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MICROBIOLOGÍA ORIGINAL ARTICLE

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noamer i Antimicrobial activity of alternatus venom

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Rev from the Northeast of Argentine Vol. 50, Nos. 3 y 4 July - September. 2008 October - December. 2008 pp. 79 - 82 Soledad Bustillo,* Laura C. Leiva,* Luis Merino,* Ofelia Acosta,* Elisa Bal de Kier Joffé,** Jorge O. Gorodner*

ABSTRACT. This study was undertaken to evaluate the antimicro- RESUMEN. La actividad antimicrobiana del veneno Bothrops al- bial activity on Gram-negative and Gram-positive bacteria of the ternatus, especie responsable de la mayoría de los accidentes ofídi- venom from Bothrops alternatus, specie responsible for most of cos en el Nordeste de , fue evaluada sobre bacterias in North-eastern Argentina. This activity was investigat- Gram-negativas y Gram-positivas. Esta actividad fue ensayada so- ed against Escherichia coli (ATCC 25922), Staphylococcus aureus bre Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853), and En- (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853), y Ente- terococcus faecalis (ATCC 29212). Inhibitory halos values and re- rococcus faecalis (ATCC 29212). Se determinaron los valores de duction of colony-forming unites (CFU) by dilution plate technique halos de inhibición y la reducción de formación de unidades for- were determined. The bactericidal activity was higher for E. coli madoras de colonias (CFU) por el método de dilución en placa. La and S. aureus, in comparison to that for P. aeruginosa. E. faecalis actividad bactericida fue mayor para E. coli y S. aureus; en compa- was the most resistant organism tested to the action of venom solu- ración con P. aeruginosa, E. faecalis fue el microorganismo eva- tions. The demonstrated bactericidal property of B. alternatus ven- luado más resistente a la acción de las soluciones de veneno. La om highlight it as a promising candidate for further studies accord- propiedad bactericida demostrada del veneno de B. alternatus es ing to detect which component, or components, are responsible of importante para poder luego determinar cuál componente, o com- this activity, in order to then investigate their potential use as anti- ponentes, son responsables de dicha actividad e investigar su poten- microbial agent. cial uso como agente antimicrobiano.

Key words: Antimicrobial, bactericidal, venom, Bothrops al- Palabras clave: Antimicrobiana, bactericida, veneno de serpiente, ternatus. Bothrops alternatus.

INTRODUCTION has been more clearly illustrated. For instance, Tashima et al. (2002), has demonstrated that and In Latin America, the majority of snakebites are caused venoms contained around 30 proteins by of the Bothrops (Cardoso, 1985; Gutiér- in the range of 7-110 kDa. rez, 1995). Analysis of venom proteins has consistently shown B. alternatus (yarará grande) and B. diporus (yarará high levels of intra and interspecific variation and there chica), that belong to the family, are re- are a small but increasing number of studies that strongly sponsible for most snake poisonings in North East of Ar- support the idea that this variation reflects local adapta- gentina, respectively (Acosta et al, 1998). tion for feeding on different prey (Tashima et al, 2008). Snake venoms are a wide mixture of proteins and pep- This active proteins and peptides are usually similar in tides (90-95%), also including amino acids, nucleotides, structure but not identical to that of prey physiological free lipids, carbohydrates and metallic elements bound to systems. In the last decade, several com- proteins (5%) (Heise et al, 1995). Viperid venoms in par- pounds were used as important tools for the understanding ticular have long been recognized for the complexity of of human physiological systems due to their similarity to their molecular composition. More recently, with the ad- physiological molecules (Meier et al, 1991, Vogel et al, vent of proteomics, the extent of thewww.medigraphic.com venom complexity 2004) and have yielded extensive important information on biological systems and insights into medical problems (Balsinde et al, 1999). Envenomation is a process associated with a low inci- dence of bacterial infection. This feature could indicate

