Natural Product Research

Formerly Natural Product Letters

ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20

Modulation of the resistance to norfloxacin in

Staphylococcus aureus by Bauhinia forficata link

Jonas Nascimento de Sousa, Aylla Beatriz Melo de Oliveira, Andressa Kelly

Ferreira, e Silva, Leide Maria Soares de Sousa, Melissa Carvalho França
Rocha, José Pinto de, Siqueira Júnior, Glenn William Kaatz, Jackson Roberto
Guedes da Silva Almeida, João Sammy Nery de Souza & Humberto Medeiros
Barreto

To cite this article: Jonas Nascimento de Sousa, Aylla Beatriz Melo de Oliveira, Andressa Kelly
Ferreira, e Silva, Leide Maria Soares de Sousa, Melissa Carvalho França Rocha, José Pinto
de, Siqueira Júnior, Glenn William Kaatz, Jackson Roberto Guedes da Silva Almeida, João
Sammy Nery de Souza & Humberto Medeiros Barreto (2019): Modulation of the resistance to
norfloxacin in Staphylococcus�aureus by Bauhinia�forficata link, Natural Product Research, DOI:
10.1080/14786419.2019.1590714

To link to this article: https://doi.org/10.1080/14786419.2019.1590714

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Published online: 02 Apr 2019.

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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2019.1590714

SHORT COMMUNICATION

Modulation of the resistance to norfloxacin in

Staphylococcus aureus by Bauhinia forficata link
Jonas Nascimento de Sousaa, Aylla Beatriz Melo de Oliveiraa, Andressa Kelly
Ferreira, e Silvaa,b, Leide Maria Soares de Sousab, Melissa Carvalho França
Rochab, Jose
Pinto de, Siqueira Ju
niord, Glenn William Kaatze,
Jackson Roberto Guedes da Silva Almeidae, Jo~ao Sammy Nery de Souzac and
Humberto Medeiros Barretoa
a
Laboratory of Research in Microbiology, Federal University of Piau
ı, Teresina, PI, Brazil;
b
Postgraduate Program in Pharmaceutical Sciences, Federal University of Piau
ı, Teresina, PI, Brazil;
c
Nucleus of Studies and Research of Medicinal Plants, Federal University of the S~ao Francisco Valley,
Juazeiro, BA, Brazil; dLaboratory of Genetics of Microorganisms, Federal University of Paraiba (UFPB),
Jo~ao Pessoa, Brazil; eDepartment of Medicine, Division of Infectious Diseases, Wayne State University
School of Medicine, Detroit, MI, USA

ABSTRACT ARTICLE HISTORY

Microdilution assays were performed in order to evaluate the anti- Received 10 January 2019
microbial activity of the ethanoic extract from the leaves of Accepted 23 February 2019
Bauhinia forficate (EEBF) against different microorganisms. The
extract did not present inner antimicrobial activities against the KEYWORDS
tested strains. However, EEBF was able to modulate the norfloxa- Bauhinia forficata; drug
resistance; efflux pump
cin-resistance against Staphylococcus aureus SA1199-B that over- inhibitors; infec-
produce the NorA efflux pump, once sub-inhibitory tious diseases
concentrations of EEBF reduced the minimal inhibitory concentra-
tio of the norfloxacin in 87.5%. This modulatory effect was also
found when the antibiotic was replaced by ethidium bromide,
suggesting that EEBF acts probably by inhibition of NorA, allow-
ing the antibiotic accumulation intracellularly, and making the
line more sensitive. These results point out the EEBF potential as
a source of NorA inhibitors that could be used in combination
with norfloxacin for treatment of infections caused by multidrug-
resistant S. aureus.

CONTACT Humberto Medeiros Barreto hmbarreto@ufpi.edu.br Laboratory of Research in Microbiology – LPM

Department of Parasitology and Microbiology – DPM, Centre of Health Sciences, Federal University of Piau
ı, Zip
code: 64049-550, Teresina, PI, Brazil.
Supplemental data for this article is available online at https://doi.org/10.1080/14786419.2019.1590714.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group
2 J. N. DE SOUSA ET AL.

