacterial cells possessing polysaccharide capsules may be explained by interference of the K antigen with polymorphonuclear leukocytes responsible for phagocytosis. The capsule may provide protection to the microorganism by masking its underlying opsonized surface and serving as a physical barrier to the phagocytic cells [3]. It is well known that subinhibitory concentrations (sub-mics) of antibiotics are able to modulate numerous bacterial characteristics, e.g., the morphology of cells, the bacterial surface components such as outer membrane proteins and lipopolysaccharides, and other virulence factors [4 6]. The aim of this investigation was to evaluate the influence of sub-mics of amikacin and ciprofloxacin on the presence of cell-surface K1 antigen of uropathogenic E. coli strains and their phagocytosis by neutrophils. In addition to these studies, the authors determined the relationship between the percentage of clones of E. coli possessing antigen K1 and the value of phagocytic indexes. Material and Methods Bacterial Strains E. coli isolates (n = 120) were isolated from the urine of children with different clinical variants of urinary tract infection, hospitalised in Korczak Paediatric Centre of Lower Silesia in Wroclaw. Antibiotics Amikacin (Biodacyna, Warsaw, Poland) and ciprofloxacin (Proxacin, Warsaw, Poland) were used. Antimicrobial Susceptibility Testing MIC assay of two antimicrobial agents for E. coli K1 strains were performed in Mueller-Hinton broth according to CLSI guidelines for broth microdilution susceptibility testing [7]. Detection of Capsular Antigen K1 The K1-specific bacteriophage (K1A) was used for detection of this antigen according to the procedure described by Devine et al. [8]. The E. coli strains were grown overnight in the absence or presence of sub-mics of amikacin or ciprofloxacin (1/2, 1/4, 1/8 of MIC). The bacterial cells were then inoculated into 4 ml of nutrient broth and incubated at 37 C for 4 h. Next a single streak of each culture was made on nutrient agar and allowed to dry. Once the streak was dry, 10 µl of bacteriophage suspension was placed in the centre. After incubation at 37 C for 18 h, the presence of K1 was indicated by a reduction in the density of bacterial growth where the bacteriophage suspension had been placed. Phage K1A and the standard strain E. coli B2095 (O2:K1) used for proliferation of the phage were from the collection of Dr. G. Schmidt (Forschungsinstitut Borstel, Institut für Experimentelle Biologie und Medizin, Germany). Phagocytic Activity of Neutrophil Granulocytes and Phagocytic Index The neutrophil phagocytic activity was observed in the in vitro experiment using whole blood taken from 4 healthy adult volunteers. The E. coli strains were grown overnight in the absence or presence of sub-mics of amikacin or ciprofloxacin (1/2, 1/4, 1/8 of MIC). Bacteria were harvested, washed and standardized spectrophotometrically to obtain suspensions containing 5 10 8 cfu/ml. Then 200 μl of blood was incubated for 1 h at 37 C with 200 μl of heat-killed E. coli cell suspension. Immediately after incubation, two May-Grünwald stained smears of blood were prepared from each sample according to the conventional methodology. The

