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REVISTA DE BIOLOGIA E CIÊNCIAS DA TERRA Suplemento Especial - Número 1 - 2º Semestre 2006 Characterization of Chromobacterium violaceum isolated from Adriana Almeida Antunes.1,2, Maria Luiza Ribeiro Brito Silva 1,2, Carlos Alberto Alves da Silva2,3, ABSTRACT
Chromobacterium violaceum is a facultative anaerobic, motile, gram-negative bacillus, inhabitant
of soil and water, and the most strains produce a violet pigment, violacein. Studies were carried out
a strain of Chromobacterium violaceum, UCP 1489 isolated from Paca river of Pernambuco,
evaluating the growth in different culture media, the biochemical characteristics, the susceptibility
to the antimicrobian, and the biosurfactant production. The isolates was demonstrated exuberant
growth on Luria Bertani medium, supplemented with glucose, negative for the following tests: the
urea, manitol, mannose, lactose, indol, and lysine; and positive for gelatinase, glucose, motility,
catalase, sucrose, oxidase and fructose. The strain showed resistant to cephalothin, imipenem,
chloramphenicol, ampicillin and amicacin. The biosurfactant production by Chromobacterium
violaceum, UCP 1489, showed reduction of superficial tension of water from 71 mNm-1 to
26mN/m-1, indicating high biotechnological potential for producing bioactive agent.
Key words: Chromobacterium violaceum, Paca river, antibiotical resistence, biosurfactant
production
RESUMO
Chromobacterium violaceum é um anaeróbio facultativo, móvel, bacilo gram-negativo, habita solos
e águas, e a maioria das espécies produzem um pigmento violeta. Estudos foram conduzidos com o
isolado Chromobacterium violaceum, UCP 1489 isolado do rio Paca, Pernmabuco; sendo avaliado
o crescimento em diferentes meios de cultura, as características bioquímicas, a susceptibilidadeaos
antimicrobianos e a produção de biossurfactante. O isolado demonstrou crescimento exuberante no
meio Luria Bertani suplementado com glicose, negativo para os seguintes testes: uréia, manitol,
manose, lactose, indol e lisina; e positivo para gelatinase, glicose, motilidade, catalase, sacarose,
oxidase e frutose. A amostra apresentou resistência para: cefalotina, imipenem, cloranfenicol,
ampicilina e amicacina. A produção de biossurfactante pelo Chromobacterium violaceum UCP
1489, apresentou uma redução da tensão superficial da água de 71 mNm-1 para 26mN/m-1,
indicando alto potencial biotecnológico para a produção de agentes bioativos.
Key words: Chromobacterium violaceum, rio Paca, resistência antibiótica, produção de
biossurfactante.
1 INTRODUCTION
subtropical regions, including soil, water and component of the Amazon Region. As a free- living microorganism, C. violaceum is exposed to a series of variable conditions, such as different sources and abundance of as oil-water or air-water interfaces (HEALY nutrients, changes in temperature and pH, property of reducing surface and interfacial potential applications in the recovery of oil, violacein, a purple pigment first isolated in identification, semi-defined medium for the been introduced as a therapeutic compound growth, and biochemical characterization of for dermatological purposes (CALDAS et al., the new strain compared with standard strain 1978). Violacein also exhibits antimicrobial of C. violaceum. The biotechnological potential of biosurfactant production of the both strains were investigate using some (SOUZA et al., 1999), Trypanosoma cruzi selected oils (corn oil, soy oil and canola oil) DESSAUX et al., 2004), Leishmania sp. (LEON et al, 2001), antiviral (DURÁN et al., 2 MATERIAL AND METHODS
2001), anticancer activity (UEDA et al., 1994; MELO et al, 2000, DIAS JR. et al., 2002), 2.1 Microorganism:
