European Review for Medical and Pharmacological Sciences
2010; 14: 739-747
Screening of extracts of algae from
Baja California Sur, Mexico as reversers of
the antibiotic resistance of some
pathogenic bacteria
M. MUÑOZ-OCHOA, J.I. MURILLO-ÁLVAREZ, L.A. ZERMEÑO-CERVANTES,
S. MARTÍNEZ-DIAZ, R. RODRÍGUEZ-RIOSMENA*
Departamento de Desarrollo de Tecnologías, Centro Interdisciplinario de Ciencias Marinas,
Instituto Politécnico Nacional, La Paz (Mexico);
*Departamento de Biología Marina, Universidad Autónoma de Baja California Sur,
La Paz (Mexico)
Abstract. – Background and Objectives: Sixty ethanol extracts of marine flora of
Baja California Sur (Mexico) were screened to
evaluate the reversing effect of the bacterial resistance to antibiotics in combination with a
sublethal concentration of ampicillin or erythromycin.
Materials and Methods: The activity was assayed by using a modification of the classical
agar-diffusion method against 3 resistant, pathogenic bacteria; Escherichia coli (ATCC BAA196),
Staphylococcus aureus (ATCC BAA42), and
Streptococcus pyogenes (ATCC BAA946).
Results: From the 60 ethanolic extracts, 12
(20%) of them in combination with ampicillin
were able to reverse the resistance of Staphylococcus aureus and 8 (13%) with erythromycin
yielded the same reversal with Streptococcus
pyogenes. An extract from Sargassum horridum
was the only one that reversed the resistance to
antibiotics against both Staphylococcus aureus
and Streptococcus pyogenes.
Conclusions: Our findings suggest that
some algae may be source of compounds with
the potential to reverse the antibiotic resistance of some bacteria. In addition, of the assayed extracts, 35 (57%) showed inhibitory activity against Staphylococcus aureus, 48 (78%)
were active against Streptococcus pyogenes,
but none was active against Escherichia coli.
The most active extracts were from Laurencia
spp., Gelidium robustum, Chnoospora implexa,
Padina mexicana, Gracilaria subsecundata, and
Dictyopteris undulata.
Key Words:
Escherichia coli, Streptococcus, Staphylococcus,
Antimicrobial.
Introduction
Today, an increasing demand of new compounds to combat the bacterial resistance to antibiotics is globally recognized. Resistance is a
phenomenon characterized by having serious
health and socioeconomic implications. Because
of the bacterial resistance, a number of infectious diseases are becoming difficult to treat,
making it necessary to be more aggressive and
using expensive treatments over a longer time1.
The problem is global, complex, and includes a
large number of bacteria of pathological importance. The multicausality of the resistance
makes it a difficult problem with which to deal2.
Highly populated areas, inadequate control of
infections, and the transference of patients between hospitals are important factors causing the
increasing of the resistance. The changes in the
ecology of infections observed since the introduction of the antimicrobial agents have been
well-documented2. In response to those changes,
bacteria have developed a number of adaptive
strategies; enzyme inactivation, efflux pumps,
target alteration, and permeability changes3,4. The
most successful mechanisms of resistance are the
expression of enzymes able to degrade the β-lactam ring of penicillins and cephalosporins5 and
efflux pumps that are able to extrude structurally
diverse compounds, including antibiotics, from
the cell. To combat the rapid spread of bacteria
expressing resistance, many Authors5-8 agree for
the need to discover new antibacterials and resistance-reversing agents. This latter involves the
Corresponding Author: Mauricio Muñoz-Ochoa, MD; e-mail: [email protected]
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M. Muñoz-Ochoa, J.I. Murillo-Álvarez, L.A. Zermeño-Cervantes, S. Martínez-Diaz, R. Rodríguez-Riosmena
discovery of compounds able to inhibit those
mechanisms that confer resistance to antibiotics
by the bacteria. With this in mind, in our research
a library of extracts from marine macroalgae
from Mexico were screened as reversers of the
resistance to antibiotics of three pathogenic bacteria to select unexplored sources of resistancereversing compounds. This choice is justified because marine algae are important sources of
metabolites, such as acetogenins, polyphenolics,
aromatic compounds, terpenes, and other related
structures9. To the best of our knowledge this is
the first time in which marine macroalgae collected from the coasts of Baja California Sur,
Mexico have been assessed as reversers of the resistance to antibiotics. Our finding has allowed
us to select a number of algae for further investigation in search for the active compounds.