Effect of Antibiotics on K1 Antigen and Phagocytosis 431 phagocytic index was calculated as the average number of bacterial cells ingested by one phagocytic cell out of fifty randomly selected neutrophils examined. Experiments were performed in duplicate on three separated occasions. Statistical Analysis All experiments were performed independently in duplicate on three separate occasions. Values were expressed as mean ± SD. The effects of antibiotics at various sub-mics were quantitatively evaluated by one-way analysis of variance (ANOVA). The expression of K1 antigen and the values of phagocytic index were compared before and after exposure of E. coli strains to antibiotics using Student s pair t-test. P values 0.05 were considered to be statistically significant. Results Table 1. MIC values of amikacin and ciprofloxacin against E. coli strains Tabela 1. Wartości MIC amikacyny i ciprofloksacyny dla badanych szczepów E. coli E. coli strains (Szczepy E. coli) MIC (µg/ml) Amikacin (Amikacyna) 1 2.0 0.031 2 0.5 0.007 3 0.5 0.007 4 0.5 0.015 5 4.0 32.0 6 1.0 0.007 7 1.0 0.031 8 0.5 0.007 9 1.0 0.031 10 0.5 0.007 11 4.0 8.0 12 1.0 0.031 13 0.5 0.007 14 0.5 0.015 15 0.5 0.015 Ciprofloxacin (Ciprofloksacyna) In the preliminary studies, the authors determined the ability of uropathogenic E. coli strains to produce K1 antigen. Fifteen of the 120 E. coli isolates synthesized this polysaccharide capsule. Then we evaluated the MICs of antibiotics suppressing the growth of E. coli strains expressing K1 antigen. For individual strains the MICs of amikacin and ciprofloxacin varied in the range 0.5 µg/ml 4.0 µg/ml and 0.007 µg/ml 32 µg/ml, respectively (Table 1). The effect of antibiotics on the loss or changes of the capsular K1 antigen by E. coli strains was estimated by examining the sensitivity to K1A phage of fifteen E. coli K1 strains exposed to sub-mics of amikacin or ciprofloxacin. The same set of strains not exposed to antibiotics was used as a control. The authors tested one hundred clones of each E. coli K1 strain, either antibiotic-treated bacteria or not (Table 2). In controls (without antibiotics), all tested E. coli K1 strains revealed the occurrence of 97 98% clones with K1 antigen. The exposure of E. coli K1 strains to sub-mics of ciprofloxacin decreased the percentage of clones with K1 antigen. The number of clones of E. coli K1 strains sensitive to K1A phage exposed to 1/2, 1/4 and 1/8 of MICs of ciprofloxacin was suppressed to 73 76%, 77 81% and 80 82%, respectively. For all studied E. coli strains a statistically significant difference was seen between the controls and bacteria treated with the sub-mics of ciprofloxacin (p < 0.05). The authors observed also that after incubation of E. coli K1 strains in 1/2, 1/4 and 1/8 of MICs of amikacin, the percentage of clones with K1 antigen was slightly lower (90 93%) compared with the control sample. Expression of K1 capsule was not significantly different between rods treated with sub-mics of amikacin in comparison with the untreated bacteria, for all studied strains (p > 0.05). In the next stage the effect of sub-mics of both antibiotics in the susceptibility of E. coli K1 strains to phagocytosis by neutrophils was determined. In preparations of the blood smear bacterial cells ingested by fifty neutrophils were counted and then the phagocytic index for each tested strain was calculated (Table 3 and 4). These values for the control samples (incubation without antibiotics) were 10.2 ± 1.6 12.2 ± 1.8 (Fig. 1). The incubation of rods in the presence of ciprofloxacin increased their susceptibility to phagocytosis, and the average number of bacterial cells, which were absorbed by phagocytes was 13.4 ± 1.4 19.2 ± 1.5. The highest values of the phagocytic index, from 17.2 ± 1.5 to 19.2 ± 1.5, were observed for all studied E. coli strains incubated at ciprofloxacin concentration equal to 1/2 of MIC (Fig. 1) and this result was found to be statistically significant (p < 0.05). Bacteria exposed to 1/4 and 1/8 of MIC of ciprofloxacin had a lower sensitivity to phagocytosis. The phagocytic indexes ranged from 14.2 ± 1.7 to 15.8 ± 1.5 and from 13.4 ± 1.4 to 14.8 ± 1.4, respec-

