In vitro additive effect of Hyptis martiusii in the resistance to aminoglycosides of methicillin-resistant Staphylococcus aureus

Abstract

Context: Bacterial infectious agents represent a risk to populations, where they are responsible for the high morbidity and mortality. In combating these pathogens, our main line of defense is the use of antibiotics. However, the indiscriminate use of these drugs select resistant strains to these same drugs.

Objective: In this study the ethanol extract of Hyptis martiusii Benth. (EEHM) (Lamiaceae) was tested for its antimicrobial activity against aminoglycoside multi-resistant Staphylococcus aureus (MRSA).

Materials and methods: In this study, the ethanol extract of H. martiusii was prepared and tested with chlorpromazine for its antimicrobial activity using the microdilution method. Chlorpromazine and the ethanol extract were used alone and also in combination with aminoglycosides against a MRSA strain resistant to these antibiotics to determine the participation of efflux systems in resistance mechanisms. The FIC index was calculated and evaluated by the checkerboard method.

Results: A potentiating effect between this extract and aminoglycosides was demonstrated. Similarly, a potentiating effect of chlorpromazine with kanamycin was detected, indicating the involvement of an efflux system in the resistance to this aminoglycoside. The checkerboard method with combinations of aminoglycosides and EEHM demonstrated additive effect with kanamycin and gentamicin. It is therefore suggested that extracts from H. martiusii could be used as a source of plant-derived natural products with resistance- modifying activity.

Conclusion: This is the first report about the modifying antibiotic activity of Hyptis martiusii, constituting a new approach against bacterial resistance to antibiotics as aminoglycosides.

Keywords::

• Aminoglycosides
• chlorpromazine
• modifying antibiotic activity
• Hyptis martiusii

Introduction

Staphylococcus genus is widely spread in nature being part of the indigenous microbiota of skin and mucosa of animal and birds. Some Staphylococcus species are frequently recognized as etiological agents of many animal and human opportunistic infections (Nostro et al., 2004). S. aureus, S. epidermidis, S. saprophyticus and S. haemolyticus are the most important species as community and nosocomial human infection causing agents. In addition to causing different kinds of intoxication, S. aureus has been the most common etiological agent of festering infections that attack different tissues and/or organs (e.g., furuncle, carbuncle, abscess, myocarditis, endocarditis, pneumonia, meningitis, bacterial arthritis) (Verhoeff et al., 1999; Pereira et al., 2004). The cell capsule, peptidoglycan, teichoic acids, adhesins and synthesis of enzymes and extracellular toxins are some virulent attributes present in/on S. aureus cells (Nostro et al., 2004).

With the increase of resistance to antibiotics, natural products could be an interesting alternative (Mbwambo et al., 2007). Many plants have been evaluated not only for direct antimicrobial activity, but also as a resistance modifying agents (Gibbons, 2004). There is a growing interest in the influence of biologically active compounds isolated from plants on the treatment of diseases caused by antibiotic-resistant microorganisms (Austin et al., 1999). One such disease is cancer, because the indirect effect of bacteria over the inflammatory process, enhancing the free radical productions, resulting in damage to cellular components such as DNA, proteins and lipid membranes (Vogelmann & Amieva, 2007). The use of plant extracts and phytochemicals, both with known antimicrobial properties, can be of great significance in therapeutic treatments. In the last few years a number of studies have been conducted in different countries to prove such efficiency (Saeed & Tariq, 2007). Many plants have been evaluated not only for direct antimicrobial activity, but also as resistance modifying agents (Coutinho et al., 2008a, 2009a, 2009b, 2009c, 2009d).

Clinicians write millions prescriptions for antimicrobials every year (Karras, 2006). Clinicians need to apply sound concepts when prescribing antimicrobials to both achieve a good outcome and avoid encouraging resistance. There is convincing evidence that inappropriate use of antibiotics directly leads to the development of resistant organisms (Malhutra-Kumar et al., 2007). To prevent this, it is necessary educate all health workers regarding healthy drug use and regarding the natural history of infection, emphasizing palliative therapies and infection control measures (Roe, 2008). Infectious diseases are the major cause of morbidity and mortality and experience has shown that an approach that seeks to “defeat” infectious diseases will not work. Long-term solutions must acknowledge this, and nurses and other healthcare professionals must take a proactive part in finding alternative solutions (Purssel, 2005).