* Universidad Nacional del Nordeste. Corrientes. Argentina. the presence of antibacterial molecules in the snake ven- ** Instituto de Oncología “Ángel H Roffo”. Área Investigación. Buenos Aires. Argentina. oms that would protect the during feeding. (De Lima et al, 2005) First version received: February 28, 2008; first version revised: May 21, 2008, September 19, 2008 and November 24, 2008 ; Second version received in revised form: December 16, 2008. Several antimicrobial studies involving venom of Accepted January 12, 2009. snakes from other regions, have described the bactericidal 80 Bustillo S et al Antimicrobial activity of Bothrops alternatus venom from the Northeast of Argentine Rev Latinoam Microbiol 2008; 50 (3-4): 79-82

activity exhibited by their enzymes, such as PLA2, from bacteria were harvested from fresh agar plates and their (Páramo et al, 1998; Lomonte et al, concentrations were adjusted to 4 x 106 colony-forming a,b 5 1999 ; Santamaría et al, 2004 and 2005); PLA2, from Bo- units (CFU)/ml. 100 µl, containing 4 x 10 CFU, were in- throps neuwiedi pauloensis (Soares et al, 2000; Rodríguez cubated for 60 min at 37 °C with different concentrations

V. et al, 2004); PLA2, from (Roberto of the crude venom solutions. The live bacteria were et al, 2004); PLA2, from (Soares et al, counted on trypticase soy agar plates. Assays were per- a 2001 ); PLA2, from (Stábeli et al, formed in triplicate. Values were expressed as mean ± SD, 2005); PLA2, from terrificus ( Soares et and statistically compared using ANOVA. al, 2001b; Toyama et al, 2003) metalloproteases, from Bo- throps jararacussu (Mazzi et al, 2004); L-amino acid oxi- RESULTS dase, from Bothrops alternatus (Stábeli et al, 2004); L- amino acid oxidase, from Bothrops pirajai (Izidoro et al, The bactericidal effects of Bothrops alternatus venom 2006). Microbicidal peptides have emerged as promising toward two Gram-positive bacteria (E. faecalis and S. au- therapeutic alternatives to cope with the increasing rates reus) and against two Gram-negative bacteria (E. coli and of antibiotic resistance encountered worldwide (Hancock P. aeruginosa) were tested by measuring the inhibition et al, 1999). halos. Our findings demonstrated that there is a dose-de- In spite of several works published on this topic, the an- pendent response of this activity to venom concentra- timicrobial activity of Bothrops alternatus venom from tions. Argentina has not been yet described. Very effective bactericidal activity was observed for S. The aim of this preliminary work was to study the bac- aureus and E. coli (Figure 1). Both strains were the most tericidal activity of Bothrops alternatus venom, against susceptible Gram-positive and Gram-negative bacteria, re- Gram-negative and Gram-positive bacteria in order to spectively. Even at the lowest concentrations tested evaluate the potential use of its components as antimicro- (0.312 mg/ml), the venom displayed significant bacteri- bial agents. cidal activity against S. aureus, but was ineffective against E coli. At concentrations of 0.625 to 10 mg/ml MATERIALS AND METHODS similar inhibition halos were observed for both strains. Intermediate potency as bactericidal agent showed B. Venom. Bothrops alternatus crude venom was collect- alternatus venom against P. aeruginosa, and inhibition ed from several snakes from the North East of Argentine, halos were observed from higher concentrations than 2.5 vacuum dried and kept at -20 °C. mg/ml. However, the bothropic venom not exhibited bac- Bacterial strains and growth conditions. The bacterial tericidal activity against E. faecalis even at the highest strains utilized were Escherichia coli (ATCC 25922), Sta- concentration assayed (Figure 1). phylococcus aureus (ATCC 25923), Pseudomonas aeu- We also studied the number of CFU after incubating ruginosa (ATCC 27853), and Enterococcus faecalis live bacteria with various concentrations of venom solu- (ATCC 29212). Culture conditions of bacteria followed tions (Figure 2). Incubation of B. alternatus venom solu- established protocols. Bacteria were harvested and sus- tions with both Gram-negative bacteria and S. aureus at pended in 0.01 M sodium phosphate, pH 7.4 and immedi- 37 °C resulted in significant bacterial killing in the 1.25- ately subjected to bactericidal assays. 10 mg/ml range of concentration. The bactericidal activi- Bactericidal-activity assays. Two bactericidal assays ty was higher for E. coli and S. aureus, in comparison to were carried out: that observed for P. aeruginosa. E. faecalis was the most 1. Inhibitory Halo Method. 50 µl of the tested bacteria resistant organism tested to the action of venom solu- suspension were homogeneously mixed on to Petri dishes tions (Figure 2). containing 15 ml of the medium. Holeswww.medigraphic.com were aseptically bored into the agar with a hollow punch and were filled up DISCUSSION with 10 µl of different concentration of crude venom solu- tions. The plates were examined after incubation at 37 ºC This work demonstrates that Bothrops alternatus ven- for 24 h. Diameters of the growth-inhibition halos were om displays dose-dependent bactericidal activity against a measured. Assays were performed in triplicate. Values variety of Gram-negative and Gram-positive bacteria. were expressed as mean ± SD, and statistically compared Both assayed methods, Inhibitory Halo Method and Di- using ANOVA. lution plate technique, demonstrated that E. coli and S. 2. Dilution Plate Technique. Bactericidal activity was aureus are the most susceptibly bacteria to B. alternatus determined as described by Páramo et al. (1998). Briefly, venom action. Bustillo S et al Antimicrobial activity of Bothrops alternatus venom from the Northeast of Argentine 81 Rev Latinoam Microbiol 2008; 50 (3-4): 79-82