432 D. Wojnicz, D. Tichaczek-Goska, A. Cisowska Table 2. The sensitivity of E. coli K1 strains, unexposed and exposed to sub-mics of amikacin and ciprofloxacin, to phage K1A a Tabela 2. Wrażliwość szczepów E. coli K1 niepoddanych i poddanych działaniu stężeń subinhibicyjnych amikacyny i ciprofloksacyny na faga K1A a E. coli strains (Szczepy E. coli) Clones sensitive to phage K1A [%] (Klony wrażliwe na faga K1A [%]) without antibiotic sub-mics of amikacin sub-mics of ciprofloxacin 1/2 1/4 1/8 1/2 1/4 1/8 1 97 91 93 93 76 80 81 2 97 91 93 93 73 80 82 3 98 91 93 93 75 81 82 4 98 90 91 93 73 80 82 5 98 91 92 92 75 78 81 6 98 91 92 92 75 78 81 7 98 90 92 93 76 78 82 8 97 91 92 92 75 80 82 9 98 91 93 93 75 79 80 10 97 90 92 92 73 80 82 11 98 92 93 93 76 78 82 12 98 91 93 93 74 77 80 13 98 91 93 93 75 78 80 14 98 90 92 92 74 80 82 15 98 91 92 92 76 79 82 a Number of clones tested: 100 for each strain. a Liczba testowanych klonów: 100 dla każdego szczepu. tively. The phagocytic indexes were significantly different only between bacteria treated with 1/4 of MIC of ciprofloxacin and the control samples, for all studied E. coli strains (p < 0.05). Moreover, changes in susceptibility to phagocytosis of E. coli strains incubated in the presence of amikacin have been observed. The exposure of rods to 1/2 of MIC of amikacin caused enhanced ingestion of bacteria by neutrophils in comparison with control samples, this effect was statistically significant for all studied E. coli strains (p < 0.05). The values of phagocytic index ranged from 14.2 ± 1.8 to 16.2 ± 1.5. The lower phagocytic indexes occurred after treatment of bacteria with 1/4 and 1/8 of MIC of amikacin. The average number of bacterial cells ingested by neutrophils were 13.2 ± 1.4 14.9 ± 1.7 and 12.8 ± 1.8 13.8±1.6, respectively. These changes were not statistically significant for the most studied E. coli strains (p > 0.05). Own results show a relationship between the average percentage of clones possessing K1 antigen and number of ingested bacteria by neutrophils observed in the case of E. coli strains unexposed as well as exposed to sub-mic of antibiotics (Fig. 2). Ciprofloxacin appeared to be more effective than amikacin in inducing changes in both respects, which depended on the antimicrobial agents concentrations. The phagocytic index values were higher for those strains which possessed less K1A phage-sensitive clones, however the observed differences were not significant for all studied strains (p > 0.05). Discussion The presence of capsular polysaccharide antigens is an important virulence factor of gram-negative and gram-positive bacteria [9, 10]. E. coli is no exception to this phenomenon. The presence of the K1 serotype capsular antigen results in reduced phagocytosis, decreased susceptibility to bactericidal action of serum and has been associated with not only neonatal meningitis but also bacteraemia