Hyptis martiusii Benth. (Labiatae) is a small shrub family with few pharmacological reports. Antitumor, cytotoxic, antibacterial, phototoxic, and insecticidal activities were identified (Araújo et al., 2003, 2006; Costa-Lotufo et al., 2004; Costa et al., 2005; Coutinho et al., 2008b, 2009e). With other plants from the genus Hyptis were isolated chemical compounds such as flavonoids (Isobe et al., 2006), diterpenes (Ohsaki et al., 2005) and sesquiterpenes (Facey et al., 2005), with antimicrobial, insecticidal, analgesic, and antiplasmodial activity (Okiemy-Andissa et al., 2004; Chukwujekwu et al., 2005; Fragoso-Serrano et al., 2005). Leaves, stems and fruits from these plants are used alone or combination by several populations as a popular remedy. Plants of this genus are used by indigenous populations of Africa, America and India as a curative or preventive antimalarial remedy and for the treatment of itching, cough, and cold (Kala, 2005; Vigneron et al., 2005).

Several chemical compounds, from synthetic or natural sources, such as the tricyclic neuroleptics and antidepressants, have direct activity against many species of bacteria, enhance the activity of specific antibiotics, reverse the natural resistance of specific bacteria to given antibiotics, promote the elimination of plasmids from bacteria such as Escherichia coli, and inhibit transport functions of the plasma membrane to given antibiotics. Inhibition of plasma membrane-based efflux pumps have been observed too (Molnar et al., 2004; Wolfart et al., 2006). The enhancement of antibiotic activity or the reversal of antibiotic resistance by natural or synthetic non-conventional antibiotics affords the classification of these compounds as modifiers of antibiotic activity.

Aminoglycosides are potent bactericidal antibiotics targeting the bacterial ribosome and the increase of bacterial resistance to aminoglycosides is widely recognized as a serious health threat (Jana & Deb, 2006). The most important mechanisms of resistance to aminoglycosides are the active efflux and enzymatic inactivation (Smith et al., 2007).

In this work, we evaluate the ethanol extract of Hyptis martiusii as a resistance modifying agent in an aminoglycoside-resistant strain of S. aureus.

Methods

Strains

The strain used was the clinical isolate Staphylococcus aureus 358 (SA358), resistant to several aminoglycosides (Freitas et al., 1999). The strain was maintained in heart infusion agar slants (HIA, Difco, Lawrence, USA) and, prior to assay, the cells were grown overnight at 37°C in brain heart infusion (BHI, Difco, Lawrence, USA).

Plant material

Leaves of H. martiusii were collected between the period of April and June of 2009 in the county of Crato, Ceará state, Brazil. The plant material was identified by the botanist Arlene Pessoa and voucher specimen has been deposited with the number 464 at Herbarium “Dárdano de Andrade Lima” of Universidade Regional do Cariri.

Preparation of ethanol extract from Hyptis martiusii

Aerial parts (200 g of leaves) were oven-dried at room temperature and powdered. The powdered material was extracted by maceration using 1 L of 95% ethanol as solvent at room temperature. The moisture was reserved for 72 h at room temperature. The extracts were then filtered and concentrated under vacuum in a rotary evaporator. For the tests, the extract was diluted in DMSO and its highest concentration remaining after dilution into broth caused no inhibition of bacterial growth.

Drugs

Chlorpromazine, gentamicin, kanamycin, tobramycin, amikacin and neomycin were obtained from Sigma. All drugs were dissolved in sterile water.

Drug susceptibility test

The minimum inhibitory concentrations (MIC) of ethanol extract from Hyptis martiusii (EEHM), antibiotics and chlorpromazine (CPZ) were determined in BHI by microdilution assay using suspensions of 10⁵ colony forming unit (CFU)/mL and a drug concentration range of 1024 to 1 μg/mL (two-fold serial dilutions) (NCCLS, 2008). MIC was defined as the lowest concentration at which no growth was observed. For the evaluation of EEHM as a modulator of antibiotic resistance, MICs of the antibiotics were determined in the presence of EEHM (32 μg/mL) and CPZ (16 μg/mL) at sub-inhibitory concentrations, and the plates were incubated for 24 h at 37°C. CPZ was used as a positive control for efflux pump inhibition.

Checkerboard method

The SA358 strain was tested by the microdilution checkerboard technique (Eliopoulos & Moellering, 1991). Suspensions of 10⁵ CFU/mL of bacterial culture were prepared and distributed into microtiter trays containing varying concentrations of the different drugs. The inoculated trays were incubated at 37°C for a period of 24 h, and then evaluated for bacterial growth. In order to evaluate the activity of combinations of drugs, fractional inhibitory concentration (FIC) indices were calculated as FIC^A + FIC^B, where FIC^A and FIC^B represent the minimum concentrations that inhibited the bacterial growth for drugs A and B, respectively: FIC^A = MIC^A combination/MIC^A alone and FIC^B = MIC^B combination/MIC^B alone. A mean FIC index was calculated based on the following equation:

and the interpretation made as follows: synergistic (< 0.5), additive (0.5-1.), indifferent (> 1), or antagonistic (> 4).