Materials and Methods
The algae studied were gathered from several
locations along the coast of Baja California Sur
(Figure 1) between June 2004 and November
2007. The collection was done by free diving at
1- to 2-m depth. Algal samples were cleaned of
epiphytes, rinsed with tap water to remove any
associated debris, and the cleaned fresh materials
were dried in the sun, ground, and stored at
–20°C until extraction. Voucher specimens were
preserved for taxonomic identification and future
reference.
The extracts were obtained by maceration.
Briefly, 100 g of each algal sample was soaked
with 250 mL of distilled ethanol at 25-35°C. After 48 h, the mixture was filtered through paper.
The residual algal tissue was extracted once
again under same conditions. After filtration,
both extracts were pooled and concentrated to
dryness under vacuum at 40°C (Yamato RE500,
San Francisco, CA, USA). The crude extracts
were kept in dark and dry conditions at –20°C
until biological testing.
A modification of the classical agar disc-diffusion method10 was used to test the effect of extracts on the reversion of the resistance of Escherichia coli (ATCC BAA-196, expressing extended spectrum β-lactamases), Staphylococcus
aureus (ATCC BAA-42, β-lactamases), and
Streptococcus pyogenes (ATCC BAA-946, efflux
pump). Each strain was first cultured on MuellerHinton agar plates at 37°C. After 18 h, the cell
740
biomass was harvested and suspended in a 0.85%
sodium chloride solution. The cell density was
adjusted by spectrophotometry (Merck SQ118,
Darmstadt, Germany) at λ 585 nm until 1.0 AU
(equivalent to 4.5 × 107 cells mL-1). The suspensions of each strain were used to inoculate the
surface of Mueller-Hinton agar plates with an
added sublethal concentration of an antibiotic.
Escherichia coli and Staphylococcus aureus were
cultured on Mueller-Hinton agar with added
ampicillin (12 mg L-1, equivalent to 75% of the
minimum inhibitory concentration (MIC)). The
medium for Streptococcus pyogenes was added
erythromycin (1.5 mg L-1, equivalent to 25% of
the MIC). The drug susceptibility pattern of
each target strain was previously determined.
The culturing conditions of strains under sublethal concentrations of antibiotic were extensively tested and optimized. The assays were
done by putting, on the inoculated surface of the
agar plate, extract-impregnated paper discs (6.5
diameter, Whathman No. 4). Each paper disc
was loaded with 100 µL of a stock solution of
each algal extract (20 mg mL-1). The impregnation of the disc was done under aseptic conditions. Solvent evaporation from the discs left
2.0 mg disc-1. Plates were incubated at 37°C.
The inhibition zone (when observed) around
discs was measured after 24 h, and expressed in
mm. Every algal extract was tested in a medium
added with antibiotic (as mentioned before) and
its effect was contrasted by testing the same extract, at the same time, in Mueller-Hinton agar
without an antibiotic. Those extracts active
against bacteria cultured on a medium with
added antibiotic, but inactive in an antibioticfree medium were considered as reversers of the
resistance. Measures of the inhibition zones are
presented as the mean values of two measurements and its SD. Ethanol was always used as a
negative control.
Statistical Analysis
We have found a great difficult to perform a
statistical analysis. However, we have taken into
mind that:
• The substances produced by each alga do not
necessarily correspond to the same chemical
group as well as we cannot assume that they
are in the same concentration, therefore a
comparison between them we could give an
erroneous interpretation of the activity of each
of the species.
Reversion of the antibiotic resistance
Figure 1. Map of Baja California peninsula (Mexico) showing the different collection locations.
• The effect of each alga on the three test bacteria cannot be compared because the mechanisms used by bacteria to resist antibiotics are
different (β-lactamases and efflux pump as
mentioned in the introduction). Thus although
in some cases there is a tendency to increase
the diameter of the halos when the algae extract is placed in the presence of the antibiotic,
it cannot be directly interpreted as an effect of
inhibition of antibiotic resistance, because the
741
M. Muñoz-Ochoa, J.I. Murillo-Álvarez, L.A. Zermeño-Cervantes, S. Martínez-Diaz, R. Rodríguez-Riosmena
extract has antibacterial activity and therefore
both mechanisms may be acting simultaneously. In future work, surely we must focus to describe the nature of the compounds responsible for the antibacterial activity and effect of
each of the molecules in the presence of antibiotics.