1. Introduction
The rise in the prevalence of infections caused by multidrug-resistant microorgan-
isms represents a significant global public health problem (Vali et al. 2015).
Different bacterial resistance mechanisms have already been elucidated, including
the production of transmembrane proteins known as efflux pumps, which are able
to perform the extrusion of different antibiotic types, reducing the intracellular
concentration needed to perform their antimicrobial effect (Hernando-Amado
et al. 2016).
Plants represent a promising source of substances with antimicrobial potential, or
that can enhance the already known antimicrobial action (Barreto et al. 2014). Natural
products from plants have been investigated for their ability to modulate the
resistance to antibiotics and potential application as adjuvants of antibiotics in the
treatment of infectious diseases caused by multi-drug microrganisms (Figueredo et al.
2013; Oliveira et al. 2011).
Bauhinia forficata Link (Fabaceae) is a native plant from South America. Previous
studies have verified that this species showed hypoglycemic, antidiabetic, antioxidant
(Ferreres et al. 2012), anti-inflammatory and hypo-cholesterolemic activities (Cunha
et al. 2010) Moreover, as well as, inhibited the formation of microbial biofilms
(Ferreira-Filho et al. 2018). The antimicrobial activity of Bauhinia species has already
been demonstrated (Savi et al. 1997; Pinheiro et al. 2016).
This study aims to evaluate the antimicrobial activity of ethanoic extract from
Bauhinia forficata (EEBF) leaves, as well as its modulatory effect on norfloxacin-
resistance in a Staphylococcus aureus strain that overproduce the NorA
efflux pump.
 NATURAL PRODUCT RESEARCH 3

2. Results and discussion

The EEBF did not show inner antimicrobial activity in the tested concentrations (MIC 
16384 lg/mL). These results differ from the ones obtained by extracts from the leaves of
different species of Bauhinia, that showed antimicrobial activity, ranging from moderate
to weak (MICs from 312 to 625 lg/mL) against S. aureus and E. coli, however, the species
B. petersiana did not show activity against C. albicans (Ahmed et al. 2012). Whereas, it
was highlighted through a diffusion test that the methanolic extract from stem bark of
B. racemosa was effective against Gram-negative and Gram-positive bacteria and fungi
(Kumar et al. 2005). The absence of some metabolites or their presence in minor concen-
trations could explain the inactivity of EEBF against the tested strains.
The modulatory activity of EEFF was assayed using the S. aureus strain SA1199-B,
that overexpress the norA gene encoding the NorA efflux pump (Figure S1, supple-
mentary material). This transmembrane protein can perform the extrusion of hydro-
philic fluoroquinolones, such as norfloxacin. Results showed that EEBF presented a
concentration-dependant modulating effect. When tested in a concentration of
512 lg/mL, EEBF decreased by 87.5% the minimum inhibitory concentration of nor-
floxacin (from 64 to 8 lg/mL).
A possible mechanism for the modulating effect observed could be the NorA inhib-
ition by the phytochemicals present in EEBF. In order to test this hypothesis, an essay
was developed, in which norfloxacin was replaced by ethidium bromide (EtBr).
Resistance to EtBr occur exclusively by efflux pumps (Markham et al. 1999), thus,
modulation of the EtBr could be an evidence of NorA inhibition by components of
EEBF. In fact, EEBF was able to reduce the MIC of the EtBr against SA1199-B (Figure
S2, supplementary material) supple, validating that B. forficata has phytochemicals
with potential modulating activity of the fluoroquinolone-resistance, acting probably
by inhibition of the NorA.
Modulatory activity of the aminoglycoside-resistance has already been reported to
Anadenanthera colubrina, another species of the Fabaceae family (Barreto et al. 2016).
This is the first study that verified the norfloxacin-resistance modulation by B. forficata
species. A previous study demonstrated the presence of condensed tanins and the fla-
vonoid kaempferitrin in the aqueous extract from the leaves of B. forficate (Marmitt
and Rempel 2016). Tannins (Santos et al. 2018) and flavonoids (Sabatini et al. 2011)
have been reported as efflux pump inhibitors and could be object of future
investigations.

3. Conclusion
The EEBF did not present antimicrobial activity against S. aureus, E. coli or C. albicans
species. On the other hand, the extract was able to modulate the norfloxacin-resist-
ance, probably by inhibition of NorA, increasing the effectiveness of the norfloxacin
against S. aureus. These results evidenced the technological potential of the B. forficata
phytochemicals combined with norfloxacin in the treatment of infections caused by
multidrug-resistant S. aureus.
4 J. N. DE SOUSA ET AL.

Acknowledgements
We thank Fundaç~ao de Amparo a Pesquisa do Estado do Piau
ı and Universidade Federal do
Piau
ı for their financial support.

Disclosure statement
No potential conflict of interest was reported by the authors.

