Influence of thiamphenicol on the primary functions of human polymorphonuclear leucocytes against Streptococcus pyogenes

International Journal of Antimicrobial Agents 24 (2004) 381–385

Influence of thiamphenicol on the primary functions of human polymorphonuclear leucocytes against Streptococcus pyogenes Vivian Tullio a,∗ , Annamaria Cuffini a , Narcisa Mandras a , Janira Roana a , Giuliana Banche a , Domenico Ungheri b , Nicola Carlone a a

Department of Public Health and Microbiology, Microbiology Division, University of Turin, Via Santena 9, 10126 Turin, Italy b Zambon Group, Lonigo, Vicenza, Italy Received 15 December 2003; accepted 21 March 2004

Abstract Current antibiotic therapy encourages the use of antibiotics that may potentiate the host’s immune defences. We therefore investigated the effect of thiamphenicol (TAP), the active principle of thiamphenicol glycinate acetylcysteinate (TGA), on human granulocyte functions, mainly phagocytosis and intracellular killing of Streptococcus pyogenes. Our findings support the use of thiamphenicol in the treatment of respiratory tract infections caused by S. pyogenes as it acts directly against the pathogen as well as in cooperation with PMNs by eliciting their intracellular killing. © 2004 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Streptococcus pyogenes; PMNs; Phagocytosis; Intracellular killing; Thiamphenicol

1. Introduction Successful management of infections requires a multidisciplinary approach in which account is taken of the kinetics and microbiological features of the drugs employed, the type of microorganism causing the infection and the host’s immune system. The differences in the way an antibiotic affects bacterial virulence and adhesiveness, as well as certain physiological activities of phagocytes (phagocytosis, intracellular killing, release of cytokines, apoptosis, etc.), must be of fundamental importance when choosing appropriate empirical therapy. Streptococcus pyogenes is one of the main causes of upper human respiratory tract infections which is usually treated with ␤-lactams or macrolides. However, the increased incidence of macrolide-resistant strains, in particular to erythromycin [1], and the fact that some patients are allergic to ␤-lactams require the use of alternative drugs whose good in vitro activity and tolerance by the patient are accompanied by an ability to stimulate, or at least not interfere with the host’s immune defences. This paper describes the in vitro evaluation of the effect of subinhibitory doses of thiamphenicol (TAP), the ac∗

Corresponding author. Tel.: +39 320 439 0067/39 001 670 5637; fax: +39 011 236 5637. E-mail address: [email protected] (V. Tullio).

tive principle of thiamphenicol glycinate acetylcysteinate (TGA), on the primary functions of human PMNs against a recently isolated clinical S. pyogenes strain. TGA has been employed for more than 30 years in the treatment of respiratory tract infections, thanks to the antimicrobial properties of TAP and the mucolytic activity of N-acetylcysteine, which makes bronchial secretions more fluid and facilitates absorption of antibiotic without altering its activity [1]. TGA possesses effective pharmacokinetic characteristics [2], good tolerability [3] and a broad spectrum of effectiveness against several Gram-positive and Gram-negative organisms, especially those involved in upper and lower respiratory tract infections (S. pneumoniae, S. pyogenes, Haemophilus influenzae, Moraxella catharralis) [1,4,5]. Moreover, in contrast to cloramphenicol, TGA has no serious haematological side-effects [6].

2. Materials and methods 2.1. Bacteria A clinical isolate of S. pyogenes was cultured to mid-exponential phase in Todd–Hewitt (TH) broth (Oxoid, Milan, Italy), supplied with 10% foetal calf serum (FCS; Gibco, Life Technologies Ltd., Paisley, Scotland), concen-