• Therefore we consider the only valid criterion
to define some attribute the extracts was attributable to the presence of antibiotic that inhibition is present whereas in the absence of antibiotic inhibition was not observed. With the
foregoing, we dismiss the ability of the extracts per se to inhibit the process of bacterial
resistance to antibiotics. In this case the values
were zero for the diameter of halo in the absence of antibiotic and values of 8 mm or
more for the diameter of halo in the presence
of antibiotic.
Results
As seem in Table I, from all the 60 algal extracts assessed, 9 extracts, 15%, were able to reverse the resistance of Staphylococcus aureus to
a sublethal concentration of ampicillin. The effect was denoted by the inhibition of growth in
combination of the antibiotic and extracts from
Padina crispata (sample ID: 04-002), Hypnea
valentiae (04-011), Sargassum horridum (04003), Rosenvingea. intricata (04-015), Spyridia
filamentosa (04-025), Liagora californica (06008), Chondracanthus canaliculatus (07-010),
Codium amplivesiculathum (04-004) and Codium cuneatum (06-011). The extracts from Sargassum horridum (04-003), Cystoseira osmundacea (06-023), Chondracanthus canaliculatus (06-026), Hypnea johnstonii (06-035),
Pterosiphonia bipinnata (07-003), Spyridia filamentosa (07-006), Gracilaria marcialana (07007), and Colpomenia sinuosa (07-008) showed
the same effect on the resistance of Streptococcus pyogenes to erythromycin. From the 3 tested
extracts of Sargassum horridum collected in different years, just the extract from the specimen
04-003 was active. In addition, Sargassum horridum (04-003) was the one that showed a reverser effect on the antibiotic resistance of
Staphylococcus aureus and Streptococcus pyogenes against ampicillin and erythromycin. Surprisingly, none of extracts tested was able to affect the growth of Escherichia coli.
742
Discussion
Objective of this study was to evaluate the
ability of extracts of marine algal species collected from several locations from Baja California
Sur (Mexico) to inhibit (or reverse) the mechanism of resistance to antibiotics of three pathogenic bacteria.
In the literature there are numerous reports
about a broad range of antimicrobial activities
from algal extracts from around the world11-20, including algae from Mexican waters21-2. A number
of algal natural products with antimicrobial activity have been described in the past few
decades9. After an extensive bibliographic review
through electronic data bases, reports of a reverser effect of the algal extracts on antibiotic-resistance of bacteria were not found. Probably our
study is a pioneer in this area.
As mentioned, just those extracts that were active against bacteria cultured in a medium with
an added sublethal concentration of antibiotic
(but necessarily an extract inactive by itself) were
considered as inhibitors of the mechanisms of
bacterial resistance. That interpretation was done
under the assumption of that some compounds
present in the extracts were able to inhibit the βlactamase activity in Staphylococcus aureus andor the efflux pump in Streptococcus pyogenes allowing the added antibiotic (ampicillin or erythromycin) to cause cell growth inhibition, denoted by inhibition zones around the discs.
In the search for inhibitors of the mechanisms
of bacterial resistance, inhibitors have been found
mainly in bacteria or by chemical synthesis. For
example, the clavulanic acid produced by Streptomyces clavuligerus acts as an inhibitor of β-lactamases, which are a defensive response to the βlactam antibiotics25. Competitive inhibition is the
mode of action of the clavulanic acid caused by a
structure identical to the natural β-lactamase substrate. We believe that competitive inhibitors of βlactamases can be found in macroalgae and that is
our explanation for the observed effect of Sargassum horridum (04-003) on the resistance of
Staphylococcus aureus against ampicillin. This
idea is supported by the isolation of sargassumlactam from Sargassum kjellmanianum26. Considering that similar compounds may be synthesized
by other brown algae, the effect shown by Padina
crispata (04-002) and Rosenvingea intricata (04015) on the resistance of Staphylococcus aureus
may be explained by the presence of structures
similar to the sargassumlactam.
