Study of the combined effect of antibacterial/antiviral agent FS-1 and antibiotics on Staphylococcus aureus in experiments in vitro

Key words: Staphylococcus aureus, halogen-containing complex compound, synergistic antimicrobial effect

Introduction 

Pyoinflammatory diseases remain a serious problem in every medical establishment [1,2]. The etiological structure of pyoinflammatory diseases is represented by a wide spectrum of opportunistic pathogens (OP), viruses, fungi and protozoa that cause infectious process. The most significant role is played by staphylococci, gram-negative OP and respiratory viruses [2-4]. In this regard the specific characteristic of nosocomial strains of microorganisms is their multidrug resistance. 

Strategy for combating infectious diseases at the present stage includes several areas that are closely interrelated with each other. Among them the main are search and practical application of new anti-infectious drugs of various nature, capable of blocking the infectious agent at the stages of physiological cycle in macroorganism. A search for drugs that prevent the possibility of microorganism influence on the cells of phagocytic system, as well as drugs that inhibit the reproduction and persistence of the pathogen is being conducted. Substances and complexes of plant, animal and microbial origin, as well as chemically synthesized and chemically modified compounds could act as antibacterial drugs [5-7].

Currently, the search for new compounds with antimicrobial, antiviral, antifungal, antiprotozoal and anthelminthic action, and developing on the their basis the therapeutic and prophylactic anti-infectious drugs is the most priority in science and one of the main tasks of medicine and veterinary medicine. 

In RSOE Scientific Center for Anti-Infectious Drugs, Almaty, Republic of Kazakhstan, a synthetic antibacterial/antiviral pharmaceutical agent FS-1 was developed, being a halogen-containing complex compound, the active substance of which is ionic nanostructured complex with intercalated iodine [8].

The objective of this study was to investigate the sensitivity of methicillin-resistant and methicillin-susceptible Staphylococcus aureus to FS-1 in experiments in vitro. 

Materials and procedures 

Microorganism cultures. Reference strains of methicillin-susceptible Staphylococcus aureus ATCC 29213 (MSSA) and methicillin-resistant S. aureus ATCC 43300 (MRSA) were obtained from the National Bank of Industrial Microorganisms and Cell Cultures, Sofia, Bulgaria. 

Clinical isolates of MSSA and MRSA were collected during the period from 2004 to 2006 by the National Reference Laboratory for Staphylococci, Sofia, Bulgaria. Isolates were identified using commercial reagents API Staph (Bio Merieux, Marcy-l'Etoile, France) and Bactident-coagulase (Merck, Germany), according to the manufacturer's instructions. 

Screening for resistance to antibiotics. Screening of clinical isolates for resistance to oxacillin/methicillin was performed using Mueller-Hinton agar (MHA) (Merck, Germany) with the addition of sodium chloride (AppliChem, Germany) at a concentration of 2%, and oxacillin (Wuhan Hezhong Bio-chemical manufature Co., Ltd, China) in the amount of 6 mg/ml. Freshly prepared suspension of the test culture S.aureus at a concentration of 106 CFU/ml was inoculated with a tampon by a imprint technique on the MHA surface. Testing was carried out under the compulsory growth control of test cultures on MHA with no sodium chloride and oxacillin. Inoculated plates were incubated at 37ºC. The results were registered following incubation during 24-48 hours. The appearance of visible growth of more than 1 colony on the site of culture application was regarded as a manifestation of this strain resistance to oxacillin and methicillin. In the absence of colony growth on the site of culture application the test strain was regarded as sensitive to oxacillin/methicillin [9]. 

Evaluation of antimicrobic effect. Evaluation of antimicrobic effect of FS-1 and antibiotics in the test cultures was carried out by macromethod of two-fold serial dilutions in Mueller-Hitton broth (MHA) (Merck, Germany) in accordance with CLSI guideline [10]. For testing FS-1 was used at the concentrations ranging from 80 to 10100 µg/ml, vancomycin – from 0.125 to 32 µg/ml, cefamandole – from 0.125 to 32 µg/ml, oxacillin – from 8 to 1024 µg/ml, which were dissolved in a sterile 0.9 % sodium chloride solution. In test tubes with the test antibacterial preparations (ABP) a freshly prepared suspension of the test culture at a concentration of 106 CFU/ml was introduced. The inoculated tubes were incubated at 37º C during 18-20 hours. As a minimum bacteriostatic concentration, the lowest concentration of the test substance that caused the apparent delay in the growth of the microorganism was specified. The minimum inhibitory (bactericidal) concentration (MIC) was determined by culture passaging from each tube without visible growth on plates containing MHA. Inoculated tubes were incubated at 370 C within 18-20 hours. MIC was determined by the lowest concentration of ABP, which completely inhibited the growth of the test organism. 