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trated 10 times in TH broth containing 30% glycerol, quick frozen in dry ice-ethanol and stored at −70 ◦ C. 2.2. Antimicrobial activity of thiamphenicol against S. pyogenes TAP was kindly provided by the Zambon Group S.p.A, Lonigo, Vicenza, Italy. Solutions, freshly prepared for each experiment, were shown to be free from endotoxin in a standard Limulus amebocyte lysate (LAL) assay (BioWhittaker, Walkersville, Md). Antibiotic susceptibility testing was performed by the standardized dilution method in TH broth, using bacterial inoculum of 107 CFU/ml, as confirmed by colony counts in triplicate. In order to evaluate the extracellular killing induced by TAP, S. pyogenes (107 CFU/ml) in 15 ml TH broth was incubated at 37 ◦ C in duplicate with or without (control) TAP at 1/2 × MIC, 1 × MIC, 2 × MIC and 10 × MIC [7]. At time zero and after 3 h of incubation, a 500 ␮l sample was removed and 10-fold dilutions were made in water and spread on TH agar plates. The mean number of CFU/ml of the duplicate samples was determined after overnight incubation of plates at 37 ◦ C in 5% CO2 . Results were reported as log CFU/ml. 2.3. PMNs PMNs were separated from lithium heparinized venous blood using Ficoll–Paque (Pharmacia S.p.A., Milan, Italy), as described previously in detail [8,9]. Using trypan blue testing, the viability of PMNs was determined as more than 95%. PMNs were suspended in RPMI 1640 medium (Gibco Laboratories, Grand Island, NY, USA), supplemented with 10% foetal calf serum (106 PMN/ml) and incubated at 37 ◦ C in a shaking water bath (150 rpm) before the addition of streptococci (107 CFU/ml).

number of survivors at time x, and dividing by the number of survivors at time zero [8–10]. According to this formula, if bacterial killing was 100% effective, the SI would be 1. To differentiate between any separate effect of TAP on the bacteria and the PMNs, the experiments were conducted after exposure of each of them to the antibiotic (one-half the MIC) for 1 h, before they were incubated together [10]. After the withdrawal of the drug, pre-exposed streptococci were added to PMNs and streptococci to pre-exposed PMNs. A control system was assayed in parallel with no antibiotic. The phagocytic and bactericidal activities of PMNs were determined as described above. Each test was performed in triplicate, and the results compared with those obtained with the control systems and expressed as the means and standard errors of the means (S.E.M.s) for 10 separate experiments. 2.5. Phenylbutazone treatment of PMNs To block the (O2 -dependent killing mechanisms by PMNs and distinguish the intracellular bactericidal activity of TAP from that of PMNs), phagocytes were treated with phenylbutazone (PB; Sigma-Aldrich, Steinheim, Germany) (1 g/l for 15 min) before the killing assay [11]. The solution of PB was prepared in phosphate saline just before use. A nitroblue tetrazolium test (NBT) was performed to check the abolition of superoxide production by PB [12]. 2.6. Statistical analysis Statistical evaluation of the differences between test and control results were performed by an analysis of variance by Tukey’s test. Extracellular killing was compared using Student’s unpaired t-test [7].

3. Results 2.4. Influence of thiamphenicol on phagocytosis and intracellular killing The effects of TAP on either the phagocytosis of radiolabelled S. pyogenes [3 H-uracil (specific activity: 1224.7 GBq/mmol; NEN Life, Milan, Italy)] or intracellular bacterial killing by PMNs were investigated by incubating the bacteria and the phagocytes (bacterium:PMN ratio was 10:1) at 37 ◦ C in a shaking water bath for periods of 30, 60 or 90 min in the presence of one-half the MIC of the drug. TAP-free controls were also included. Phagocytosis and intracellular killing were assessed by the methods described previously [8–10]. Radioactivity was expressed as the counts per min/sample. The percentage of phagocytosis at a given sampling time was calculated as follows: percent phagocytosis = [(cpm in PMNs pellet)/(cpm in total bacterial pellet)] × 100 [8–10]. The PMN killing values were expressed as the survival index (SI), which was calculated by adding the number of surviving bacteria at time zero to the

3.1. Antimicrobial activity of thiamphenicol against S. pyogenes The minimal inhibitory concentrations (MIC) of TAP for S. pyogenes was 8 mg/l with an inoculum of 107 CFU/ml. At one-half the MIC (4 mg/l), the activity of TAP was only bacteriostatic as indicated by a slight decrease in live bacteria (0.1 log) after 3 h incubation. In the presence of 1 × MIC (8 mg/l), 2 × MIC (16 mg/l) and 10 × MIC (80 mg/l), of the drug the reduction was 0.5, 1 and 2.1 log, respectively, indicating that bactericidal activity of TAP was proportional to its concentration (data not shown). 3.2. Effect of thiamphenicol on phagocytosis and intracellular killing In all experiments, the viability of PMNs remained unchanged throughout the experiments. Table 1 illustrates the