Effect of Antibiotics on K1 Antigen and Phagocytosis 433 Table 3. Effect of sub-mics of amikacin on phagocytosis of E. coli rods by neutrophils. Phagocytotic indexes marked with asterix present statistically significant change with respect to the control value (without antibiotic), p < 0.05 Tabela 3. Wpływ stężeń subinhibicyjnych amikacyny na fagocytozę pałeczek E. coli przez neutrofile. Wartości indeksów fagocytarnych oznaczone gwiazdką są statystycznie istotne w odniesieniu do grupy kontrolnej (bez antybiotyku), p < 0,05 E. coli strains (Szczepy E. coli) Phagocytic index (± SD) (Indeks fagocytarny) without antibiotic sub-mics of amikacin 1/2 1/4 1/8 1 10.8 ± 1.7 16.0 ± 1.5* 13.8 ± 1.4 13.5 ± 1.7 2 10.2 ± 1.6 15.9 ± 1.4* 13.8 ± 1.4 13.2 ± 1.4 3 10.7 ± 1.5 15.8 ± 1.5* 13.4 ± 1.5 12.8 ± 1.4 4 11.0 ± 1.8 16.2 ± 1.5* 14.2 ± 1.7* 13.2 ± 1.3 5 10.9 ± 1.8 15.6 ± 1.7* 13.4 ± 1.6 13.4 ± 1.6 6 12.0 ± 1.7 15.9 ± 1.8* 13.8 ± 1.8 13.2 ± 1.6 7 12.2 ± 1.8 16.0 ± 1.5* 14.9 ± 1.7* 13.4 ± 1.7 8 11.7 ± 1.7 14.2 ± 1.5* 13.8 ± 1.8 13.6 ± 1.8 9 11.5 ± 1.8 14.4 ± 1.4* 13.3 ± 1.8 12.8 ± 1.8 10 11.6 ± 1.6 14.8 ± 1.5* 13.2 ± 1.4 13.0 ± 1.5 11 11.1 ± 1.8 15.1 ± 1.7* 14.0 ± 1.5* 13.6 ± 1.6 12 11.5 ± 1.8 14.8 ± 1.8* 13.4 ± 1.5 12.8 ± 1.7 13 11.1 ± 1.8 15.8 ± 1.7* 13.7 ± 1.3 13.4 ± 1.8 14 11.4 ± 1.8 15.3 ± 1.7 * 13.9 ± 1.3 13.8 ± 1.6 15 11.6 ± 1.7 14.8 ± 1.7 * 13.9 ± 1.7 13.8 ± 1.6 in adults or urinary tract infections in children or adults [11]. The overall frequency of E. coli Kl observed in this study (12.5%) is similar to the results of other investigations [12, 13]. Fünfstück et al. [14] have shown that 14% E. coli strains isolated from the urine of patients with non-obstructive chronic pyelonephritis possessed K1 antigen. Slightly higher percentage of E. coli rods with K1 polysaccharide capsule (17%) was found by Devine et al. [8] and Evans et al. [15]. Czirok et al. [16] reported only 7.3% of urinary E. coli isolates carried the K1 antigen. It has frequently been assumed that antibiotics used at sub-mics might have an influence on the composition of the cell surface of gram-negative bacteria [4, 6, 17]. In this study, the authors investigated the influence of sub-mics of amikacin and ciprofloxacin on the presence of cell-surface K1 antigen of uropathogenic E. coli strains and their phagocytosis by neutrophils. The authors found that these antibiotics reduced amount of E. coli clones possessing polysaccharide capsule. Own findings are consistent with those reported by Raponi et al. [18], who described a reduction in capsular material after exposure of E. coli rods to sub-mics of netilmicin, ciprofloxacin, and fleroxacin. Suerbaum et al. [19] found a dose-dependent reduction of Kl production under the influence of imipenem, cephaloridine, and ciprofloxacin. Also Taylor et al. [20] and Kadurugamuwa et al. [21] observed an influence of antibiotics on the production of capsular polysaccharides in E. coli and Klebsiella pneumoniae strains. Taylor et al. [20] found that treatment of E. coli cells with mecillinam caused a reduction of the extractable amount of Kl. Similarly Kadurugamuwa et al. [21] reported that various cephalosporins decreased the formation of capsule by iron-depleted K. pneumoniae. The effects of sub-mics of penicillin or clindamycin were evaluated in Streptococcus pneumoniae isolates [22]. Clindamycin appears to be superior to penicillin in reducing the expression of the capsule by S. pneumoniae. After incubation in one-half of the MIC of clindamycin, 17.5% of isolates retained a capsule, compared to 87.5% after incubation with one-half of the MIC of penicillin. Brook et al. [23] showed that the exposure of S. pneumoniae to

