Introduction

Allocasuarina littoralis (black she-oak; previously known as Casuarina littoralis and Casuarina suberosa), a member of the genus Allocasuarina (family Casuarinaceae) are native trees to Australia, Southern Asia and the Pacific islands (Yasodha et al., 2004). Some members of the Casuarinaceae family (Casuarina cunninghamiana, Casuarina equisetifolia, Casuarina glauca) have also been introduced to other areas such as USA and the Caribbean (Staples et al., 2000) and Southern Africa (Henderson, 1995) where they are considered invasive species.

Members of the Casuarinaceae family were important as a food source to the Aboriginal people of Australia prior to European settlement. The Aborigines in the Canberra region are known to have eaten the leaves and young cones of Allocasuarina verticellara as a food (Nash, 2004). The Casuarinaceae family also had a role as traditional bush medicines for Australian Aborigines. The bark of Casuarina equisetifolia was used as an astringent and to treat diarrhoea, dysentery, headache and fever (Maiden, 1913; Maiden, 1889). On Groote Eylandt in the Northern Territory of Australia, the twigs were soaked in water and used as a mouthwash (Maiden, 1889). No evidence was found of Australian Aborigines, nor any other ethnic group, using Casuarinaceae as antimicrobial agents.

A recent report has demonstrated the antibacterial activity of a related Casuarinaceae species (Casuarina equisetifolia) against a variety of bacterial species (Parekh et al., 2005). Studies within this laboratory (Cock, 2008) have also found antibacterial activity in methanolic extracts of A. littoralis leaves against a limited panel of bacteria. The current study was undertaken to validate and extend these observations against a wider panel of bacteria and fungi.

Materials and Methods

Plant Collection and Extraction

Test Microorganisms

Evaluation of Antimicrobial Activity

Bacterial Growth Time Course Assay

Results and Discussion

A. littoralis leaf extract was diluted to a 25 mg/ml concentration. 10 µl of the extract was tested in the disc diffusion assay against 17 microorganisms (table 1). The A. littoralis leaf extract inhibited the growth of 8 of the 14 bacteria tested (57%). The antibacterial activity was relatively strong against A. faecalis, P.fluorescens, S. Salford and S. aureus (as determined by the diameter of the zone of inhibition).

Figure 1

Table 1: Antibacterial activity of extract. Numbers indicate the mean diameters of inhibition of triplicate experiments ± standard deviation. – indicates no growth inhibition. Chl indicates chloramphenicol (10 µg) was used as the positive control. Amp indicates ampicillin (2 µg) was used as the positive control. Cip indicates ciprafloxicin (2.5 µg) was used as the positive control. . Nys indicates nystatin nystatin discs (100 µg) was used as the positive control.

Both Gram-positive and Gram-negative bacterial growth were inhibited by A. littoralis leaf extract although a greater susceptibility of Gram-positive bacteria was apparent. Of the 11 Gram-negative bacteria tested, 5 (45%) were inhibited by A. littoralis extract. The extract inhibited the growth of all of Gram-positive bacteria tested. The greater susceptibility of Gram-positive bacteria has been previously reported for South American (Paz et al., 1995), African (Kudi et al., 1999; Vlietinck et al., 1995) and Australian (Palombo and Semple, 2001) plant extracts. Susceptibility differences between Gram-positive and Gram-negative bacteria may be due to cell wall structural differences between these classes of bacteria. The Gram-negative bacterial cell wall outer membrane appears to act as a barrier to many substances including antibiotics (Tortora et al., 2001). No antifungal activity was seen in the present study, although a limited panel of fungi was tested.

The antibacterial activity of the A. littoralis leaf extract was further investigated by bacterial growth time course assays in the presence and absence of the extract. The concentration of the extract used in these assays was 41.7 µg/ml. A. littoralis leaf extract was able to significantly inhibit B. cereus (figure 1a), P. fluorescens (figure 1c) and B. subtilis (figure 1d) growth within 1 h indicating a rapid antimicrobial action. Aeromonas hydrophilia (figure 1b) growth was not inhibited greatly by A. littoralis leaf extract, in agreement with previously reported results (Cock, 2008).

Figure 2

Figure 1: Inhibition of bacterial growth by methanolic extract of leaves against (a) , (b) , (c) , (d) . For all graphs, ? represent measured bacterial growth values for test cultures (with extract) and ? represent control bacterial growth values (no extract). Values are the mean of triplicate determinations.

In summary, these studies and previous studies within this laboratory (Cock, 2008) are the first studies that have demonstrated the antibacterial activity of A. littoralis leaf extracts. Both Gram-positive and Gram-negative bacteria were susceptible to A. littoralis leaf extract. The broad range of microbial susceptibilities indicates the potential of A. littoralis leaf extract as a surface disinfectant as well as for medicinal purposes and possibly as food additives to inhibit spoilage. However, further studies are needed before these extracts can be applied to these purposes. In particular, toxicity studies are needed to determine the suitability of these extracts for the use as antiseptic agents and as a food additive.

Acknowledgements