and is reported to have other bactericidal 1983; DURÁN, 1990). Otherwise, C. violaceum was first described by Bergonzini, river located in the city of Camaragibe, (1881 in Johnson et al, 1971). The first recognized human cases occurred in Malaysia kept in bottle amber and submitted to the in 1927 and reported by Johnson et al., (1971) microorganisms. Technique of the multiple countries and regions where infection with C. pipes was used, being carried through the violaceum has been recognized include presumptive assay, using 10 mL of the sample Argentina, Brazil, India, Malaysia, Senegal, and inoculated in Petri dishes containing Singapore, Thailand, Vietnam, and Northern nutritive agar, and incubated at 37 oC, for 24 Australia. In the past, it is likely that C. violaceum was isolated but not recognized as microorganisms isolated from contaminated water produced violet colonies, characterized 1941; SNEATH et al., 1953). The bacteria are as Chromobacterium violaceum. The strain the largest responsible for the production of was deposited in the Bank of cultures of the have been isolated of the soil, sea water, Sciences of UNICAP/PE, Brazil, maintained Growth and Biochemical Characterization:
have both clearly defined hydrophilic and molecular film, orderly in the interfaces, it dishes for testing different culture media: reduces the tension interfacial and superficial being responsible for the only properties of gelatin – 10,5g/L; brain heart infusion g/L; the surfactants. These moieties partition peptone of meat – 11g/L; dextrose – 2g/L; preferentially at the interface between fluid sodium chloride – 5g/L; phosphate dibasic of phases with different degrees of polarity, such [Triptone – 10g/L; sodium chloride – 5g/L; hours and the reading of the halos formed yeast extract – 5g/L; agar – 15g/L], LB with around the disks was accomplished with a glucose [Triptone – 10g/L; sodium chloride – halometer, expressed in sensible or resistant. 5g/L; yeast extract – 5g/L; glucose – 5g/L; All tests were compared with C. violaceum agar – 15g/L], Müeller Hinton agar [infusion of meat – 2g/L; casein hydrolyzed – 17,5g/L; starch – 1,5g/L; agar – 15g/L], King A agar [peptone – 20g/L; magnesium chloride – 1,4g/L; sulfato of ammonium – 10g/L; agar – The strains C. violaceum UCP 1489 15g/L], Mac Conkey ágar (MC) [peptone – and was maintained and transferred to Luria 19g/L; lactose – 10g/L; sais biliares – 1g/L; sodium chloride – 5g/L; crystal violet – biosurfactant production was carried out in 0,001g/L; red neutro – 0,03g/L; agar – 15g/L], M9 agar [NH4Cl – 1g/L, Na2HPO4 – containing 50mL of LB liquid Luria Bertani medium [triptona - (10g/L); yeast extract - glycerol ou asparagine – 5g/L; magnesium (5g/L); NaCl - (5g/L); glucose - (5g/L). sulfate 1M – 1mL; calcium chloride 0,01M – Glucose (5g/L), added of corn oil, soy oil and 10mL; agar – 15g/L] Cled agar [peptone – canola oil, maintained in orbital shaker 150 4g/l; meat extract – 3g/L; tryptone – 4,0g/L; rpm, at 30°C, in different periods of time lactose – 10g/L; l-cystine – 1,128 g/L; blue of bromothimol – 0,02g/L; agar – 15g/L], BTB Lactose agar [meat extract – 5g/L; peptone – 10g/L; lactose – 10g/L; blue of bromothimol – 0,008g/L; agar – 15g/L] and incubated for 48 hours at 30°C. For characterization of the strain, tests of fermentative activity were accomplished (glucose, mannose, fructose, 3 RESULTS AND DISCUSSIONS
sucrose, lactose and manitol), and urea, indol, lysine, catalyses, gelatinize and oxidize tests disk CECON, and motility test (BALOWS et showed that the isolated of C. violaceum, UCP 1489, presented growth in the following glucose and LB with glucose, Müeller Hinton, Infusion), as well as in the introduction of new medium as: Cled agar, King A and BTB described by Kirby-Bauer. Initially the isolate Lactose. It was still observed, that the lineage was grown in LB solid medium for 48 hours of C. violaceum, UCP 1489, grew in all of the to 30oC. The isolated was transferred for test tubes containing 5mL of the nutritious broth tested means, the LB medium with glucose (TSB), and incubated for 2 hours at 30oC. stood out as middle of larger growth for the After this period