434 D. Wojnicz, D. Tichaczek-Goska, A. Cisowska Table 4. Effect of sub-mics of ciprofloxacin on phagocytosis of E. coli rods by neutrophils. Phagocytotic indexes marked with asterix present statistically significant change with respect to the control value (without antibiotic), p < 0.05 Tabela 4.Wpływ stężeń subinhibicyjnych ciprofloksacyny na fagocytozę pałeczek E. coli przez neutrofile. Wartości indeksów fagocytarnych oznaczone gwiazdką są statystycznie istotne w odniesieniu do grupy kontrolnej (bez antybiotyku), p < 0,05 E. coli strains (szczepy E. coli) Phagocytic index ( ± SD) (Indeks fagocytarny) without antibiotic sub-mics of ciprofloxacin 1/2 1/4 1/8 1 10.8 ± 1.7 18.9 ± 1.4* 15.4 ± 1.4* 14.2 ± 1.4 2 10.2 ± 1.6 17.2 ± 1.5* 14.3 ± 1.4* 13.8 ± 1.5 3 10.7 ± 1.5 18.3 ± 1.5* 14.2 ± 1.7* 13.4 ± 1.4 4 11.0 ± 1.8 19.0 ± 1.6* 14.7 ± 1.7* 13.6 ± 1.3 5 10.9 ± 1.8 19.2 ± 1.5* 14.8 ± 1.6* 14.2 ± 1.3* 6 12.0 ± 1.7 18.6 ± 1.5* 15.3 ± 1.4* 14.8 ± 1.4 7 12.2 ± 1.8 17.6 ± 1.7* 14.8 ± 1.4* 13.9 ± 1.6 8 11.7 ± 1.7 17.3 ± 1.8* 15.3 ± 1.7* 14.7 ± 1.7 9 11.5 ± 1.8 17.8 ± 1.7* 15.6 ± 1.8* 14.6 ± 1.7 10 11.6 ± 1.6 17.8 ± 1.5* 14.3 ± 1.8* 13.9 ± 1.8 11 11.1 ± 1.8 19.0 ± 1.5* 14.7 ± 1.5* 13.9 ± 1.5 12 11.5 ± 1.8 18.7 ± 1.6* 14.2 ± 1.7* 13.4 ± 1.5 13 11.1 ± 1.8 18.4 ± 1.7* 15.3 ± 1.4* 14.7 ± 1.6 14 11.4( ± 1.8) 18.2 ± 1.3* 15.6 ± 1.4* 14.1 ± 1.7 15 11.6( ± 1.7) 18.5 ± 1.4* 15.8 ± 1.5* 14.1 ± 1.4 Fig. 1. Microscopic appearance of phagocytic ingestion of E. coli cells grown: (a) in the absence of antibiotic, (b) in the presence of 1/2 of MIC of ciprofloxacin. Magnification 1000 (arrow points to the ingested bacteria) Ryc. 1. Mikroskopowy obraz sfagocytowanych komórek E. coli rosnących: (a) bez antybiotyku, (b) w obecności 1/2 MIC ciprofloksacyny. Powiększenie 1000 (strzałka wskazuje sfagocytowane bakterie) telithromycin at 1/2 of MIC commonly suppressed the formation of the capsule, whereas a sub-mic of penicillin was less effective in this aspect. Several studies indicate that capsular polysaccharides have antiphagocytic activity and suggest that bacterial strains possessing K antigens have enhanced virulence [24 26]. The encapsulated strains are more resistant to phagocytosis than their unencapsulated mutants. Capsules are thought to impair phagocytosis by several mechanisms: their

Effect of Antibiotics on K1 Antigen and Phagocytosis 435 120 20 97.73 100 90.8 92.4 94.6 18 16 % clones sensitive to phage K1A % klonów wrażliwych na faga K1Av 81.4 80 74.73 79.06 60 40 20 14 12 10 8 6 4 value of phagocytic index wartość indeksu fagocytarnego 11.28 15.29 13.76 13.3 18.3 14.95 14.08 0 control 1/2AN 1/4AN 1/8AN 1/2CIP 1/4CIP 1/8CIP grupa kontrolna 2 0 Fig. 2. Comparison of the influence of sub-mics of amikacin and ciprofloxacin on E. coli K1 rods in regards to: the percentage of clones susceptible to phage K1A (white bars) and phagocytic index (grey bars). Numbers over bars represent percentage of clones, numbers on grey bars represent phagocytic index. The results are average values derived from all examined E. coli strains. Abbreviations: AN amikacin, CIP ciprofloxacin Ryc. 2. Porównanie wpływu działania sub-mics amikacyny i ciprofloksacyny na pałeczki E. coli K1 w odniesieniu do: odsetka klonów szczepów E. coli wrażliwych na faga K1A (białe słupki) i indeksu fagocytarnego (szare słupki). Liczby nad słupkami odpowiadają odsetkowi klonów, liczby na szarych słupkach są wartościami indeksów fagocytarnych. Przedstawione dane są średnimi wartościami wyników pochodzących od wszystkich badanych szczepów E. coli. Skróty: AN amikacyna, CIP ciprofloksacyna hydrophilic nature, negatively charged character but also by serving as camouflage and masking the underlying opsonized surface [24 26]. The results of own study confirmed that strains that possessed lower amounts of clones with K antigen were more sensitive to phagocytosis. Adinolfi et