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 wereinvestigate 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),
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 livein 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).
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,
and indicated high biotechnological potential
financiadoras CNPq (Processos Nº141158/02-6 e Nº3096610/2003-6), CTPETRO, FINEP
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2Núcleo de Pesquisas em Ciências Ambientais – UNICAP,  Doutorado em Ciências Biológicas, UFPE, Recife, PE;  DNúcleo de Pesquisas em Ciências Ambientais, UNICAP, Recife, PE;  Prof. Dr. Adjunto I, Departamento de Química da Universidade Católica de Pernambuco, 50.050-900 Recife-PE, Brasil  Profa. Dra. Adjunto III e Prof. Adjunto I Departamento de Química da Universidade Católica de Pernambuco, 50.050-900 Recife-PE, Brasil
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