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370 EWA WITKOWSKA-BANASZCZAK et al.serial dilution of Bronchosol on the right medium. Inthe case of the anaerobic bacteria, Brucella agar with5% of defibrinated sheep blood, menadione andhemin was used, while Mueller-Hinton agar was usedfor the aerobic bacteria. The following concentrationsof the preparation were tested: 6.2, 12.5, 25.0, 50.0,100.0 and 200.0 mg/mL. A suspension containing 10 6CFU per drop was placed on the surface of the rightagar (depending on the bacteria type) with Steersreplicator, after previous addition of the appropriateconcentration of the preparation to the agar. Agarwhich did not contain Bronchosol was used to controlthe strain growth. The inoculated media and the controlmedia were incubated in either anaerobic (inanaerobic jars) or aerobic conditions, at 37 O C for theright period of time depending on the bacteria type:for 48 h in the case of the anaerobic bacteria and for24 h in the case of the aerobic bacteria. The MIC wasdefined as the lowest concentrations of the preparationthat completely inhibited the growth of the testedanaerobic or aerobic bacteria strains.The yeast-like fungi strains cultured from thematerials obtained from patients with infections ofthe upper respiratory tract were inoculated inSabouraudís agar and incubated at 37 O C for 24-72 h.Antifungal activityDetermination of minimal fungicidal concentrationIn order to assess the fungicidal activity (MFC- minimal fungicidal concentration), 0.1 mL of asuspension of the culture of the tested strain containing10 6 CFU in 1 mL was added to 1 mL ofBronchosol (undiluted preparation). After 15 and 30min, samples of 0.1 mL were taken and inoculatedinto 2 mL of BHI broth (Merck). The BHI brothinoculated with 0.1 mL of the fungal culture constitutedthe growth control of a given strain. The inoculatedand control media were incubated at 37 O C for24 h in aerobic conditions. A lack of any yeast-likefungi growth in the medium proved fungicidal activityof the preparation.Table 2. Bactericidal activity (MBC - minimal bactericidal concentration) of Bronchosol at concentration recommended by produceragainst reference strains of aerobic bacteria and bacteria isolated from patients with respiratory infections.Number ofNumber of strains sensitiveAerobic bacteria strains to preparationtested after 15 min after 30 minGram-positive cocci:Staphylococcus aureus* 1 1 1Staphylococcus epidermidis* 1 1 1Enterococcus faecalis* 1 0 0Streptococcus anginosus* 1 1 1Streptococcus pneumoniae* 1 1 1Streptococcus pyogenes* 1 1 1Total Gram-positive cocci 6 5 5Gram-negative baccilli:Acinetobacter baumannii* 1 1 1Citrobacter freundii* 1 0 1Escherichia coli* 1 1 1Haemophilus influenzae* 1 0 1Klebsiella pneumoniae* 1 0 1Pseudomonas aeruginosa* 1 0 0Serratia marcescens* 1 1 1Total Gram-negative baccilli 7 3 6Standard strains:Staphylococcus aureus ATCC 25923 1 1 1Streptococcus pneumoniae ATCC 49619 1 1 1Streptococcus pyogenes ATCC 19615 1 1 1Haemophilus influenzae ATCC 49274 1 1 1Moraxella catarrhalis ATCC 25238 1 1 1Klebsiella pneumoniae ATCC 13883 1 0 1Pseudomonas aeruginosa ATCC 27853 1 0 0Total standard strains 7 5 6Total aerobic bacteria 20 13 17* - aerobic bacteria strains isolated from patients with respiratory infections, 0 - no effect of the preparation.
In vitro antimicrobial activity of bronchosol 371Table 3. Sensitivity (MIC - minimal inhibitory concentration) to Bronchosol of anaerobic bacteria isolated from patients with respiratoryinfections and that of reference strains.NumberMIC [mg/mL]Anaerobic bacteriaof strainstested ≥ 200.0 100.0 50.0 25.0 12.5 6.2Gram-positive cocci:Peptostreptococcus anaerobius* 2 1 1 0 0 0 0Finegoldia magna* 2 1 1 0 0 0 0Micromonas micros* 2 1 1 0 0 0 0Total Gram-positive cocc i 6 3 3 0 0 0 0Gram-positive baccilli:Actinomyces odontolyticus* 1 0 1 0 0 0 0Bifidobacterium breve* 1 0 1 0 0 0 0Propionibacterium acnes* 1 1 0 0 0 0 0Propionibacterium granulosum* 1 1 0 0 0 0 01Total Gram-positive baccilli 4 2 2 0 0 0 0Gram-negative baccilli:Prevotella intermedia* 3 3 0 0 0 0 0Prevotella levii* 1 1 0 0 0 0 0Prevotella loescheii* 1 1 0 0 0 0 0Porphyromonas asaccharolytica* 1 1 0 0 0 0 0Porphyromonas gingivalis* 1 1 0 0 0 0 0Fusobacterium nucleatum* 1 1 0 0 0 0 0Fusobacterium necrophorum* 1 1 0 0 0 0 0Bacteroides fragilis* 1 1 0 0 0 0 0Total Gram-negative baccilli 10 10 0 0 0 0 0Standard strains:Finegoldia magna ATCC 29328 1 0 1 0 0 0 0Peptostreptococcua anaerobiusATCC 27337 1 1 0 0 0 0 0Propionibacterium acnes ATCC 11827 1 1 0 0 0 0 0Bifidobacterium breve ATCC 25286 1 1 0 0 0 0 0Fusobacterium nucleatum ATCC 25586 1 1 0 0 0 0 0Bacteroides fragilis ATCC 25285 1 1 0 0 0 0 0Total standard strains 6 5 1 0 0 0 0Total anaerobic bacteria 26 20 6 0 0 0 0* - anaerobic bacteria strains isolated from patients with respiratory infections, 0 - no effect of the preparation.The sensitivity test (MIC) of the yeast-like fungito Bronchosol was conducted by the method of serialdilution in Sabouraudís agar (38). The following dilutionsof the preparation were tested: 6.2, 12.5, 25.0,50.0, 100.0 mg/mL. An inoculum containing 10 6 CFUper drop was placed on the surface of the agar withSteers replicator. A medium without the preparationwas used to control the growth of the tested fungistrains. Incubation of the inoculated and controlmedia was performed at 37 O C for 24 h in aerobic conditions.The MIC was defined as the lowest concentrationsof Bronchosol which completely inhibited thegrowth of the tested strains of yeast-like fungi.RESULTS AND DISCUSSIONThe aim of the study was to determine theantimicrobial activity of Bronchosol (Phytopharm,KlÍka) against anaerobic and aerobic bacteria, andfungi that cause respiratory infections most frequently,isolated from patients with respiratoryinfections, and reference ones.The study was conducted in in vitro conditionsslightly different from in vivo conditions. All testswere performed at a temperature of 37 O C, similar tothe temperature found in the oral cavity and upperrespiratory tract of a healthy organism. A bacterial,viral or fungal infection usually causes a rise in thetemperature, which can be controlled with the use ofantipyretics. The neutral pH found in the oral cavityand upper respiratory tract is similar to the pH reactionof the applied media (6.4 ñ 7.4), except forSabouraudís agar ñ pH 5.4, whose acidic reactioncorresponds to pH of the oral cavity during and aftera meal. The exposure of the microorganisms toBronchosol in the experimental conditions lasted 48
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PHARMACEUTICAL TECHNOLOGY347. £uka
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