al. [27] observed that phagocytosis after treatment with sub-mics of imipenem or piperacillin was significantly higher compared to bacteria unexposed to antibiotics. Pretreatment with the β-lactam antibiotics rendered Staphylococcus aureus more liable to phagocytosis. Also the values of phagocytic index were lower after the exposure of E. coli strains to sub-mics of gatifloxacin [28]. Presumably the exposure of bacteria to antibiotics leads to changes in the surface components, altering among others, the hydrophilic character of surface into hydrophobic one, which contributes to enhanced bacterial uptake by granulocytes and monocytes. The loss of antigen K1 facilitates the interaction between the specific antibody and complement component C3 associated with the outer membrane of bacteria and their receptors on phagocytic cells. In conclusion, sub-mics of amikacin and ciprofloxacin interfere with the assembly of Kl capsular polysaccharide of encapsulated E. coli, leading to an increase in phagocytic index. References [1] Bonacorsi S, Houdouin V, Mariani-Kurkdjian P, Mahjoub-Messai F, Bingen E: Comparative prevalence of virulence factors in Escherichia coli causing urinary tract infection in male infants with and without bacteremia. J Clin Microbiol 2006, 44, 1156 1158. [2] Korzeniowska-Kowal A, Witkowska D, Gamian A: Mimikra cząsteczkowa bakteryjnych antygenów polisacharydowych i jej rola w etiologii chorób infekcyjnych i autoimmunologicznych. Post Hig Med Dośw 2001, 55, 211 232. [3] Wilson JW, Schurr MJ, LeBlanc CL, Ramamurthy R, Buchanan KL, Nickerson CA: Mechanisms of bacterial pathogenicity. Postgrad Med J 2002, 78, 216 224. [4] Wojnicz D, Jankowski S: Effect of subinhibitory concentrations of amikacin and ciprofloxacin on the hydrophobicity and adherence to epithelial cells of uropathogenic Escherichia coli strains. Int J Antimicrob Agents 2007, 29, 700 704. [5] Wojnicz D, Kłak M, Adamski R, Jankowski S: Influence of subinhibitory concentrations of amikacin and ciprofloxacin on morphology and adherence ability of uropathogenic strains. Folia Microbiol 2007, 52, 429 436.

436 D. Wojnicz, D. Tichaczek-Goska, A. Cisowska [6] Wojnicz D, Cisowska A: Composition of the outer membrane proteins of Escherichia coli strains in relation to serum susceptibility after exposure to subinhibitory concentrations of amikacin and ciprofloxacin. Int J Antimicrob Agents 2009, 33, 579 582. [7] National Committee for Clinical Laboratory Standards 2003. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 5th ed. Approved standard M7 A6. National Committee for Clinical Laboratory Standards, Wayne, Pa. [8] Devine DA, Roberts AP, Rowe B: Simple technique for detecting K1 antigen of Escherichia coli. J Clin Pathol 1990, 43, 76 78. [9] Lukacova M, Barak I, Kazar J: Role of structural variations of polysaccharide antigens in the pathogenicity of Gram-negative bacteria. Clin Microbiol Infect 2008, 14, 200 206. [10] O Riordan K, Lee JC: Staphylococcus aureus capsular polysaccharides. Clin Microbiol Rev 2004, 17, 218 234. [11] Johnson JR, Delavari P, Kuskowski M, Stell AL: Phylogenetic distribution of extraintestinal virulence-associated traits in Escherichia coli. J Infect Dis 2001, 183, 78 88. [12] Jankowski S, Grzybek-Hryncewicz K, Żarczyńska H, Sarowska J, Franiczek R, Mróz E: Występowanie chorobotwórczych pałeczek Escherichia coli z antygenem powierzchniowym K1w zakażeniach układu moczowego. Diagn Lab 1992, 29, 31 34. [13] Drulis-Kawa Z: Charakterystyka uropatogennych szczepów z rodziny Enterobacteriaceae izolowanych w latach 1996 1997 