1.61 ± 0.09 1.88 ± 0.09 (12%) 1.93 ± 0.12(7%) 29.8 ± 1.30 31.0 ± 1.65 32.0 ± 1.98 30.4 ± 1.9 31.3 ± 1.1 33.4 ± 1.6 31.8 ± 1.8 35.8 ± 1.3 40.8b ± 1.2 29.1 ± 1.9 30.2 ± 2.0 32.3 ± 2.15 30 60 90

A,A1 : addition of streptococci and 1/2 MIC of thiamphenicol to PMNs; B,B1 : addition of streptococci to PMNs following 1 h pre-exposure of streptococci to 1/2 MIC of thiamphenicol; C,C1 : addition of streptococci to PMNs following 1 h pre-exposure of phagocytes to 1/2 MIC of thiamphenicol. a Values are mean + S.E.M. b Significantly different (P < 0.05) from the controls. c Significantly different (P < 0.01) from the controls. d Percentages of initial bacterial population killed by PMNs in absence and in presence of the antibiotic.

1.51b ± 0.05 (49%) 1.60c ± 0.04 (40%) 1.90 ± 0.01 (10%) ± 0.1 (75%) 1.58c ± 0.05 (42%) 1.70c ± 0.08 (30%) ± 0.13 (71%) 1.24c ± 0.08 (76%) 1.23c ± 0.10 (77%) 1.29c

1.25c

B1 A1

(39%d )

Controls C B A Controls

Survival index (%) Phagocytosis (%) Time (min)

Table 1 Effect of one-half the MIC of thiamphenicol on human PMN phagocytosis and intracellular killing of S. pyogenesa

C1

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in vitro effects of one-half the MIC of TAP on PMN phagocytosis and intracellular killing of S. pyogenes. Until the first hour of contact, the drug had no significant effect on phagocytosis and the percent phagocytosis was only slightly higher than that in the controls (column A). However, it increased with time when the drug was present and the difference became significant after 90 min compared with the controls (40 versus 32.3%; P < 0.05). Conversely, TAP, in the same experimental conditions, had a marked effect on intracellular killing, resulting in increased numbers of killed streptococci for all three incubation times compared with those for the antibiotic-free system controls. During the 90-min period, the intracellular bacterial load was reduced by 77% (SI = 1.23) (P < 0.01; column A1). To investigate the direct effect of thiamphenicol on the phagocyte functions, bacteria and human PMNs were pre-incubated for l h with one-half the MIC of the drug. After withdrawal of the antibiotic, bacterial uptake and microbicidal activity were determined. Drug pre-treatment of the bacteria during their growth phase had no effect on phagocytosis itself. In fact, PMNs engulfed the bacteria at the same rate as the untreated ones (column B). However, drug-pretreated streptococci were killed far more efficiently by the PMNs during the 90 min incubation compared with bacteria not treated: 75–42–30% versus 39–12–7% (P < 0.01) (column Bl). Pre-exposure of the PMNs to one-half the MIC of TAP for 60 min had no effect on phagocytosis: streptococci were phagocytosed at the same rate as controls (column C). In contrast, there was enhanced intracellular killing, leading to a significant decrease in SIs for up 60 min, compared with the controls (P < 0.01; column Cl). 3.3. Effect of pre-incubation of PMNs with PB on the killing of intracellular bacteria The bactericidal activity of PMNs was completely abolished by the PB treatment: in fact, SIs were consistently >2, indicating intracellular surviving and absence of killing (Table 2). The NBT test showed that PB-pre-treatment abolished the production of the superoxid anion in 90% of PMNs and hence the oxidative microbicidal mechanisms. When TAP was added at one-half the MIC to PB-treated PMNs, there was restored intracellular killing (P < 0.01), mean Table 2 Effect of one-half the MIC of thiamphenicol on intracellular killing of S. pyogenes by PB-treated PMNsa Time (min)

30 60 90

Survival index Controls

Thiamphenicol

>2 >2 >2

1.61b ± 0.15 (39%c ) 1.54b ± 0.05 (46%) 1.52b ± 0.14 (48%)

Values are mean + S.E.M. Significantly different (P < 0.01) from the controls. c Percentages of initial bacterial population killed by PMNs in presence of the antibiotic. a

b

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values 39% at 30 min, 46% at 60 min and 48% at 90 min corresponding to SIs = 1.61, 1.54 and 1.52, respectively (Table 2).