References
Ahmed AS, Elgorashi EE, Moodley N, Mcgaw LJ, Naidoo V, Eloff JN. 2012. The antimicrobial, anti-
oxidative, anti-inflammatory activity and cytotoxicity of different fractions of four South
African Bauhinia species used traditionally to treat diarrhoea. J Ethnopharm. 143(3):826–839.
Barreto HM, Coelho KMRN, Ferreira JHL, dos Santos BHC, de Abreu APL, Coutinho HDM, da Silva
RAC, de Sousa TO, Cito
A. M D GL, Lopes JAD, et al. 2016. Enhancement of the antibiotic
activity of aminoglycosides by extracts from Anadenanthera colubrine (Vell.) Brenan var.cebil
against multi-drug resistant bacteria. Nat Prod Res. 30(11):1289–1292.
Barreto HM, Fontinele FC, Oliveira AP, Arcanjo DDR, Santos BHC, Abreu APL, et al. 2014.
Phytochemical prospection and modulation of antibiotic activity in vitro by Lippia origanoides
H.B.K. in methicillin resistant Staphylococcus aureus. BioMed Res Int. 2014:1–7.
Cunha AM, Menon S, Menon R, Couto AG, Burger C, Biavatti MW. 2010. Hypoglycemic activity of
dried extracts of Bauhinia forficata Link. Phytomed. 17:37–41.
Ferreira-Filho JCC, Marre A. T D O, Almeida J. S D S, Lobo LA, Farah A, Valença AMG, Fonseca-
Gonçalves A. 2018. Treatment of dental biofilm with a tincture of Bauhinia forficata leaves: an
ex-vivo study. Nat Prod Res. 1.
Ferreres F, Gil-Izquierdo A, Vinholes J, Silva ST, Valentao P, Andrade PB. 2012. Bauhinia forficata
link authenticity using flavonoids profile: relation with their biological properties. Food Chem.
134(2):894–904.
Figueredo FG, Ferreira EO, Lucena BFF, Torres CMG, Lucetti DL, Lucetti ECP, Silva JMFL, Santos
FAV, Medeiros CR, Oliveira GMM, et al. 2013. Modulation of the antibiotic activity by extracts
from Amburana cearensis AC Smith and Anadenanthera macrocarpa (Benth.) Brenan. BioMed
Res Int. 2013:1–5.
Hernando-Amado S, Blanco P, Alcalde-Rico M, Corona F, Reales-Caldero
n JA, S
anchez MB,
Mart
ınez JL. 2016. Multidrug efflux pump as main players in intrisic and acquired resistance
to antimicrobials. Drug Resist Updat. 28:13–27.
Kumar RS, Sivakumar T, Sunderam RS, Gupta M, Mazumdar UK, Gomathi P, Rajeshwar Y,
Saravanan S, Kumar MS, Murugesh K, et al. 2005. Antioxidant and antimicrobial activities of
Bauhinia racemosa L. stem bark. Braz J Med Biol Res. 38(7):1015–1024.
Markham PN, Westhaus E, Klyachko K, Johnson ME, Neyfakh AA. 1999. Multiple novel inhibitors
of the NorA multidrug transporter of Staphylococcus aureus. Antimicrob Agents Chemother.
43(10):2404–2408.
Marmitt DJ, Rempel C. 2016. An
alise fitoqu
ımica das folhas de tr^es esp
ecimes de Bauhinia forfi-
cata link comparando com um esp
ecime de Bauhinia variegata L. RUVRV. 14:229–237.
Oliveira DR, Brito-Junior FE, Bento EB, Matias EFF, Sousa ACA, Costa JGM, Coutinho HDM,
Kerntopf MR, Menezes IRA. 2011. Antibacterial and modulatory effect of Stryphnodendron
rotundifolium. Pharm Biol. 49(12):1265–1270.
Pinheiro EAA, Pina JRS, Feitosa AO, Carvalho JM, Borges FC, Marinho PSB. 2016. Bioprospecting
of antimicrobial activity of extracts ofendophytic fungi from Bauhinia guianensis. Rev Arg
Microb. 49:3–6.
Savi AOS, Breviglieri E, Cruz AB, Yunes RCF, V. 1997. Antibacterial activity of Bauhinia splendens
leaves (Leguminosae). Rev Biol Trop. 44:601–603.
 NATURAL PRODUCT RESEARCH 5

Vali L, Dashti AA, Jadaon MM, El-Shazly S. 2015. The emergence of plasmid mediated quinolone
resistance qnrA2 in extended spectrum b-lactamase producing Klebsiella pneumoniae in the
Middle East. Daru. 23:34–40.
Sabatini S, Gosetto F, Manfroni G, Tabarrini O, Kaatz GW, Patel D, Cecchetti V. 2011. Evolution
from a natural flavones nucleus to obtain 2-(4-Propoxyphenyl) quinoline derivatives as potent
inhibitors of the S. aureus NorA efflux pump. J Med Chem. 54(16):5722–5736.
Santos JFS, Tintino SR, Freitas TS, Campina FF, Menezes IRA, Siqueira-Ju
nior JP, et al. 2018. In
vitro e in silico evaluation of the inhibition of Staphylococcus aureus efflux pumps by caffeic
and gallic acid. Comp Immunol Microbiol Infect Dis. 57:22–28.