od dzieci z zakażeniami dróg moczowych. Praca doktorska. Uniwersytet Wrocławski 2000. [14] Fünfstück R, Tschäpe H, Stein G, Vollandt R, Schneider S: Virulence of Escherichia coli strains in relation to their hemolysin formation, mannose-resistant hemagglutination, hydroxamate production, K 1-antigen and the plasmid profile in patients with chronic pyelonephritis. Clin Nephrol 1989, 32, 178 184. [15] Evans DJ, Evans DG, Höhne C, Noble MA, Haldane EV, Lior H, Young LS: Hemolysin and K antigens in relation to serotype and hemagglutination type of Escherichia coli isolated from extraintestinal infections. J Clin Microbiol l981, 13, 171 178. [16] Czirok E, Milch M, Csiszar K, Csik M: Virulence factors of Escherichia coli. III. Correlation with Escherichia coli pathogenicity of haemolysin production, haemagglutinating capacity, antigens K1, K5, and colicinogenicity. Acta Microbiol Hung 1986, 33, 69 83. [17] Dal Sasso M, Bovio C, Culici M, Braga PC: Interference of sub-inhibitory concentrations of gatifloxacin on various determinants of bacterial virulence. J Chemother 2002, 14, 473 482. [18] Raponi G, Keller N, Overbeek BP, Rozenberg-Arska M, van Kessel KP, Verhoef J: Enhanced phagocytosis of encapsulated Escherichia coli strains after exposure to sub-mics of antibiotics is correlated to changes of the bacterial cell surface. Antimicrob Agents Chemother 1990, 34, 332 336. [19] Suerbaum S, Leying H, Kroll HP, Gmeiner J, Opferkuch W: Influence of beta-lactam antibiotics and ciprofloxacin on cell envelope of Escherichia coli. Antimicrob Agents Chemother 1987, 31, 1106 1110. [20] Taylor PW, Kroll HP, Tomlinson S: Effect of subinhibitory concentrations of mecillinam on expression of E. coli surface components associated with serum resistance. Drugs Exp Clin Res 1982, 8, 625 631. [21] Kadurugamuwa JL, Anwar H, Brown MRW, Zak O: Protein antigens of encapsulated Klebsiella pneumoniae surface exposed after growth in the presence of subinhibitory concentrations of cephalosporins. Antimicrob Agents Chemother 1985, 28, 195 199. [22] Brook I, Gober AE: In vitro effects of penicillin and clindamycin on the expression of Streptococcus pneumoniae capsule. J Med Microbiol 1996, 45, 505 506. [23] Brook I, Hausfeld JN: In vitro effects of penicillin and telithromycin on the expression of Streptococcus pneumoniae capsule. J Antimicrob Chemother 2006, 58, 678 679. [24] Hyams C, Camberlein E, Cohen JM, Bax K, Brown JS: The Streptococcus pneumoniae capsule inhibits complement activity and neutrophil phagocytosis by multiple mechanisms. Infect Immun 2010, 78, 704-715. [25] Corbett D, Roberts IS: Capsular polysaccharides in Escherichia coli. Adv Appl Microbiol 2008, 65, 1 26. [26] Taylor CM, Roberts IS: Capsular polysaccharides and their role in virulence. Contrib Microbiol 2005, 12, 55 66. [27] Adinolfi LE, Bonventre PF: Enhanced phagocytosis, killing, and serum sensitivity of Escherichia coli and Staphylococcus aureus treated with sub-mics of imipenem. Antimicrob Agents Chemother 1988, 32, 1012 1018. [28] Braga PC, Dal Sasso M, Bovio C, Zavaroni E, Fonti E: Effects of gatifloxacin on phagocytosis, intracellular killing and oxidant radical production by human polymorphonuclear neutrophils. Int J Antimicrob Agents 2002, 19, 183 187. Address for correspondence: Dorota Wojnicz Department of Biology and Medical Parasitology Wroclaw Medical University Mikulicza-Radeckiego 9 50-367 Wrocław Poland Tel.: + 48 71 784 15 12 E-mail: wojnicz@biolog.am.wroc.pl Conflict of interest: None declared Received: 23.03.2010 Revised: 30.06.2010 Accepted: 27.07.2010