462

Revista de Biología Tropical, ISSN electrónico: 2215-2075 Vol. 69(2): 462-471, April-June 2021 (Published Apr. 01, 2021)

Antimicrobial activity of bacteria isolated from tissue of the coral Palythoa

caribaeorum (Zoantharia: Sphenopidae) from Paraíba, Brazil coastal reefs

Jalcinês C. Pereira

Krystyna Gorlach-Lira

Bruno O. de Veras

1. Federal University of Paraiba, Post-graduation Program in Cell and Molecular Biology, João Pessoa, Brazil;

jalcinescosta@gmail.com

2. Federal University of Paraiba, Departament of Molecular Biology, João Pessoa, Brazil; kglira@yahoo.com

3. Federal University of Pernambuco, Post-graduation Program in Tropical Medicine, Recife, Brazil;

brunooveras@hotmail.com; *Correspondence

Received 22-II-2020. Corrected 21-X-2020. Accepted 11-II-2021.

ABSTRACT

Introduction: The coral-associated bacteria with antimicrobial activity may be important to promote the health

of their host through various interactions, and may be explored as a source of new bioactive compounds.

Objective: To analyze the antimicrobial activity of bacteria associated with the zoanthid Palythoa caribaeorum

from the coral reefs of Carapibus, Paraiba state, Brazil. Methods: The phylogenetic analysis of the bacteria was

conducted based on partial sequences of the 16S rRNA gene using molecular and bioinformatics tools. The anti-

microbial activity of the 49 isolates was tested against four bacterial strains and one yeast strain: Bacillus cereus

(CCT0198), Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa

and Candida albicans (ATCC 10231). The antibiosis and antibiogram assays were conducted and the Minimal

Inhibitory Concentration (MIC) was determined by the microdilution method. Results: The bacterial isolates

belonged to Firmicutes phylum (84 % of the isolates) and the Proteobacteria phylum (16 % of the isolates).

Among the 49 isolates five genera were found, with the Bacillus genus being the most abundant (82 % of the iso-

lates), followed by Vibrio (10 %), Pseudomonas (4 %), Staphylococcus (2 %) and Alteromonas (2 %). Antibiosis

test revealed that 16 isolates (33 %) showed antimicrobial activity against one or more of five tested reference

strains. The highest number of antagonistic bacteria were found in the Bacillus genus (12 isolates), followed

by Vibrio (three isolates) and Pseudomonas (one isolate) genera. The B. subtilis NC8 was the only isolate that

inhibited all tested strains in the antibiosis assay. However, antibiogram test with post-culture cell-free superna-

tant of NC8 isolate showed the inhibition of only B. cereus, S. aureus and C. albicans, and the lyophilized and

dialyzed material of this isolate inhibited only B. cereus. The lyophilized material showed bacteriostatic activity

against B. cereus, with a MIC value of 125 μg/μl, and in the cytotoxicity assay, the hemolysis value was of 4.8

%, indicating its low cytotoxicity. Conclusions: The results show the antimicrobial potential of some bacterial

isolates associated with the P. caribaeourum tissue, especially those belonged to Bacillus genus.

Key words: antimicrobial substances; marine bacteria; Bacillus; zoanthid; Palythoa caribaeorum.

Pereira, J.C., Gorlach-Lira, K. & de Veras, B.O. (2021).

Antimicrobial activity of bacteria isolated from tissue of

the coral Palythoa caribaeorum (Zoantharia: Sphenopidae)

from Paraíba, Brazil coastal reefs. Revista de Biología

Tropical, 69(2), 462-471. DOI 10.15517/rbt.v69i2.40809

Reef environments are restricted to the

tropical regions and they are spread over 3 000

km along the coast in Brazil, showing high rate

of endemic coral species (Francini-Filho et al.,

2013; Leão et al., 2016). Coral reefs are dis-

tributed in the state of Paraiba over the entire

coastal stretch of 138 km (Costa, Sassi, Costa,

& Brito, 2007).

DOI 10.15517/rbt.v69i2.40809

463

Revista de Biología Tropical, ISSN electrónico: 2215-2075, Vol. 69(2): 462-471, April-June 2021 (Published Apr. 01, 2021)

Palythoa caribaeorum is a species of typi-

cally sessile colonial zoanthid found frequently

in coral reefs along the coast of the Atlantic

Ocean and oceanic islands, being one of the

most representative species of the several coral

reefs of Brazil, Caribbean and Florida. Zoan-

thids, such as P. caribaeorum, may occupy

large surface area of disturbed reefs since their

high physiological tolerance and reproductive

rates (Francini-Filho et al., 2013; Silva et al.,

2015; Durante, Cruz, & Lotufo, 2018).