brackets of the nutritious lineage UCP 1489, being confirmed by all of the lineages of C. violaceum used by Antunes plates containing Müeller Hinton ágar, with et al, (2006). It was still observed, that the the aid of sterile swab. Afterwards, disks of 6 isolated of C. violaceum, UCP 1489, grew in millimeters of diameter of the antibiotics all of the culture medium used (Table 1). The (CECON) amicacin, ampiciclina, aztreonam, isolated studied, UCP 1489, presented white gentamicina, imipenem, nitrofurantoina, and tetraciclina, were deposited in the surface of Trabulsi et al., (1999), Myers et al., (1992) the medium in a halfway. The plates were and Sorenson et al, (1985), not all stains are incubated to the temperature of 30oC for 18 pigmented and nonpigmented colonies on the et al, 1983). Creczynski-Pasa and Antonio, Table 2. Result of the biochemical tests accomplished
(2004), tell that the results of the good growth with the isolated of Chromobacterium violaceum, UCP 1489 compared with C. violaceum IUCP 1471 C. violaceum in the different culture medium are leaning for the literature and they consider the microorganism as a bacterium no demanding. The authors say that C. violaceum is able to live in both aerobic and anaerobic conditions. In aerobic conditions, C. violaceum is able to grow in a minimal medium with simple sugars, such as glucose, Meyerhoff, tricarboxylic acid and glyoxylate Like this, the obtained results confirm Luria Bertani medium with glucose, and was Table 1. . Growth of Chromobacterium
violaceum, UCP 1489 compared with C. violaceum IUCP1471 (standard), on different culture media. resistance to the ampicillin and cephalothin, this isolated also presented resistance to amicacin (Table 3). The largest sensibility halos were observed for ciprofloxacin, with Sorenson et al., (1985); Kaufman et al., (1986); Suarez et al., (1986); Georghiou et al, (1989); Ponte e Jenkins, (1982) ; Hassan et al, (1993), C. violaceum should be considered cephalothin, cefamandole, and vancomycin. resistance is observe by C. violaceum to susceptible to chloramphenicol, tetracycline, presented in the table 3, being confirmed in and the aminoglycosides (Yo et al, 1999), the literature by several authors. (BAILEY corroborating with the obtained results by Antunes et al., (2006), with 12 of the studied strains, except for the isolated UCP 1489, from Pernambuco, that was shown resistant to the chloramphenicol, tetracycline, and the Chromobacterium violaceum, ATCC 12472 (standard) local de origem do isolado e as condições using different substrates and the superficial tensions of each condition for fermentation. ambientais apresentam uma relação direta com a susceptibilidade aos antimicrobianos. Table 3. Result of the antimicrobial activity with the
isolated of Chromobacterium violaceum, UCP 1489 compared with C. violaceum IUCP1471 (standard). 4 CONCLUSIONS
confirmed to be Chromobacterium violaceum and shown similar characteristics with C. violaceum UCP1471, and is the first description of isolation of C. violaceum in different profile of antimicrobial resistence, evidencing genetic variability. C. violaceum The superficial tensions of the strain UCP 1489 showed higher ability of biosurfactant standard strain UCP 1471, and besides being mN/m respectively to soy oil, glucose, corn a good surfactant, has attractive properties as oil and canola substrates, during 24 hours. an emulsifier, two characteristics that are not easily found together in other kinds of biosurfactants. Chromobacterium violaceum UCP 1489 show the best results of reduction of superficial tension of water from 71 mN m-1 to 26mN/m, AGRADECIMENTOS
and indicated high biotechnological potential financiadoras CNPq (Processos Nº141158/02-6 e Nº3096610/2003-6), CTPETRO, FINEP Chromobacterium violaceum, (UCP 1489) using different substrates and the superficial tensions of each REFERENCES
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