POPULATION ANALYSIS OF SUSCEPTIBILITY TO PROPOLIS IN REFERENCE STRAINS OF Staphylococcus aureus AND Escherichia coli

FERNANDES Jr., A.; LOPES, C. A. M.; SFORCIN, J. M.; FUNARI, S.R.C.

doi:10.1590/S0104-79301997000200005

Abstract

Susceptibility to several ethanolic extracts of propolis (EEP) concentrations was tested with the population analysis technique in reference strains of Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 35218). The results of these tests showed that all bacteria were killed by EEP concentrations approximately equal or higher (2.0% v/v- S. aureus; 10.0% v/v - E. coli) than the respective minimal inhibitory concentrations (MIC). Regarding the susceptibility to propolis, there was a homogeneity of data with the respective time kill curves showing a clear bactericidal effect during 6 to 9 h of exposition.

propolis; population analysis; susceptibility; Staphylococcus aureus; Escherichia coli

Original paper

POPULATION ANALYSIS OF SUSCEPTIBILITY TO PROPOLIS IN REFERENCE STRAINS OF Staphylococcus aureus AND Escherichia coli

A. FERNANDES Jr. , C. A. M. LOPES, J. M. SFORCIN , S.R.C. FUNARI

1 Department of Microbiology and Immunology of the Institute of Bioscience of Botucatu - UNESP, Botucatu, State of São Paulo, Brazil, 2 Department of Production and Animal Exploration of the School of Veterinary Medicine and Animal Husbandry of Botucatu - UNESP, Botucatu, State of São Paulo, Brazil, 3 Center for the Study of Venoms and Venomous Animals - CEVAP - UNESP, Botucatu, State of São Paulo, Brazil.

ABSTRACT. Susceptibility to several ethanolic extracts of propolis (EEP) concentrations was tested with the population analysis technique in reference strains of Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 35218). The results of these tests showed that all bacteria were killed by EEP concentrations approximately equal or higher (2.0% v/v- S. aureus; 10.0% v/v - E. coli) than the respective minimal inhibitory concentrations (MIC). Regarding the susceptibility to propolis, there was a homogeneity of data with the respective time kill curves showing a clear bactericidal effect during 6 to 9 h of exposition.

KEY WORDS: propolis, population analysis, susceptibility, Staphylococcus aureus, Escherichia coli.

INTRODUCTION

Propolis is a natural resinous product collected by honeybees and used to wax, seal and strengthen hives against cracks and to protect them by covering unwanted objects inside them (5). Through its biochemical constituents including a variety of flavonoids, chalcones, benzoic acid and derivatives, terpenes, aliphatic hydrocarbons, minerals, sterols, sugars and amino acids (1,4,7,9,10), propolis has shown several biological properties such as antiulcerative, antibacterial, antiviral, fungicidal, local anaesthestic and as an immunostimulating agent(2,5,6).

The first systematic investigation on propolis antibacterial properties was made by Kivalkina in 1948(5). He detected a bacteriostatic activity on Staphylococcus aureus, typhoid bacillus and other pathogens. Recently, a number of investigators have studied this antimicrobial action mainly when the product is prepared from an ethanolic extract(2,3,8). Due to lack of information on the susceptibility of bacterial pathogens to propolis with a population analysis technique, this survey was conducted to study this susceptibility in reference strains of Staphylococcus aureus and Escherichia coli.

MATERIAL AND METHODS

MICROORGANISMS: A strain of Staphylococcus aureus ATCC 25923 and of Escherichia coli ATCC 35218 obtained from the American Type Culture Collection, Rocksville Md., USA showing a minimal inhibitory concentration (MIC) of propolis of 0.6% v/v and of 10.5% v/v, respectively, were used.

PROPOLIS ETHANOLIC EXTRACT (EEP): Samples of propolis were obtained from hives maintained under controlled conditions at the Department of Production and Animal Exploration of the School of Veterinary Medicine and Animal Husbandry of Botucatu, UNESP. The EEP solutions for testing were prepared by stirring propolis in ethanol (Synth) 96º GL (50 g in 100 mL ethanol) and submitting to filtration. The final EEP solution yielded a matrix standard concentration of 50% v/v.

POPULATION ANALYSIS: Population analysis was done using the standard matrix EEP for preparation of serial concentrations of propolis in the range of 0.5 - 2.0% v/v for S. aureus and of 0.8 to11% v/v for E. coli, in tubes containing Brain Heart Infusion Broth EEP/BHI (Difco). A control tube of BHI without EEP but added with the respective amount of ethanol to achieve each EEP concentration was included in each series of EEP/BHI tubes. The inoculum of each EEP/BHI and control tubes was made with an aliquot of 0.1 mL of a bacterial suspension obtained from a BHI tube incubated at 37°C and yielding a bacterial concentration of approximately 4 x 10 colony-forming units per milliliter (CFU/mL). After the inoculation of the tubes, an aliquot of 1 mL from each EEP/BHI and control tubes was inoculated in plates of Mueller Hinton Agar MHA-(Difco) and these were incubated at 37ºC/24 h. Counts were made from the MHA plates corresponding the results to the zero time determination. After this procedure, all EEP/BHI and control tubes were incubated at 37ºC for 1, 2 ,3 ,6, 9 and 24 h and the respective colony counts determined. Results were expressed as CFU/mL- Log10 and percentage of CFU.

RESULTS AND DISCUSSION

The population analysis in S. aureus ATCC 25923 (Table 1 and Figure 1), showed that all bacteria were killed by an EEP concentration of 2.0% v/v during 6 to 9 h of exposition. EEP concentrations of 0.5 to 1.5% v/v determined a significant bacteriostatic effect inducing a reduction of 96 to 98% on the bacterial population regarding the initial viable count. For S. aureus ATCC 25923, an exposition to EEP 0.5% v/v longer than 9 h showed a reversion in the bacteriostatic effect characterized by an increase of 26 to 28% of CFU in relation to the initial inoculum.

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TABLE 1. Population analysis of susceptibility to EEP concentrations in S. aureus (ATCC 25923). Viable count (CFU/ml - log10 ) and percentages of viable bacteria in time.

On the other hand, the E. coli ATCC 35218 population analysis (Table 2 and Figure 2) compared with that of S. aureus ATCC 25923 showed an effective EEP concentration for a bactericidal effect in the range of 10 to11% v/v achieved with expositions during 6 to 9 h. Excluding the EEP concentration of 0.5% v/v for S. aureus ATCC 25923, identical profiles in the time kill curves (Figure 3 and Figure 4) were observed for both reference strains.

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TABLE 2. Population analysis of susceptibility to EEP concentrations in E. coli (ATCC 35218). Viable count (CFU/ml - log10) and percentages of viable bacteria in time.

In summary, the population analyses described here showed that the EEP concentrations approximately equal or higher than the respective MICs induced a marked bactericidal effect, expressing a similarity in the time kill curve profiles. However, elucidation of the differences and variations in the susceptibility to propolis between the Gram-negative and Gram-positive bacteria still remains an important subject for further investigations.

Received 04 September 1996

Accepted 05 October 1996

References

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CORRESPONDENCE TO:

C.A.M. LOPES - Departamento de Microbiologia e Imunologia, Instituto de Biociências de Botucatu, UNESP, CEP 18.618-000, Caixa Potal 510, Botucatu, São Paulo, Brasil.