Evaluation of combined effect. Studying the antimicrobic activity of FS-1 in combination with antibiotics was conducted in a reference methicillin-resistant strain of S.aureus ATCC 43300 using the technique Checkerboard [11]. For the first series of experiments vancomycin (glycopeptide) was selected, as the most commonly used to treat infections caused by MRSA [12]. For testing vancomycin was used at the concentrations ranging from 0.125 to 8 µg/ml, and FS-1 – from 310 to 5080 µg/ml. For the second series of experiments cefamandole (second-generation cephalosporin) was used in concentrations ranging from 0.125 to 128 µg/ml, with FS-1 in concentrations from 310 to 5080 µg/ml. For the third series of experiments a beta-lactam antibiotic oxacillin (penicillinase-resistant penicillin) was selected to which MRSA are resistant at concentrations ranging from 0.25 to 1024 µg/ml, with FS-1 at concentrations ranging from 310 to 5080 µg/ml. Antibiotics were arranged in a checkerboard pattern in a final volume of 0.5 ml (0.25 ml of each antibiotic). 0.5 ml of freshly prepared bacterial inoculum of S.aureus at a concentration of  106 CFU/ml was added to each tube. The inoculated tubes with macrodilutions were incubated at 37º C within 18-20 hours. The results were interpreted following the calculation of the fractional inhibitory concentration index (FIC) using the following formula: 

where A and B - two tested antibiotics; Аа and Ва – MICs obtained when testing antibiotics separately, Ас and Вс - the concentration of each compound at the lowest effective combination. FIC index was calculated for each series of dilutions. The lowest values were used for the interpretation. Synergism was evaluated at FIC index of ≤ 0.5, partial synergism was ascertained against FIC > 0.5, but < 1. Additivity – when FIC = 1. Indifference – in the absence of changes in MIC, or when testing antibiotics separately or in combination (FIC>1, but < 4), and antagonism – when FIC > 4 [11]. 

Results 

Screening for antibiotic resistance. As a result of screening for resistance to oxacillin/methicillin it was found that of 114 clinical isolates of S.aureus 58 strains belong to methicillin-resistant and 56 strains – to methicillin-susceptible. 

Evaluation of antimicrobic action. As a result of the experiments it was established that FS-1 possesses antimicrobic activity against both MSSA isolates and MRSA isolates (Table 1). The minimum bacteriostatic activity was detected at a concentration of 620 µg/ml. The highest antibacterial activity was recorded at 2540 µg/ml, although for the majority of the tested staphylococcal strains the minimum bacteriostatic activity was manifested at a concentration of 1270 µg/ml (Table 1). A comparison of methicillin-resistant and methicillin-susceptible clinical isolates found that 45 (77.6 %) of MRSA strains revealed minimal bacteriostatic activity at concentrations of 1270 µg/ml against 51 (88 %) of MSSA strains (Table 1).

Table 1 

Summarized characteristics of FS-1 bacteriostatic and bactericidal activity when attacking Staphylococcus aureus

The submitted in Table 1 data show that the minimum bactericidal activity of FS-1 was in the range of 2540-1270 µg/ml. At the same time for the majority of strains it has been demonstrated that the bactericidal concentration of FS-1 was equal to the bacteriostatic or exceeding it per one dilution. For example, among MRSA strains the bactericidal activity of FS-1 for 45 (77.6 %) isola-tes was 1270 µg/ml and for 13 (22.4 %) – 2540 µg/ml. Among the MSSA strains the bactericidal activity of FS-1 was uniformly distributed. Therefore, FS-1 reveals an antibacterial action equally effective against both MSSA and MRSA. 

MIC of FS-1 varied in the range of 2540-1270 µg/ml (Table 2). Moreover, FS-1 was equally effective against both the museum cultures and clinical isolates, including the resistant ones. 

Table 2 

MIC of the drug FS-1 when attacking staphylococcal clinical isolates in comparison with the reference strains

Thus, FS-1 possesses an expressed antimicrobic activity and manifests similar efficacy with respect to the reference and clinical MSSA strains, as well as to the reference and clinical MRSA strains. 

