a Eyteen Ltd, Rad Ramot Biotechnology Incubator, Tel-Hashomer b Vulcani Centre, Agricultural Research Organization, Bet-Dagan c Department of Pathology, Sackler School of Medicine, Tel-Aviv University, Ramat Aviv, Israel d Departments of Pharmacology e and Microbiology, Federal University of Minas Gerais, Belo Horizonte, Brazil f Susanne Levy-Gertner Oncogenetics Unit, Institute of Genetics, Chaim Sheba Medical Centre, Tel-Hashomer 52621, Israel
Sir,
Exposure of higher eukaryotes to pathogenic microorganisms stimulates a complex host defence response that includes the synthesis and secretion of naturally occurring antimicrobial peptides.1 The existence of these peptides was first recognized in invertebrates, with the discovery of cecropins in the silkmoth,2 and was subsequently confirmed by the isolation of magainin from frog skin.3 Since these discoveries, more than 200 antibacterial compounds have been detected in many different species, including humans.
The emergence of multidrug-resistant bacterial pathogens and the corresponding decline in the number of antibiotics available as treatment of patients with infections caused by them underscores the need to identify novel antimicrobial agents. Yet, no new class of antibacterials has been introduced into clinical practice during the past two decades.4 Naturally occurring peptides with antibacterial properties have exhibited impressive in-vitro activities against microorganisms resistant to conventional antibiotics5 and may therefore represent a new therapeutic modality. In the present study, we have screened a range of tissues from wild and domestic birds and reptiles for the presence of antibacterial activities.
The animals studied were vultures (Coragyps atratus foetens), ravens (Corvus corax), crocodiles (Crocodilus niloticus), ostriches (Struthio camelus), turkeys (Meleagris gallopavo) and chickens (Galus galus); these animals were chosen on the basis that, in their natural habitats, they are constantly exposed to a wide range of pathogenic bacteria. The tissues that were assessed included the following: stomach, duodenum, jejunum, colon, lung, liver, brain, heart, adrenal gland, muscle and blood. Following dissection (and manual cleansing of gastrointestinal tract organs), the tissues were frozen at -70°C. They were subsequently lyophilized and pulverized with a mortar and pestle. The resultant powder was suspended in 10% acetic acid and the suspension boiled for 15 min, followed by cooling to room temperature and centrifugation at 23,000g for 30 min at 10°C. The supernatant was applied to a solid phase extraction column LC-18 (5 gr-Supelco, Bellafonte, PA, USA) and the cartridges were washed with 20 mL of 0.1% trifluoroacetic acid (TFA) in water, followed by 20 mL of 0.1% TFA in 10% acetonitrile. Elution was performed with 0.1% TFA, first in 40% acetonitrile and then 80% acetonitrile. The fractions were collected in 1 mL volumes and vacuum dried. At the time of testing, they were resuspended in 80 µL of sterile water. Antibacterial activity was detected and semi-quantified by a zone inhibition assay described previously6 with Micrococcus luteus as the indicator organism; muscle tissue was used as a negative control.
The results of the screen are summarized in the Table; only data for those tissues exhibiting antibacterial activities are shown. At least one tissue from each of the avian species tested exhibited some antibacterial activity, the raven possessing the largest number of tissues with antibacterial activities, as well as the tissues exhibiting the most potent activities. This may be because the food on which ravens live tends to be highly contaminated. Unexpectedly, only a small number of the tissues from vultures, which also exist on highly contaminated food, demonstrated antibacterial activities and these activities were comparatively modest. For the most part, the tissues which most consistently exhibited antibacterial activities and which exhibited the most potent activities were those most likely to be exposed to pathogenic bacteria, i.e. lung and gastrointestinal tract tissues.
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To the best of our knowledge, this is the first study that has demonstrated the presence of naturally occurring antibacterial agents in avian tissues. Focusing on species that are normally exposed to large numbers of potentially pathogenic bacteria appears to be a good starting point in the search for such compounds. Our observations also suggest that future searches should concentrate on the tissues of those organs that normally tend to be extensively exposed to bacterial pathogens, i.e. those of the lungs and the gastrointestinal tract.
Notes
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References
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