10.0 ± 0
18.0 ± 0
14.5 ± 0.7
9.5 ± 0.7
14.5 ± 0.7
10.5 ± 0.7
0±0
9±0
15.5 ± 0.7
Corallinaceae
07-001, Amphiroa valonioides Yendo, 1902 (7)
06-025, Corallina vancouveriensis Yendo, 1902 (4)
06-027, Corallina sp. (2)
Dictyotaceae
06-016, Dictyota flabellata Setchell & Gardner, 1924 (6)
06-028, Dictyopteris undulata Holmes, 1896 (2)
06-029, Dictyopteris delicatula Lamouroux, 1809 (2)
04-002, Padina mexicana Thivy, 1945 (8)
04-021, Padina mexicana Thivy, 1945 (8)
06-018, Padina concrescens Thivy, 1945 (2)
8.0 ± 0
0±0
10.0 ± 0
9.5 ± 2.1
9.5 ± 0.7
Codiaceae
04-004, Codium amplivesiculatum Setchell & Gardner, 1924 (8)
06-012, Codium amplivesiculatum Setchell & Gardner, 1924 (6)
04-024, Codium cuneatum Setchell & Gardner, 1924 (8)
06-011, Codium cuneatum Setchell & Gardner, 1924 (6)
06-010, Codium simulans Setchell & Gardner, 1924 (6)
0±0
8.0 ± 0
10.0 ± 1.4
Cladophoraceae
06-001, Cladophora sp. (7)
Cystocloniaceae
06-035, Hypnea johnstonii Setchell & Gardner, 1924 (2)
04-011, Hypnea valentiae (Turner) Montagne, 1841 (8)
10.0 ± 0
0±0
Chnoosporaceae
04-018, Chnoospora implexa Agardh, 1848 (8)
Bangiaceae
06-020, Porphyra perforata Agardh, 1883 (2)
Family
Sample ID, species name, and authority (collection place)
MAWA
S. aureus
7.5 ± 0.7
14.0 ± 1.4
11.5 ± 1.4
0±0
7.0 ± 0
7.0 ± 0
7.0 ± 0
0±0
0±0
11.0 ±0
9.5 ± 0.7
0±0
0±0
9.5 ± 0.7
0±0
7.0 ± 0
7.0 ± 0
7.0 ± 0
0±0
CWA
14.0 ± 0
21.5 ± 0.7
13.0 ± 1.4
11.0 ± 0
16.5 ± 0.7
12.5 ± 0.7
10.0 ± 0
12.5 ± 0.7
11.0 ± 0
9.5 ± 0.7
15.5 ± 0.7
13.0 ± 0
11.5 ± 0.7
14.5 ± 0.7
15.0 ± 0
11.5 ± 0.7
16.0 ± 1.4
19.5 ± 07
0±0
MAWE
S. pyogenes
18.5 ± 0.7
18.5 ± 0.7
11.0 ± 0
10.0 ± 0
10.0 ± 0
11.0 ± 0.7
0±0
10.5 ± 0.7
17.5 ± 2.1
13.5 ± 0.7
10.5 ± 0.7
12.0 ± 0
10.0 ±
11.0 ± 0
12.5 ± 0.7
8.0 ± 0
22.0 ± 1.4
12.0 ± 0
0±0
CWA
Diameter of the inhibition zone, in mma
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
MAWA
E. coli
(Continued)
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0(
0±0
0±0
0±0
0±0
0±0
0±0
0±0
CWA
Table I. Diameter of inhibition zone around the discs individually impregnated with sixty algal extracts collected from Baja California Sur (Mexico). Bacteria were cultured on
Mueller Hinton agar plates add with a sublethal concentration of antibiotic.