In various regions of Northeastern Brazil,

including the Paraiba coast, P. caribaeorum

is one of the most abundant zoanthid in the

reef environments (Costa, Sassi, Gorlach-Lira,

Lajeunesse, & Fitt, 2013; Melo, Lins, & Eloy,

2014; Araújo, Gorlach-Lira, Medeiros, & Sassi,

2015; Silva, 2015). The occupational success

of this species is mainly due to competitive

strategies and rapid growth, even in unfavor-

able conditions such as high sedimentation

(Castro, Segal, Negrão, & Calderon, 2012).

The microbiota associated with P. caribaeo-

rum is still little known. According to Pereira,

Palermo, Carlos, & Ottoboni (2017), the Alp-

haproteobacteria were abundant in the mucus

of this species, while Silva (2015) revealed

that the majority of bacterial isolates from P.

caribaeorum tissue belonged to the Bacilli

class of Firmicutes phylum, followed by the

Gammaproteobacteria.

Recent works has demonstrated the impor-

tance of the bacterial community for the health,

development and resilience of various spe-

cies of corals and zoanthids (Pham, Wiese,

Wenzel-Storjohann, & Imhoff, 2016; Pereira

et al., 2017). According to these studies, the

antimicrobial activity performed by associ-

ated bacteria promotes the health of its host

through ecological interactions, and may repre-

sent a source for obtaining new bioactive com-

pounds, which can be used in the production

of new drugs. Bacillus was found to be one of

the leading genera with antimicrobial activity

among bacteria associated with corals (Pham

et al., 2016; Pereira et al., 2017). The bioactive

compounds with antimicrobial properties of

marine Bacillus species have been extensively

reviewed by Mondol, Sin, & Islam (2013).

Since several studies (Li et al., 2011; Li et

al., 2012; Pereira et al., 2017; Mickymaray et

al., 2018) show that bacteria associated with

corals might be promissory producers of bio-

active compounds, we aimed in this work to

perform phylogenetic analysis and to analyze

antimicrobial activity of bacteria isolated from

the tissue of P. caribaeorum from the reefs of

Carapibus, Paraiba state, Brazil.

MATERIALS AND METHODS

Bacterial isolates: The bacteria were iso-

lated from healthy and necrotic tissue from

the zoanthid Palythoa caribaeorum from the

coastal reefs of Carapibus, Paraíba state, Brazil

(7º17’59.14” S & 34º47’45” W). The isolation

procedure was described by Silva (2015).

DNA extraction and amplification: Bac-

terial isolates were incubated in Brain and

Heart Infusion Broth (BHI) at 37 ºC for 48

hours. The extraction of genomic DNA from

bacterial isolates was performed using the KIT

HiPura TM Miniprep (HiMedia), according to

the manufacturer’s instructions. The 16S rRNA

gene was amplified in the thermocycler (Pri-

mus, USA) using the following universal prim-

ers (50 pmol): forward 26F: 5′- GAG TTT GAT

CMT GGC TCA G - 3′ and reverse 1492R:

5′ - ACG GCT ACC TTG TTA CGA CTT - 3′

(Lane, 1991), 200 ng of genomic DNA and the

Master Mix PCR kit (Promega), according to

the manufacturer’s instructions. The amplifica-

tion and purification of the 16S rRNA were

done as described by Silva (2015).

Phylogenetic sequencing and analysis:

The sequencing of the samples was carried

out at the Federal University of Pernambuco

Sequencing Platform, Recife, Brazil, using

the automatic sequencer ABI-PRISM 3100

Genetic Analyzer (Applied Biosystems). The

generated sequences were submitted to a query

for similarity with the data deposited in the

GenBank accessed through the NCBI (National

464

Revista de Biología Tropical, ISSN electrónico: 2215-2075 Vol. 69(2): 462-471, April-June 2021 (Published Apr. 01, 2021)

Center for Biotechnology Information) using

the program BLAST-“Basic Local Alignment

Search Tools” (Altschul et al., 1997). Sequenc-

es with more than 97 % similarity were con-

sidered valid. The multiple alignment of the

sequences and the construction of the phylo-

genetic tree were performed using the MEGA

version 6 program (Tamura, Stecher, Peter-

son, Filipski, & Kumar, 2013). The sequences

of antagonistic isolates used to construct the

phylogenetic tree were deposited in the NCBI

sequence database (GenBank access numbers:

MT071323-MT071338).

Antibiosis test: The antimicrobial activity

of isolates was analyzed against the follow-

ing standard strains: Escherichia coli (ATCC

25922), Pseudomonas aeruginosa, Bacillus

cereus (CCT0198), Staphylococcus aureus

(ATCC 25923) and Candida albicans (ATCC

10231). The isolates and standard strains were

grown in the Brain and Heart Infusion Agar

(HiMedia) at 37 ºC for 48 hours. The B. cereus

was incubated for 24 hours due to its rapid

formation of endospores. For the antibiosis

test, the cross-streak method was used, where

each tested isolate was inoculated in a central

line of a Petri dish containing Mueller Hinton

Agar and incubated for 48 hours at 37 ºC. After

this period, the standard strains were inoculated

perpendicularly to the central streak culture.

