JAC Advance Access published online on June 13, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn239
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Research letter |
Lysates of Locusta migratoria brain exhibit potent broad-spectrum antibacterial activity
School of Biological and Chemical Sciences, Birkbeck College, University of London, London WC1E 7HX, England, UK
* Corresponding author. Tel: +44-207-631-6237; Fax: +44-207-631-6246; E-mail: n.khan{at}sbc.bbk.ac.uk
Key Words: insects , antibiotic , orthoptera
In recent years, the burden of infectious diseases has exacerbated with the emergence of antimicrobial resistance and lessening efficacy of the available antimicrobial compounds.1 Vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus (MRSA) have recently emerged as major threats to public health. In fact, the drug of choice to treat MRSA is vancomycin, but it has emerged recently that vancomycin-intermediate S. aureus is exhibiting some levels of resistance against vancomycin. In addition, there are reports from the USA of MRSA showing high-level resistance to glycopeptides due to acquisition of the vanA gene complex.2
Thus, new antimicrobial agents are required to meet the challenge posed by the emergence of multidrug-resistant microorganisms. The search for new antibiotic compounds originating from natural resources is an important research area. Insects are the largest (80% of all fauna) and most widespread group within the Animal Kingdom, and synthesize a variety of antibacterial compounds, including the defensins and cecropins, as part of their innate immune response to infection.3–5 Up to 50% of the reported antimicrobial substances are identified in invertebrates (predominately in insects).3–5 We investigated the possible presence of antimicrobial activity in the various tissues of a locust, Locusta migratoria.
Briefly, locust brains were isolated by making a sagittal cut through the base of the left antenna as described previously.6 Brains were pooled in batches of 30, resuspended in 500 µL PBS and subjected to four cycles of freeze–thaw. The brains were disrupted using a Cole-Parmer cup-horn sonicator and tested for antimicrobial activity. In addition, fat body and dorsal longitudinal flight muscle tissue (equivalent to that of 30 pooled brains) were removed and lysates prepared. A 100 µL aliquot of brain lysate (equivalent to six brains) or lysate from muscle or fat body was incubated with 
106 bacterial cells [Escherichia coli K1 (a cerebrospinal fluid isolate from a meningitis patient; 018:K1:H7), Staphylococcus epidermidis, S. aureus and MRSA]. All bacteria tested here are clinical isolates that were obtained from Birkbeck culture collection (available upon request). Tubes were incubated for 2 h at 37°C, and bacterial counts were determined by plating on nutrient agar plates. For controls, bacteria were incubated in PBS without the brain lysates. The percentage bactericidal effect was determined as the percentage of bacteria surviving relative to the control as follows: 100 – (cfu in brain lysates/cfu in PBS x 100). The results revealed that brain lysates killed more than 99% E. coli K1, while muscle and fat body lysates had no effect (Table 1), suggesting that the active compound(s) are specific to the brain tissue. The brain lysates had broad-spectrum activity as demonstrated by potent bactericidal effects against E. coli K1, MRSA, S. aureus and S. epidermidis (Table 1).
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To determine the potency of the compound(s) in the locust brain lysates, serial dilutions of the brain lysates were made and tested against E. coli K1. Aliquots of brain lysate ranging from 5, 10, 50 and 100 µL were bactericidal, i.e. 99.82%, 99.98%, 99.99% and 99.997%, respectively, whereas volumes of brain lysate <1 µL did not show bactericidal activity. When the brain lysates are heated at 100°C for 10 min, the potency remains the same. Perhaps, there is more than one compound present in the brain lysates, and heating activates one or more of these compounds, or heating increases the efficiency of extraction, thus increasing the antimicrobial potency of the lysates above that of non-heated brain lysates. When brain lysates were treated with 1% SDS and boiled as above, the bactericidal activity disappeared (Table 1), suggesting that the active components of brain lysates are proteinaceous in nature.
To determine the effect of the locust brain lysates on human brain microvascular endothelial cells (HBMECs), cytotoxicity assays were performed using a cytotoxicity detection kit (Roche Applied Science) as described previously.7 HBMECs grown in 24-well plates were incubated with different volumes of brain lysate from 25 to 200 µL for 24 h at 37°C in a 5% CO2 incubator. Next, the supernatant of each well was collected and percentage cell death determined. It was demonstrated that the locust brain lysates had no cytotoxic effect on HBMECs (data not shown), suggesting that the putative target(s) for the active component(s) may be absent in eukaryotes. In support, our preliminary studies suggest that the brain lysates have no amoebicidal effects against Acanthamoeba castellanii and Balamuthia mandrillaris (N. A. K., K. O. and G. J. G., unpublished data).
Future work will include identification of the nature of the compound(s), such as the chemical structure and properties, as well as its mode of action, and whether this is a known or novel compound.
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None to declare.
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This work was supported by grants from the Faculty Research Fund, Birkbeck, University of London, and The Royal Society.
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The authors are grateful to Mary Lightfoot (Birkbeck, University of London) for providing skilled technical assistance.
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1 . World Health Organization. Mortality Report. (2002) (1 October 2007, date last accessed).
2 . Tenover FC, McDonald LC. Vancomycin-resistant staphylococci and enterococci: epidemiology and control. Curr Opin Infect Dis (2005) 18:300–5.[Web of Science][Medline]
3 . Salzet M. Neuropeptide-derived antimicrobial peptides from invertebrates for biomedical applications. Curr Med Chem (2005) 12:3055–61.[CrossRef][Web of Science][Medline]
4 . Meylaers K, Cerstiaent A, Vierstraete E, et al. Antimicrobial compounds of low molecular mass are constitutively present in insects: characterisation of β-alanyl-tyrosine. Curr Pharm Des (2003) 9:159–74.[CrossRef][Web of Science][Medline]
5 . Vizoli J, Salzet M. Antimicrobial peptide from animals: focus on invertebrates. Trends Pharmacol Sci (2002) 23:494–6.[CrossRef][Medline]
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Mokri-Moayyed B, Goldsworthy G, Khan NA. Development of a novel ex vivo insect model for studying virulence determinants of Escherichia coli K1. J Med Microbiol (2008) 57:106–10.
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Sissons J, Kim KS, Stins M, et al. Acanthamoeba castellanii induces host cell death via a phosphatidylinositol 3-kinase-dependent mechanism. Infect Immun (2005) 73:2704–8.
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