Reversion of the antibiotic resistance
743
744
S. aureus
Rhodymeniaceae
06-003, Rhodymenia californica Kylin, 1931 (7)
06-032, Rhodymenia californica Kylin, 1931 (2)
Liagoraceae
04-022, Ganonema farinosum Fan & Wang, 1974 (8)
06-008, Liagora californica Zeh, 1912 (8)
06-013, Liagora californica Zeh, 1912 (6)
7.0 ± 0
0±0
12.5 ± 2.1
9.0 ± 0
0±0
0±0
Lessoniaceae
06-022, Eisenia arborea Areschoug, 1876 (2)
0±0
0±0
0±0
13.5 ± 0.7
Gracilariaceae
07-007, Gracilaria marcialana Setchell & Gardner, 1924 (8)
04-007, Gracilaria veleroae Dawson, 1944 (8)
04-010, Gracilaria vermiculophylla (Ohmi) Papenfuss, 1967 (5)
04-023, Gracilaria subsecundata Setchell & Gardner, 1924 (8)
9.0 ± 0
9.5 ± 0.7
9.0 ± 0
Gigartinaceae
06-026, Chondracanthus canaliculatus Harvey, 1993 (2)
07-010, Chondracanthus canaliculatus Harvey, 1993 (1)
Laminariaceae
06-019, Macrocystis pyrifera Agardh, 1820 (2)
11.0 ± 1.4
14.0 ± 1.4
12.5 ± 2.1
Gelidiaceae
04-008, Gelidium robustum Hollenberg & Abbott, 1965 (3)
06-024, Gelidium robustum Hollenberg & Abbott, 1965 (4)
06-033, Gelidium robustum Hollenberg & Abbott, 1965 (2)
MAWA
7.0 ± 0
0±0
9.0 ± 0
0±0
0±0
0±0
10.0 ± 0
0±0
0±0
0±0
11.0 ± 1.4
7.5 ± 0.7
0±0
11.5 ± 0.7
8.5 ± 0.7
10.5 ± 0.7
CWA
14.0 ± 0
0±0
17.0 ± 2.1
13.5 ± 1.4
0±0
0±0
11.0 ± 0
11.0 ± 0
0±0
11.0 ± 0
14.0 ± 0
10.0 ± 1.4
0±0
22.5 ± 0.7
14.0 ± 1.4
14.5 ± 0.7
MAWE
S. pyogenes
13.0 ± 0
0±0
17.0 ± 0
10.5 ± 0.7
0±0
0±0
18.5 ± 0.7
0±0
0±0
11.0 ± 0
9.0 ± 1.4
0±0
0±0
19.0 ± 0
7.0 ± 0
11.0 ± 0.7
CWA
Diameter of the inhibition zone, in mma
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
MAWA
E. coli
(Continued)
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
CWA
Table I. Diameter of inhibition zone around the discs individually impregnated with sixty algal extracts collected from Baja California Sur (Mexico). Bacteria were cultured on
Mueller Hinton agar plates add with a sublethal concentration of antibiotic.
M. Muñoz-Ochoa, J.I. Murillo-Álvarez, L.A. Zermeño-Cervantes, S. Martínez-Diaz, R. Rodríguez-Riosmena
0±0
9.0 ± 0
9.0 ± 0.7
9.5 ± 0.7
10.5 ± 0.7
9.5 ± 0.7
11.5 ± 0.7
7.5 ± 0.7
9.0 ± 0
10.5 ± 2.1
13 ± 1.4
11.0 ± 0
10.5 ± 0.7
11.0 ± 0
0±0
11.0 ± 0.7
Sargassaceae
06-023, Cystoseira osmundacea (Turner) Agardh, 1820 (4)
04-003, Sargassum horridum Setchell & Gardner, 1924 (8)
06-005, Sargassum horridum Setchell & Gardner, 1924 (8)
06-009, Sargassum horridum Setchell & Gardner, 1924 (8)
Scytosiphonaceae
04-017, Hydroclathrus clathratus (Agardh) Howe, 1920 (8)
06-006, Hydroclathrus clathratus (Agardh) Howe, 1920 (8)
04-015, Rosenvingea intricata (Agardh) Børgesen, 1914 (8)
04-020, Rosenvingea intricata (Agardh) Børgesen, 1914 (8)
06-004, Rosenvingea intricata (Agardh) Børgesen, 1914 (8)
06-017, Colpomenia tuberculata Saunders, 1898 (6)
06-030, Colpomenia tuberculata Saunders, 1898 (2)
07-008, Colpomenia sinuosa Derbés & Solier, 1851 (8)
Spyridiaceae
04-025, Spyridia filamentosa (Wulfen) Harvey, 1833 (5)
07-006, Spyridia filamentosa (Wulfen) Harvey, 1833 (5)
Ulvaceae
04-001, Ulva rigida Agardh, 1823 (8)