The cultures were analyzed after 24 hours of

incubation in order to verify possible inhibition

of the growth of standard strains.

Antibiogram test: The diffusion method

in solid medium (antibiogram) on Mueller-Hin-

ton Agar (HiMedia) was used to evaluate anti-

microbial activity of the cell-free supernatant,

lyophilized material and dialysate of the NC8

isolate against the five standard strains men-

tioned above. The NC8 isolate was grown in

Marine Broth (sea water 1 l, peptone 5 g, yeast

extract 2 g) and Mueller Hinton Broth (HiMe-

dia) at 37 ºC for 48 h, and after incubation a

1.5 ml aliquot of the culture was centrifuged

for 10 minutes at 12 000 rpm. 50 µl aliquots of

cell-free supernatant were placed in the wells

on Mueller Hinton Agar previously inocu-

lated with standard strains. After the incubation

period for 24 hours at 37

C, the diameter of

the inhibition halo was measured. All analyzes

were done in duplicate. An antibiogram test

was also conducted using the lyophilized mate-

rial (1.0 g/ml) obtained after lyophilization

process of 400 ml of NC8 isolate supernatant

The lyophilized material was also subjected to

dialysis with a cellulose membrane with a flat

width of 10 mm and 6 mm in diameter (Sigma),

that retain most proteins of molecular weight

12 000 or greater, obtaining the material with

concentration of 0.01 g/ml.

Determination of Minimum Inhibitory

Concentration (MIC): The antimicrobial sus-

ceptibility test performed for the NC8 isolate

was based on the reference method for broth

microdilution tests for aerobic growth bacte-

ria (M27-A6) (NCCLS, 2003). The MIC of

the lyophilized material of the NC8 isolate

was determined against the standard strain

B. cereus in a 96-well microplate, using BHI

broth and nine dilutions (1.95-500 μg/μl) of

the lyophilized material. Each well received

10 μl (3 x 10

CFU/ml) of the standard strain

cell suspension of B. cereus. The aliquots of 10

μl of antibiotic streptomycin sulfate (0.1 g/ml)

was used as a control for the relative evaluation

of the level of inhibition of the tested samples.

Controls were also carried out for the viability

of the tested microorganism and the sterility of

the culture medium. The test was performed in

triplicate. The microplates were incubated at 37

°C in the Thermo Scientific™ Multiskan™ GO

Microplate Spectrophotometer and the optical

density measurements (540 nm) were recorded

every 1hour during 24 hours of incubation. The

Minimum Bactericidal Concentration (MBC)

was determined using 10 μl aliquots collected

from each CIM assay well and inoculated on

Mueller-Hinton agar medium. After incubation

at 37 °C for 24 hours the growth of bacte-

rial colonies was observed. The CBM value

was considered as the lowest concentration of

lyophilized material in which microbial growth

was not detected.

465

Revista de Biología Tropical, ISSN electrónico: 2215-2075, Vol. 69(2): 462-471, April-June 2021 (Published Apr. 01, 2021)

Hemolysis test: Hemolytic activity was

measured by determining human erythrocyte

lysis (hRBCs), provided by the hospital of the

Federal University of Rio Grande do Norte,

Natal, Brazil. Hemolytic activity was tested by

incubating the material subjected to lyophiliza-

tion and dialysis (0.01 g/ml) with erythrocytes

at 2 % of the O- group washed three times

with PBS (phosphate buffered saline), pH 7.2.

Saline solution (NaCl 0.9 %) was used as a

negative control and Triton X-100 (1 %) as a

positive control. The samples were incubated

for 4.8 and 12 h at 37 °C and then centrifuged

at 2 500 rpm for 5 min. Hemolysis was mea-

sured by spectrophotometry at a wavelength of

540 nm in 96 wells microplate, using 200 μl of

samples. All tests were performed in triplicate

and expressed as a percentage (Ahmad, Khan,

Manzoor, & Khan, 2010).

RESULTS

Phylogenetic analysis of bacteria: The

bacterial isolates obtained from healthy (19

isolates) and necrotic tissue (30 isolates) of

the zoanthid P. caribaeorum were identified

on the basis of partial sequences of 16S rRNA.