Evaluation of combined effect. To study the combined effect of FS-1 with antibiotics against MRSA ATCC 43300, MICs for the selected antibiotics were established. It was found that MIC of vancomycin comes to 2 µg/ml, cefamandole – 64 µg/ml, oxacillin – 256 µg/ml. 

A study of the combined action of FS-1 and vancomycin demonstrated that MIC of the latter was not changed (Fig. 1). At the same time, the bactericidal concentration of FS-1 in the combined testing decreased per one dilution as compared to MIC and was 1270 µg/ml. The calculated FIC index for this combination came to 1.5, which can be interpreted as an indifference.  

Fig. 1. Combined effect of FS-1 and vancomycin on MRSA ATCC 43300

A study of the combined action of FS-1 and cefamandole demonstrated a decrease in bactericidal concentrations of both antimicrobial agents (Fig. 2). A decrease in cefamandole bactericidal concentrations from 64 µg/ml to 8 µg/ml and in FS-1 bactericidal concentrations from 2540 µg/ml to 1270 µg/ml was recorded. FIC index for this combination came to 0.62, which characterizes the interaction of these antimicrobial agents as a partial synergism.

Fig. 2. Combined effect of FS-1 and cefamandole on MRSA ATCC 43300

In the study of the combined effect of FS-1 and oxacillin the decrease in bactericidal concentrations of both antimicrobial agents was established (Fig. 3). Reduced bactericidal concen-trations for oxacillin ranged from 256 µg/ml to 64 µg/ml and for FS-1 from 2540 µg/ml to 1270 µg/ml. FIC index for this combination was 0.75, which characterizes the interaction of these antimicrobial agents, as a partial synergism. 

Fig. 3. Combined effect of FS-1 and oxacillin on MRSA ATCC 43300

Consequently, the combined use of FS-1 is characterized by a synergistic effect against MRSA ATCC 43300 for beta-lactam antibiotics by the example of oxacillin, and for II generation cephalosporins by the example of cefamandole. The combined action of FS-1 and glycopeptides by the example of vancomycin does not change the antibacterial activity. 

Discussion

In the clinical practice for the treatment of methicillin-resistant staphylococcal infections a preference is given to flucloxacillin, cefamandole, clindamycin, fusidic acid, teicopla-nin/vancomycin, and linezolid [13]. According to the literary data a highly active against aurococ-cus and coagulase-negative staphylococci is oxacillin, but it has no effect on methicillin or oxacillin-resistant staphylococci. Therefore, a drug of choice for the treatment of staphylococcal infections today is vancomycin. Its use as a tool for empirical therapy led to a selection of vancomycin-resistant strains [14]. In 2002 new strains with high-level resistance to vancomycin (MIC> 128 µg/ml) appeared [15]. Therefore, at the present time the widely used previously antibiotics as an agent of empirical therapy, have lost their importance. 

This paper demonstrates a pronounced antimicrobial activity of a synthetic pharmaceutical agent FS-1 with respect to MSSA reference and clinical strains and MRSA reference and clinical strains. 

The use of combinations of antibacterial agents with different mechanism of action to accelerate the eradication of the pathogen in severe infections has extended since the second half of the 70ies in the last century [16]. There are clinically effective combinations of II-III generation cephalosporins and macrolides, cefamandole and aminoglycoside, oxacillin and aminoglycoside, amikacin and vancomycin, daptomycin and oxacillin [17,18]. 

In this study it was shown that the combined use of FS-1 and beta-lactam antibiotics (by the example of oxacillin), as well as II generation cephalosporins (by the example of cefamandole) leads to a synergistic effect against MRSA ATCC 43300. 

It can be concluded that the combined effect of FS-1 and antibiotics leads to a synergistic effect against staphylococcal infections caused by resistant S.aureus in vitro. This necessitates the study of the combined action of two agents in the system in vivo, in animals. In case of confirmation of the results, the agent FS-1 could be used in the treatment of staphylococcal infections caused by resistant S.aureus in humans. 

Acknowledgement 

The author expresses her deep appreciation to Dr. Dumitr Nashev, the researcher of the Reference Laboratory for Staphylococci, National Centre of Infectious and Parasitic Diseases (Sofia, Bulgaria), for scientific advice in carrying out this work. 

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