06-002, Ulva dactylifera Setchell & Gardner, 1920 (7)
0±0
7.0 ± 0
0±0
9.0 ± 0
7.0 ± 0
7.0 ± 0
0±0
7.0 ± 0
7.0 ± 0
8.0 ± 0
13 ± 0
9.0 ± 0
0±0
0±0
7.0 ± 0
7.0 ± 0
0±0
0±0
30.5 ± 0.7
11.5 ± 0.7
18.0 ± 0.7
12.0 ± 0.7
14.5 ± 0.7
12.0 ± 0
0±0
CWA
15 ± 0
14.5 ± 0.7
15.0 ± 0
11.5 ± 0.7
13.0 ± 0
12.5 ± 0.7
15.0 ± 0
13.0 ± 0.7
14.0 ± 0
20.5 ± 13.4
13 ± 0
10.5 ± 0.7
11.0 ± 0.7
12.0 ± 0
13.5 ± 0.7
15.5 ± 0.7
12.0 ± 0
10.5 ± 0.7
38.0 ± 0.7
9.0 ± 1.4
31.5 ± 2.1
21.5 ± 2.1
30.0 ± 0
11.0 ± 0
10.5 ± 0.7
MAWE
S. pyogenes
7.0 ± 0
11.0 ± 0
7.0 ± 0
0±0
12.0 ± 0
15.0 ± 0
10.0 ± 0
13.0 ± 0
11.5 ± 0.7
9.5 ± 0.7
10 ± 0
0±0
0±0
0±0
14.0 ± 1.4
14.5 ± 0.7
10.5 ± 0.7
8.5 ± 0.7
26.5 ± 1.4
10.5 ± 0.7
25.0 ± 0.7
19.5 ± 0.7
30.5 ± 0.7
13.0 ± 0
0±0
CWA
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
MAWA
E. coli
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
0±0
CWA
Mean values (n = 2) ± standard deviation. MAWA = cultured on medium added with 75% of the MIC of ampicillin; CWA = cultured without antibiotic; MAWE = cultured on
medium added with 25% of the MIC of erythromycin.
a
0±0
0±0
30.0 ± 0
12.0 ± 0
19.5 ± 0.7
21.0 ± 0
19.0 ± 0.7
12.0 ± 1.4
0±0
Rhodomelaceae
07-004, Acanthophora spicifera (M. Vahl) Børgesen, 1910 (8)
07-002, Laurencia gardneri Hollenberg, 1943 (7)
04-005, Laurencia johnstonii Setchell & Gardner, 1924 (8)
07-009, Laurencia johnstonii Setchell & Gardner, 1924 (8)
06-007, Laurencia pacifica Setchell & Gardner, 1924 (8)
06-015, Laurencia pacifica Setchell & Gardner, 1924 (6)
07-005, Laurencia sp. (8)
06-031, Neorhodomela larix (Turner) Masuda, 1982 (2)
07-003, Pterosiphonia bipinnata Falkenberg, 1901 (7)
MAWA
S. aureus
Diameter of the inhibition zone, in mma
Table I. Diameter of inhibition zone around the discs individually impregnated with sixty algal extracts collected from Baja California Sur (Mexico). Bacteria were cultured on
Mueller Hinton agar plates add with a sublethal concentration of antibiotic.
Reversion of the antibiotic resistance
745
M. Muñoz-Ochoa, J.I. Murillo-Álvarez, L.A. Zermeño-Cervantes, S. Martínez-Diaz, R. Rodríguez-Riosmena
The synthetic inhibitors of a bacterial efflux
pump have been investigated in the activity of the
NorA pump expressed in some species of the
genus Staphylococcus and Streptococcus 27,28.
Among the compounds investigated as pump inhibitors are the indole alkaloid reserpine, and
some tyrosine derivatives such as MC207110 and
INF240. Other compounds containing bromophenol as structural moieties have been investigated29,28. The literature shows that compounds
related to those inhibitors of the NorA pump are
present in extracts from Hypnea johnstonii (06035), Chondracanthus canaliculatus (06-026),
Gracilaria marcialana (07-007), Pterosiphonia.
bipinnata (07-003), Colpomenia osmundacea
(06-023), Sargassum horridum (04-003),
Colpomenia sinuosa (07-008), and Spyridia filamentosa (07-006)9, and we believe that may explain the effect observed by the extracts on the
resistance of Streptococcus pyogenes.