The isolates showed 98-100 % similarity with

the sequences deposited in the GenBank and

belonged to the phyla: Firmicutes with 84 %

and Proteobacteria with 16 % of the isolates

(Table 1). The Firmicutes phylum was rep-

resented by two families, Bacillaceae and

Staphylococcaceae, distributed in Bacillus and

Staphylococcus genera, respectively (Table

1). The isolates of Proteobacteria phylum

belonged to the families of Pseudomonadace-

ae, Vibrionaceae and Alteromonadaceae, dis-

tributed in genera of Pseudomonas, Vibrio and

Alteromonas, respectively (Table 1).

Antibiosis: The antibiosis test revealed

that among 49 tested isolates, 16 (33 %) exhib-

ited antagonistic activity against at least one of

the five standard strains tested (Fig. 1). Among

the healthy tissue isolates, only two (Bacillus

sp. PS1, Vibrio sp. PS11) showed antagonistic

activity, while 14 isolates from the necrotic tis-

sue were positive in this test, with 11 isolates

belonging to the Bacillus genus, 2 to the Vibrio

genus and 1 to the Pseudomonas genus.

Among antagonistic isolates, the Vibrio

isolates (PS11, PN13, PN35) showed similarity

with the species of V. harveyi and V. campbel-

lii (Table 1, Fig. 1) and Bacillus isolates were

phylogenetically related to B. zhangzhouen-

sis, B. pumilus and B. safensis (PN29, PN70,

PN71, PN73, PN78, PN85, PN89, PN91,

PN92, PN94), one to B. aerius (PS1) and one

to B. subtilis (NC8) (Table 1, Fig. 1).

Among the tested microorganisms, C. albi-

cans and E. coli were more sensitive to the

TABLE 1

Classification of bacterial isolates from the tissue of P. caribaeorum based on partial sequences of 16S rRNA.

The E values were 0.0 and the maximal identity 98-100 %

Phylum/Family/Genus/Species Number of Isolates

Firmicutes/Bacillaceae/Bacillus/B. aerius (NR 118439.1)

B. zhangzhouensis (NR 148786.1)

B. subtilis (NR 04324.1)

B. pumilus (NR 113945.1)

Firmicutes/Staphylococcaceae/Staphylococcus/S. epidermidis (NR 113957.1)

Proteobacteria/Pseudomonadaceae/Pseudomonas/P. stutzeri (NR 103934.2)

Proteobacteria/Vibrionaceae/Vibrio/V. campbellii (NR 119050.1) and V. harveyi (NR 102976.1)

V. proetolyticus (NR118095.1)

Proteobacteria/Alteromonadaceae/Alteromonas/A. macleodii (NR 074797.1)

Total 49

NNR: GenBank access code.

466

Revista de Biología Tropical, ISSN electrónico: 2215-2075 Vol. 69(2): 462-471, April-June 2021 (Published Apr. 01, 2021)

antimicrobial action of most isolates. C. albi-

cans growth was inhibited by 13 isolates, and

among them 10 isolates were Bacillus spp. and

three isolates Vibrio spp. The E. coli growth

was inhibited by seven isolates of Bacillus spp.,

two of Vibrio spp. and one isolate of Pseudo-

monas sp. Among these antagonistic isolates,

eight were obtained from necrotic and two

from healthy tissue of P. caribaeorum.

The growth of S. aureus was inhibited by

four Bacillus isolates of the necrotic tissue of

the zoanthid, and P. aeruginosa growth was

weakly inhibited by two isolates of Bacil-

lus spp. The B. cereus was inhibited only by

Fig. 1. Phylogenetic tree of antagonistic bacteria isolated from the healthy and necrotic tissue of Palythoa caribaeorum

and from bacterial strains of the GenBank based on the comparison of the sequences of RNAr 16S using neighbor-joining

analysis and the Tamura 3-parameter model. The bootstrap values shown in the tree were obtained based on 1 000 replicates.

Access numbers for GenBank strains are shown in parentheses.

467

Revista de Biología Tropical, ISSN electrónico: 2215-2075, Vol. 69(2): 462-471, April-June 2021 (Published Apr. 01, 2021)

the isolate Bacillus subtilis NC8. Among the

bacterial isolates tested in the antibiosis assay,

the NC8 isolate was the only one that showed

growth inhibition of all standard strains in the

antibiosis test.

Antimicrobial activity of Bacillus subti-

lis NC8: The cell-free supernatant of the NC8

isolate showed antimicrobial action against

standard strains of B. cereus, S. aureus and C.

albicans (Table 2). However, the activity of

the lyophilized material (1.0 g/ml) and mate-

rial subjected to dialysis (0.01 g/ml) showed

antibacterial activity in the antibiogram test

only against B. cereus, with no antimicrobial

action against other standard strains. Therefore,

the tests to determine the MIC and MBC were

conducted only against the strain of B. cereus

using lyophilized material.