4. Discussion The ability of certain species of bacteria to survive and even multiply within phagocytic cells can pose serious clinical problems, since they can escape antibiotics such as ␤-lactams, which have poor intracellular penetration [13,14]. Recent evidence that strains of S. pyogenes may locate intracellularly might explain the failure of ␤-lactams in the treatment of streptococcal pharyngotonsillitis [15,16]. This also questions the role of macrolides intracellularly [16]. However the increased clinical isolation of macrolide-resistant strains has directed attention to alternative drugs, such as thiamphenicol. Current trends require the use of antibiotics which combine good antimicrobial properties with the capacity to act in concert with the immune system in a way that potentiates the host’s defence mechanisms [17,18]. Therefore, we looked at the effects of TAP on human granulocyte functions, mainly phagocytosis and intracellular killing of S. pyogenes. When TAP was added to the medium containing PMNs and bacteria, percentage phagocytosis increased with time (Table 1, column A). In the same experimental conditions, TAP also stimulated intracellular killing (Table 1, column Al), suggesting possible penetration of the phagocyte and direct intracellular killing or cell damage. Comparison of these results with others is not possible as only bovine PMNs have been used previously [19]. To determine whether the increase in microbicidal activity induced by TAP was due to its direct action on the bacteria or on the PMNs, both types of cells were separately exposed to the drug for 1 h prior to phagocytosis and killing tests. The results showed that TAP does not depress bacterial uptake, since the percentage of intracellular bacteria was similar to that observed in the controls (Table 1, columns B and C). There was more intracellular killing of streptococci that had been pre-exposed to TAP (Table 1, column Bl). The direct action of TAP makes the bacteria more susceptible to PMN lytic mechanisms, probably related to an impairment of protein synthesis. When proteins, lipids and carbohydrates sources are limited, the surface hydrophobicity of S. pyogenes is also decreased and PMN killing is more efficient [20]. Pre-treatment of PMNs with the drug also significantly increased intracellular microbicidal activity over the course of 1 h (Table 1, column Cl). This may provide indirect evidence of TAP penetration of PMNs, and the antibiotic remaining effective within PMNs thus increasing intracellular killing. The literature does not report any work on the intraphagocytic penetration of TAP. It is possible, however, to hypothesize a behaviour similar to that of chloramphenicol, a structurally-related drug, which is accumulated in phagocytes at intracellular concentrations 2–10 times higher than

those extracellular, although without having activity against the intracellular bacteria [21,22]. In order to simulate clinical conditions more effectively, we also looked at extracellular killing of S. pyogenes induced by subinhibitory, inhibitory and superinhibitory concentrations of TAP. In the presence of PMNs, one-half the MIC of TAP resulted in intracellular killing whereas there was only a bacteriostatic effect extracellularly at the same concentration. This difference can be explained by hypothesizing that TAP may enhance microbicidal activity of PMN, either by making intracellular bacteria more sensitive to PMN lytic action or through accumulation to bactericidal concentrations intracellularly. Further investigations are required to look at these possibilities. It is well known that PMNs kill phagocytosed bacteria in various ways, usually by O2 -dependent and -independent mechanisms. In chronic granulomatous disease, AIDS and other immunodeficiencies, alteration or inefficiency of the oxidative microbicidal mechanisms increase the risk of infection [11]. In the final part of the study, PMNs were treated with PB to both simulate the situation in PMNs with altered oxidative function and to distinguish their intracellular killing from that attributable to TAP. In the absence of the antibiotic there was virtually no bactericidal activity, while the addition of one-half the MIC of TAP restored intracellular killing, probably due to the drug activity within the phagocyte. These findings suggest that TAP would be efficient in the treatment of S. pyogenes respiratory tract infections due to its ability to act both directly against the pathogen and to boost bacterial intracellular killing in PMNs. These immunomodulating properties along with its broad spectrum, excellent pharmacokinetics, no haematological side-effects and good tolerability make TAP suitable for the treatment of respiratory tract infections especially in immunocompromised patients with defective defence mechanisms.

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