In this study, a different response was measured by extracts from same species collected in
different locations and-or year. Temporal and
spatial variation of the biological responses of
crude extracts of marine organisms as a consequence of biotic and nonbiotic conditions has
been extensively documented30-34.
One can note that 80% of the extracts were active against one, and 51% were active against
two target organisms.
Conclusions
The results clearly suggest that macroalgae
collected from Baja California Sur (Mexico) are
an interesting resource for the search for compounds that may be developed as potential reversers of some mechanisms of bacterial resistance to antibiotics. The finding of new reversers
of resistance may have important implications
for public health.
3) DAVIES J. Inactivation of antibiotics and the dissemination of resistance genes. Science 1994;
264: 375-382.
4) TENOVER FC. Mechanisms of antimicrobial resistance in bacteria. Am J Med 2006; 119: S3-S10.
5) LIVERMORE DM. Evolution of β-lactamase inhibitors.
Intens Care Med 1994; 20: S10-S13.
6) MAHAMOUD A, CHEVALIER J, ALBERT-FRANCO S, KERN
WV, PAGÈS JM. Antibiotic efflux pumps in Gramnegative bacteria: the inhibitor response strategy.
J Antimicrob Chemother 2007; 59: 1223-1229.
7) STAVRI M, PIDDOCK LJV, GIBBONS S. Bacterial efflux
pump inhibitors from natural sources. J Antimicrob Chemother 2007; 59: 1247-1260.
8) DZIDIC S, SUSKOVIC J, BLAZENKA K. Antibiotic resistance mechanisms in bacteria: biochemical and
genetic aspects. Food Technol Biotechnol 2008;
46: 11-21.
9) BLUNT JW, COPP BR, HU WP, MUNRO MHG, NORTHCOTE PT, PRINSEP MR. Marine natural products. Nat
Prod Rep 2007; 24: 31-86.
10) BAUER AW, KIRBY WM, SHERRIS JC, TURK M. Antibiotic
susceptibility testing by standardized single disk
method. Am J Clin Pathol 1966; 45: 493-496.
11) NAQVI SWA, KAMAT SY, FERNANDES L, REDDY CVG.
Screening of some marine plants from the Indian
coast for biological activity. Bot Mar 1980; 24: 5155.
12) CACCAMESE S, TOSCANO RM, FURNARI G, CORMACI M.
Antimicrobial activities of red and brown algae
from southern Italy coast. Bot Mar 1985; 28: 505507.
13) HODGSON LM. Antimicrobial and antineoplastic activity in some south Florida seaweeds. Bot Mar
1984; 27: 387-390.
14) BALLANTINE DL, GERWICK WH, VELEZ SM, ALEXANDER
E, GUEVARA P. Antibiotic activity of lipid-soluble extracts from Caribbean marine algae. Hydrobiologia 1987; 151/152: 463-469.
15) CAMPOS-TAKAKI GM, DIU MBS, KOENING ML, PEREIRA
EC. Screening of marine algae from Brazilian
northeastern coast for antimicrobial activity. Bot
Mar 1988; 3: 375-377.
16) BALLESTEROS E, MARTIN D, URIZ MJ. Biological activity of extracts from some Mediterranean macrophytes. Bot Mar 1992; 35: 481-485.
1) ANONYMOUS. The world health report 2007. Geneve: WHO Press; 2007.
17) KUMAR AK, RENGASAMY R. Evaluation of antibacterial
potential of seaweeds occurring along the coast
of Tamil Nadu, India against the plant pathogenic
bacterium Xanthomonas or yzae pv. or yzae
(Ishiyama) dye. Bot Mar 2000; 43: 409-415.
2) SMITH PA, ROMESBERG FE. Combating bacteria and
drug resistance by inhibiting mechanisms of persistence and adaptation. Nat Chem Biol 2007; 3:
549-556.
18) BHOSALE SH, NAGLE VL, JAGTAP TG. Antifouling potential of some marine organisms from India
species of Bacillus and Pseudomonas. Mar
Biotechnol 2002; 4: 111-118.
References
746
Reversion of the antibiotic resistance
19) RODRIGUES E, SUPRIYA T, NAIK CG. Antimicrobial activity of marine organisms collected off the coast
of South East India. J Exp Mar Biol Ecol 2004;
309: 121-127.