On the basis of the microdilution test, the

MIC value of lyophilized material was 125 μg/

μl (Fig. 2). The optical density values (540 nm)

revealed that the B. cereus did not show growth

during 24 hours of the test in the presence

of 500.0 μg/μl, 250.0 μg/μl and 125.0 μg/μl

lyophilized material (OD 540 nm: 0.09) (Fig.

2). Concentrations of 62.5 μg/μl, 31.25 and

15.63 μg/μl inhibited the growth of B. cereus

up to 6 hours of incubation, and after this

period the growth of the isolate was detected

Fig. 2. Growth kinetics of standard strain of B. cereus in the presence of lyophilized material (1.96-500 μg/ml) of the

B. subtilis NC8 isolate in the microdilution test. The growth was measured by spectrophotometry at 540 nm in 96 wells

microplate during 24 hours of incubation. Control-the culture medium without the addition of lyophilized material.

TABLE 2

Antibiogram test of cell-free supernatant and lyophilized and dialyzed material from B. subtilis NC8 isolate

Material

E. coli P. aeruginosa S. aureus B. cereus C. albicans

Inhibition zone (mm)

Post-culture liquid - - 11.00 (MHB)

13.5 (MB)

20.5 (MHB)

Lyophilized material - - - 14.6 (MB)

Dialyzed material - - - 13.3 (MB)

1. MHB-Mueller Hinton Broth;

MB-Marine Broth.

468

Revista de Biología Tropical, ISSN electrónico: 2215-2075 Vol. 69(2): 462-471, April-June 2021 (Published Apr. 01, 2021)

(OD 540 nm: 0.14-0.19). Concentrations below

15.63 μg/μl lyophilized material did not reduce

the growth of B. cereus.

The result of the MBC test showed the

microbial growth in all concentrations of

lyophilized material (500 μg/μl-1.96 μg/μl)

tested, demonstrating that the antimicrobial

activity of the lyophilized material of NC8 iso-

late had bacteriostatic activity.

The hemolytic rate of the lyophilized mate-

rial subjected to dialysis was 4.8 % in the con-

centration of 0.01 mg/ml during the 12 hours

of incubation (Fig. 3). Hemolysis obtained by

Triton X-100 (1 %) (positive control) was con-

sidered 100 % hemolysis.

DISCUSSION

In the coastal reefs of Carapibus Beach

of Conde (Paraiba state, Brazil), the colonies

of P. caribaeorum are wide spread and some

colonies are affected by a tissue necrosis (Silva,

2015). Among the bacteria associated with the

tissue of P. caribaeorum the Bacillus genus

was the most abundant (84 % of the isolates),

followed by genera of Vibrio, Pseudomonas,

Staphylococcus and Alteromonas.

When studying the biodiversity of bac-

teria associated with the soft coral Alcionium

digitatum, abundant in the Baltic Sea, Pham

et al. (2016) also identified the genus Bacillus

as the most abundant and diverse group, with

17 species. The genus Bacillus was also found

in other species of corals, however, in smaller

proportions in relation to other taxa found.

For example, Eiahwany, Ghozlan, Eisharif, &

Sabry (2013) found that bacteria associated

with soft coral Sarcophyton glaucum the Red

Sea reefs were representatives of the Gamma-

proteobacteria, Actinobacteria and Firmicutes,

and 11 species of Bacillus were found. The

authors reported a high proportion of bacteria

with antimicrobial and antifungal activities,

especially those belonging to the Bacillus

genus that showed higher antimicrobial activ-

ity. They suggested that these bacteria may play

an important protective role, helping their host

in the defense against marine pathogens.

In our study, the class of gram-negative

bacteria Gammaproteobacteria was found in

a smaller proportion (16 %), however, other

works has reported that Gammaproteobacteria

dominate the microbial community, although

there is a variation of genera associated with

zoanthids and corals (Moreira et al., 2014;

Pereira et al., 2017).

In our study, some isolates, mostly from

the necrotic tissue of P. caribaeorum, were

phylogenetically related to such as V. camp-

bellii, known to be a pathogen of aquatic

Fig. 3. Hemolytic activity of the material subjected to lyophilization and dialysis (0.01 g/ml) obtained from isolate B.

subtilis NC8. Hemolysis of erythrocytes at 2 % of the O- group was measured by spectrophotometry at 540 nm in 96 wells

microplate after 4.8 and 12 hours of incubation. Positive control-1 % of Triton X-100; negative control-0.9 % of NaCl.

469

Revista de Biología Tropical, ISSN electrónico: 2215-2075, Vol. 69(2): 462-471, April-June 2021 (Published Apr. 01, 2021)

organisms. Among the isolates were also found

Staphylococcus epidermidis, disease-causing

species in humans, and P. stutzeri, considered

an opportunistic pathogen in clinical settings.