10 * * 9 * * 8 * Significantly different to untreat- * * * ed control cultures, (p < 0.001). 7 * * * 6 * Figure 1. Bactericidal activity was 5 * estimated indirectly, by comparing * * the diameters of inhibition halos 4 caused by the B. alternatus ven- 3 om. against Staphylococcus au- Inhibition halos (mm) reus (ATCC 25923), Escherichia 2 coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853) and En- 1 terococcus faecalis (ATCC 29212). 0 Values are means ± S.E. (3 repli- 10 5 2.5 1.25 0.625 0.3125 0 cas/treatment). Data were analyzed Venom (mg/ml) by one way ANOVA, Tukey test and p < 0.001 was considered sig- E. coli S. aureus P. aeruginosa E. faecalis nificant.

100 90 80 * 70 60 * Significantly different to untreat-

Survival) ed control cultures, (p < 0.001). 50 *

(% 40 * * Figure 2. Bactericidal activity of 30 B. alternatus venom was estimated CFU * * * by the dilution plate technique. Sur- 20 viving bacteria were counted. CFU, 10 * ** colony-forming units. Bars repre- 0 sents mean ± SD of triplicate 5 2.5 1.25 0.625 counts. Venom (mg/ml) Data were analyzed by one way ANOVA, Tukey test and p < 0.001 E. coli S. aureus P. aeruginosa E. faecalis was considered significant.

On the other hand, B. alternatus venom showed an in- bacterial and antifungal peptides from natural sources termediate potency as bactericidal agentwww.medigraphic.com against P. aerug- have attracted the attention in recent years. The impor- inosa, and neither against E. faecalis, being the most resis- tance of bactericidal action exhibited by snake toxins is tant bacteria. not only for a better understanding of the ophidian en- Differences in individual susceptibilities were evi- venomation mechanism, but also due to their biotech- dent, in that sense, B alternatus venom exhibited nological potential as model for therapeutic agents. strongly bactericidal activity on S. aureus, while it was In conclusion, the present study provides the first ex- not able to kill E. faecalis in spite of both being Gram- perimentalESTE DOCUMENTO evidence ES that ELABORADO B. alternatus POR venom MEDIGRA- from Ar- positive bacteria. gentinaPHIC trigger antibacterial functions in vitro. Further It is has become increasingly clear that due to the de- studies will be required in order to elucidate the mecha- velopment of antibiotic-resistant microbes, new anti- nism/s involved in this toxicity. 82 Bustillo S et al Antimicrobial activity of Bothrops alternatus venom from the Northeast of Argentine Rev Latinoam Microbiol 2008; 50 (3-4): 79-82

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Asp49 myotoxic phospholipases A2 from Bothrops asper snake venom. Synthetic Lys49 myotoxin II-(115-129)-peptidewww.medigraphic.com iden- Soledad Bustillo tifies its bactericidal region. Eur. J. Biochem. 253:452-461. Tel.: 54-03783-457996-Int. 112; 15. Roberto, P. G., Kashima, S., Marcussi, S., Pereira, J., Astol.-Fil- fax: 54-03783-473930 ho, S., Nomizo, A., Giglio, J. R., Fontes, M. R. M., Soares, A. E-mail address: [email protected]