20) BANSEMIR A, BLUME M, SCHRÖDER S, LINDEQUIST U.
Screening of cultivated seaweeds for antibacterial
activity against fish pathogenic bacteria. Aquaculture 2006; 252: 79-84.
21) DE LARA-ISASSI G, ÁLVAREZ-HERNÁNDEZ S. Actividad biológica de las macroalgas marinas mexicanas.
Rev Soc Mex Hist Nat 1994; 45: 51-60.
22) D E L ARA -I SASSI G, Á LVAREZ -H ERNÁNDEZ S, L OZANO RAMÍREZ C, HERNÁNDEZ-SOTO N. Nuevas adiciones al
conocimiento de la actividad antibiótica de
macroalgas marinas mexicanas. Hidrobiologica
1999; 9: 159-169.
23) FREILE PY, MORALES JL. Antibacterial activity in marine algae from Yucatan coast, Mexico. Bot Mar
2004; 47: 140-146.
24) ORANDAY CMA. Componentes químicos de algas
del estado de Tamaulipas y su aplicación farmacológica. Ph.D. Thesis. Universidad Autónoma de
Nuevo León. Mexico, 1998.
25) HALL GB, SALIPANTE SJ, BARLOW MM. Independent
origins of subgroup Bl+B2 and subgroup B3 metallo-lactamases. J Mol Evol 2003; 59: 133-141.
26) NOZAKI H, FUKUOKA Y, MATSUO A, SOGA 0, NAKAYAMA M.
Structure of sargassumlactam, a new β, γ-unsaturated-γ-lactam, from the marine alga Sargassum kjellmanianum. Chem Lett 1980; 9: 1453-1454.
27) KAATZ WG, SEO MS. Inducible NorA-mediated multidrug resistance in Staphylococcus aureus. Antimicrob Agents Chemother 1995; 39: 2650-2655.
28) MARKHAM NPE, KLYACHKO KW, JONHNSON ME, NEYFAKH AA. Multiple novel inhibitors of the NorA
multidrug transporter of Staphylococcus aureus.
Antimicrob Agents Chemother 1999; 43: 24042408.
29) GIBBONS S, OLUWATUYI M, KAATZ GW. A novel inhibitor of multidrug efflux pumps in Staphylococcus aureus. J Antimicrob Chemother 2003; 51:
13-17.
30) CHUNG HY, M A WCJ, A NG PO, K IM JS, C HEN F.
Seasonal variations of bromophenols in brown
algae (Padina arborescens, Sargassum siliquastrum and Lobophora variegata) collected
in Hong Kong. J Agric Food Chem 2003; 51:
2619-2624.
31) MARECHAL JP, CULIOLI G, HELLIO C, THOMAS-GUYON H,
CALLOW ME, CLARE AS, ORTALO-MAGNÉ A. Seasonal
variation in antifouling activity of crude extracts of
the brown alga Bifurcaria bifurcata (Cystoseiraceae) against cyprids of Balanus amphitrite
and the marine bacteria Cobetia marina and
Pseudoalteromonas haloplanktis. J Exp Mar Biol
Ecol 2004; 313: 47-62.
32) STIRK AW, REINECKE LD, STADEN J. Seasonal variation
in antifungal, antibacterial and acetylcholinesterase activity in seven South African
seaweeds. J Appl Phycol 2007; 19: 271-276.
33) DAR A, BAIG HS, SAIFULLAH SM, AHMAD VU, YASMEEN
S, NIZAMUDDIN M. Effect of seasonal variation on
the anti-inflammatory activity of Sargassum
wightii growing on the N. Arabian Sea coast of
Pakistan. J Exp Mar Biol Ecol 2007; 351: 1-9.
34) AMSLER DC. Algal Chemical Ecology. Heidelberg:
Springer; 2008.
––––––––––––––––––––
Acknowledgements
This study was done in partial fulfillment of the requirements of a Doctor in Sciences degree for MMO, with
grants from IPN (ref. SIP 20070020, SIP 20080216),
and CONACyT (ref. SEP-47942/A-1). Stephanie Castro
is acknowledged for technical assistance during the extractions. Authors thank COFAA-IPN and EDI-IPN for
the incentives granted. Thanks also to Dr. Ellis Glazier
for editing this English-language text.
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