Potentially pathogenic bacteria for humans

have also been found in the microbiota of P.

caribaeorum on the coral reef of Ponta Verde

in Maceio, Alagoa state, Brazil, exposed to

untreated sewage dumping (Paulino, 2017).

This work reported changes in the microbial

community associated with P. caribaeorum

due to anthropogenic effect, studied, show-

ing that about 25 % of sequences obtained by

pyrosequencing techniques belonged to the

Streptococcus, Staphylococcus and Propioni-

bacterium genera.

The antimicrobial activity was evidenced

in 33 % of the tested bacterial isolates, and

higher number of antagonistic bacteria was

obtained from the necrotic tissue of P. carib-

aeorum. The genus Bacillus presented a greater

number of isolates with antimicrobial activity,

and among them the B. subtilis NC8 was the

only one that inhibited the growth of the five

standard strains in antibiosis test. The hemo-

lysis assay of the lyophilized and dialyzed

material of the NC8 isolate was less than 5 %,

indicating that the antimicrobial compounds

has probably no cytotoxic activity, that is very

important characteristic for the potential use of

this isolate for the production of antimicrobials.

Several studies report that marine Bacillus

species with potential action and broad anti-

microbial spectrum were isolated from several

species of corals (Pham et al., 2016; Pereira et

al., 2017).

Li et al. (2011) studied the bogorol A, a

new peptide antibiotic produced by B. subtilis

isolated from a reef in Papua, New Guinea,

and found its high activity against methicillin-

resistant S. aureus (MRSA), vancomycin resis-

tant enterococcus (VRE) and E. coli. In a later

study, Li et al. (2012) reported that the amide

group C-12 amikoumarkin produced by marine

bacterium B. subtilis from the Red Sea exhib-

ited antimicrobial activity against B. subtilis, S.

aureus and Laribacter hongkongensis.

Various species of Bacillus produce bacte-

riocins, for example Bacillus sp. SM01, isolat-

ed from mangrove sediments which produced

bacteriocin Bac-SM01 with long-range antimi-

crobial activity, strongly inhibiting the growth

of S. aureus methicillin resistant (MRSA),

Acinetobacter baumannii, P. aeruginosa and E.

coli (Mickymaray et al., 2018).

Antimicrobial compounds are promising

sources for the production of new drugs and

can be used to fight infectious diseases. In our

study various marine isolates showed antimi-

crobial activity against a range of pathogenic

microorganisms, and particularly one isolate

B. subtilis NC8, that show a potential to be

explored in the future studies.

ACKNOWLEDGMENTS

We thank Coordination for the Improve-

ment of Higher Education Personnel (CAPES)

for the scholarship granted to Jalcinês da Costa

Pereira. We acknowledge the Cell and Molecu-

lar Biology Post-Graduate Program of the

Federal University of Paraiba for institutional

support and Laboratory of Reef Environments

and Biotechnology of Microalgae (LARBIM)

of the Federal University of Paraiba for techni-

cal support.

RESUMEN

Actividad antimicrobiana de bacterias aisladas

del tejido del coral Palythoa caribaeorum (Zoantharia:

Sphenopidae) de los arrecifes costeros de Paraíba, Bra-

sil. Introducción: La actividad antimicrobiana realizada

por las bacterias asociadas con los corales, además de pro-

mover la salud de su huésped, representa una fuente para

obtener nuevos compuestos bioactivos. Objetivo: Analizar

la actividad antimicrobiana de las bacterias asociadas con

el zoantario Palythoa caribaeorum de los arrecifes de

Carapibus, Paraíba, Brasil. Metodología: El análisis filo-

genético de la bacterias se realizó con base en secuencias

parciales del gen RNAr 16S utilizando herramientas mole-

culares y de bioinformática. La actividad antimicrobiana

de las cepas se probó contra cuatro cepas bacterianas y una

cepa de levadura: Bacillus cereus (CCT0198), Escherichia

coli (ATCC 25922), Staphylococcus aureus (ATCC 25923),

Pseudomonas aeruginosa y Candida albicans (ATCC

10231), utilizando ensayos antibiosis y antibiograma, y la

concentración inhibitoria mínima (CIM) que se determinó

470

Revista de Biología Tropical, ISSN electrónico: 2215-2075 Vol. 69(2): 462-471, April-June 2021 (Published Apr. 01, 2021)

por el método de microdilución. Resultados: Las cepas

bacterianas pertenecían a Firmicutes (84 %) y Gamma-

proteobacteria (16 %). Entre 49 cepas se encontraron

cinco géneros de bacterias: Bacillus, Vibrio, Pseudomonas,

Staphylococcus y Alteromonas. Un total de 19 cepas exhi-

bieron actividad antimicrobiana, siendo el género Bacillus

el responsable del mayor número de bacterias antagonistas,

con 12 cepas positivas en el ensayo de antibiosis y cuatro

en la prueba de antibiograma. El mayor número de bacte-

rias antagonistas se encontró en Bacillus (12 aislamientos),

seguido por Vibrio (tres aislamientos) y Pseudomonas (un

aisladmiento). El NC8, clasificado como Bacillus subtilis,

inhibió todas las cepas estándar en el ensayo de antibiosis

y las cepas de B. cereus, S. aureus y C. albicans en la prue-

ba de antibiograma. El material liofilizado del B. subtilis

NC8 mostró acción bacteriostática contra B. cereus, con

un valor de CIM de 125 μg/μl. En la prueba de citotoxici-

dad, el grado de hemólisis fue del 4.8 % para el material

liofilizado a las concentraciones probadas, lo que indica su

baja citotoxicidad. Conclusión: Los resultados muestran

el potencial antimicrobiano de algunos aislamientos bac-

terianos asociados al P. caribaeourum, especialmente los

pertenecientes al género Bacillus.

Palabras clave: sustancias antimicrobianas; bacterias

marinas; Bacillus; zoantario; Palythoa caribaeorum.

REFERENCES

Ahamad, A., Khan, A., Manzoor, N., & Khan, L.A. (2010).

Evolution of ergosterol biosynthesis inhibitors as fun-

gicidal against Candida. Microbial Pathogenesis, 48,

35-41. DOI: 10.1016 / j.micpath.2009.10.001

Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J.,

Zhang, Z., Miller, W., & David, J.L. (1997). Gapped

BLAST and PSI-BLAST: a new generation of protein

database search programs. Nucleic Acids Research,

25, 3389-3402. DOI: 10.1093/nar/25.17.3389

Araújo, G.H., Gorlach-Lira, K., Medeiros, D.S., & Sassi,

C.F.C. (2015). Physicochemical and bacteriological

seawater quality and sustainability of Cabo Branco

(Brazil) coral reef. Pan-American Journal of Aquatic

Sciences, 10(2), 94-104.

Castro, C.B., Segal, B., Negrão, F., & Calderon, E.N.

(2012). Four-year monthly sediment deposition on

turbid Southwestern Atlantic coral reefs, with a

comparison of benthic assemblages. Brazilian Jour-

nal of Oceanography, 60(1), 49-63. DOI: 10.1590/

S1679-87592012000100006

Costa, C.F., Sassi, R., Costa, M.A.J., & Brito, A.C.L.

(2007). Recifes costeiros da Paraíba, Brasil: usos,

impactos e necessidades de manejo no contexto da

sustentabilidade. Gaia Scientia, 1(1), 37-45. Retrie-

ved from https://periodicos.ufpb.br/ojs/index.php/

gaia/article/view/2226

Costa. C.F., Sassi, R., Gorlach-Lira, K., Lajeunesse, T.C.,

& Fitt, W.K. (2013). Seasonal changes in zooxanthe-

llae harbored by zoanthids (Cnidaria, Zoanthidea)

from coastal reefs in Northeastern Brazil. Pan-

American Journal of Aquatic Sciences, 8(4), 253-264.

Durante, L.M., Cruz, I.C.S., & Lotufo, T.M.C. (2018).

The effect of climate change on the distribution of

a tropical zoanthid (Palythoa caribaeorum) and its

ecological implications. Peer J-the journal of live and

Environmental Sciences, 6, e4777. DOI: 10.7717/

peerj.4777

Eiahwany, A.M.D., Ghozlan, H.A., Eisharif, H.A., & Sabry,

S.A. (2013) Phylogenetic diversity and antimicrobial

activity of marine bacteria associated with the soft

coral Sarcophytonglaucum. Journal of Basic Micro-

biology, 55(1), 2-10. DOI: 10.1002/jobm.201300195

Francini-Filho, R.B., Coni, E.O.C., Meirelles, P.M.,

Amado-Filho, G.M., Thompson, F.L., Pereira-Filho,

G.H., … Moura, R. (2013). Dynamics of coral reef

benthic assemblages of the Abrolhos bank, eastern

Brazil: inferences on natural end anthropogenic dri-

vers. Plos One, 8(1), e54260. DOI: 10.1371/journal.

pone.0054260

Lane, D.J. (1991). 16S/23S rRNA sequencing. In E. Stac-

kebrandt & M. Goodfellow (Eds.), Nucleic acid tech-

niques in bacterial systematics (pp. 115-175). New

York, USA: John Wiley & Sons, Inc.

Leão, Z.M.A.N., Kikuchi, R.K.P., Ferreira, B.P., Neves,

E.G., Sovierzoski, H.H., Oliveira, M.D.M., …

Johnson, R. (2016). Brazilian coral reefs in a period

of global change: A synthesis. Brazilian Journal

of Oceanography, 64(sp2), 97-116. DOI: 10.1590/

S1679-875920160916064sp2

Li, D., Carr, G., Zang, Y., Williams, D.E., Amlani, A.,

Bottriell, H., ... Andersen, R. (2011). Turnagainolides

A and B, cyclic depsipeptides produced in culture by

a Bacillus sp.: isolation, structure elucidation, and

synthesis. Journal of Natural Products, 74(5), 1093-

1099. DOI: 10.1021/np200033y

Li, Y., Xu, I., Liu, L., Han, Z., Lai, P.Y., Guo, X., …

Pei-Yuan, Q. (2012). Five new amicoumacins iso-

lated from a marine-derive bacterium Bacillus sub-

tilis. Marine Drugs, 10(2), 319-328. DOI:10.3390/

md10020319

Melo, R.S., Lins, R.P.M., & Eloy, C.C. (2014). O Impacto

do turismo em ambientes recifais: caso praia Seixas-

Penha, Paraíba, Brasil. REDE - Revista Eletrônica do

Prodema, 8(1), 67-83.

Moreira, A.P.B., Luciane, A., Tonon, C., Pereira, C.V.P.,

Alves, N., Amado-Filho, G.M., ... Thompson, F.L.

(2014). Culturable heterotrophic bacteria associated

with healthy and bleached scleractinian Madracis

decactis and the fireworm Hermodice carunculata

from the remote St. Peter and St. Paul Archipelago,

471

Revista de Biología Tropical, ISSN electrónico: 2215-2075, Vol. 69(2): 462-471, April-June 2021 (Published Apr. 01, 2021)

Brazil. Current Microbiology, 68(1), 38-46. DOI:

10.1007/s00284-013-0435-1

Mickymaray, S., Alturaiki, W., Al-Aboody, M.S.,

Mariappan, P., Rajenderan, V., Alsagaby, S.A., ...

Alarfajj, A.A. (2018). Anti-bacterial efficacy of bac-

teriocin produced by marine Bacillus subtilis against

clinically important extended spectrum beta-lactama-

se strains and methicillin-resistant Staphylococcus

aureus. International Journal of Medical Research &

Health Sciences, 7(2), 75-83.

Mondol, M.A.M., Sin, H.J., & Islam, M.T. (2013). Diver-

sity of secondary metabolites from marine Bacillus

species: chemistry and biological activity. Marine

Drugs, 11(8), 2846-2872. DOI: 10.3390/md11082846

NCCLS. (2003). Methods for dilution antimicrobial sus-

ceptibility tests for bacteria that grow aerobically.

Approved standard (Sixth Edition). Pensylvania,

USA: NCCLS.

Paulino, G.V.B. (2017). Diversidade de bactérias não-

cultiváveis associadas ao zoantídeo Palythoa cari-

baeorum (Dissertação de Mestrado). Universidade

Federal de Alagoas, Maceió, Brasil. Retrieved from

http://www.repositorio.ufal.br/handle/riufal/5412

Pereira, L.B., Palermo, B.R.Z., Carlos, C., & Ottoboni,

L.M.M. (2017). Diversity and antimicrobial activity

of bacteria isolated from different Brazilian coral

species. FEMS Microbiology Letters, 364(16). DOI:

10.1093/femsle/fnx164

Pham, T.M., Wiese, J., Wenzel-Storjoann, A., & Imhoff,

J.F. (2016). Diversity and antimicrobial potential

of bacterial isolates associated with the soft coral

Alcyonium digitatum from the Baltic sea. Antonie

Van Leeuwenhoek, 109(1), 105-119. DOI: 10.1007/

s10482-015-0613-1

Silva, R.M.S. (2015). Diversidade de bactérias cultiváveis

associadas ás colônias sadias e necrosadas do

zoantídeo Palythoa caribaeorum (Cnidaria, Antho-

zoa) dos recifes costeiros de Carapibus, Paraíba

(Dissertação de Mestrado). Universidade Federal da

Paraíba, João Pessoa, Brasil. Retrieved from https://

repositorio.ufpb.br/jspui/handle/tede/9442

Silva, J.F., Gomes, P.B., Santana, E.C., Silva, J.M., Lima,

E.P., Santos, A.M.M., & Pérez, C.D. (2015). Growth

of the tropical zoanthid Palythoa caribaeorum (Cni-

daria: Anthozoa) on reefs in Northeastern Brazil.

Brazilian Academy of Sciences, 87(2), 985-996. DOI:

10.1590/0001-3765201520140475

Tamura, K., Stecher, G., Peterson, D., Filipski, A., &

Kumar, S. (2013). MEGA6: Molecular evolutionary

genetics analysis version 6.0. Molecular Biology and

Evolution, 30(12), 2725-2729. DOI: 10.1093/molbev/

mst197