Results

The addition of EEHM to the growth medium at 32 μg/ mL produced a dramatic reduction in the MIC for kanamycin, gentamicin and neomycin in the strain S. aureus 358, demonstrating a synergistic effect of EEHM with aminoglycosides (Table 1).

Table 1.  MIC values (μg/mL) of aminoglycosides in the absence and presence of EEHM and CPZ in Staphylococcus aureus (SA) 358.

Antibiotics	SA 358
	MIC alone	Combined MIC
		EEHM (32 μg/mL)	CPZ (16 μg/mL)
EEHM	64	–	–
Amykacin	8	8	8
Gentamicin	8	1	8
Kanamycin	≥ 1024	2	8
Neomycin	16	≤  1	16
Chlorpromazine	16	–	–

MIC, minimal inhibitory concentration; EEHM, ethanol extract of Hyptis martiusii; CPZ, chlorpromazine.

A MIC reduction for kanamycin was also observed when CPZ was added to the growth medium at 16 μg/ mL, which indicates the involvement of an efflux pump in the resistance to this antibiotic (Table 1). The effect was also observed when EEHM was added to the medium, suggesting that EEHM is a putative inhibitor of an efflux pump (Table 1), but additional experiments are needed to confirm such mechanisms.

Synergistic effect of CPZ with amikacin, gentamicin or neomycin was not observed, which suggests the occurrence of other resistance mechanisms or of a CPZ-insensitive efflux pump that can be blocked by EEHM (Table 1).

Table 2 shows the results of the combinations of antibiotics and the EEHM, at or below their MIC. An additive effect was observed with the combination of EEHM with kanamycin and gentamicin. The combinations of EEHM and amikacin or neomycin showed a FIC index of 3 and the combinations of the extract with kanamycin and gentamicin showed a FIC index of 0.5 and 0.625, respectively. Natural products of Hyptis martiusii having an additive effect on aminoglycosides resistance have not been previously reported using the checkerboard method.

Table 2.  MIC of antibiotics and the effect of combinations with EEHM against Staphylococcus aureus 358.

Antibiotic + EEHM	MIC (μg/mL)	FIC index (Type of interaction)
EEHM	64
amikacin	8
kanamycin	≥ 1024
gentamicin	8
neomycin	16
EEHM/amikacin	128/16	4 (indifferent)
EEHM/kanamycin	32/32	0.53 (additive)
EEHM/gentamicin	32/1	0.625 (additive)
EEHM/neomycin	64/32	3 (indifferent)

MIC, minimal inhibitory concentration; EEHM, ethanol extract of Hiptys martiusii; FIC, fractional inhibitory concentration.

Discussion

Phenothiazines such as chlorpromazine probably act on the plasma membrane of bacteria, affecting the efflux pumps. This modification of drug permeability could enhance the activity of antibiotics that act within the cell, such as the aminoglycosides (Kristiansen & Amaral 1997).

Combining natural products with synthetic drugs to improve efficacy has been investigated for other plants of the Lamiaceae family. The essential oil of Agastache rugosa Kuntze (Labiatae) and its main constituent estragole both showed synergism with ketoconazole against Blastoschizomyces capitatus (Shin & Kang, 2003). Estragole showed synergism too with amphotericin B against Candida species (Shin & Pyun, 2004). Against bacteria, data from the checkerboard titer test confirmed synergism between norfloxacin (a quinolone) and Thymus magnus (Nakai) Nakai (Labiatae) essential oil or thymol and the extracts of Mentha arvensis L. (Labiatae) and H. martiusii were effective to enhance the antibiotic activity of aminoglycosides against Escherichia coli and Staphylococcus aureus (Shin & Kim, 2005; Coutinho et al., 2008a, 2009a, 2009c). This study demonstrates the additive effect of H. martiusii against Gram positive bacteria.

The results obtained indicate that Hyptis martiusii (and broadly Lamiaceae) could serve as a source of plant-derived natural products with modifying antibiotic activity, an interesting alternative to combat antibiotic multi-resistance.

Declaration of interest

This work was granted by the Brazilian research agencies: CNPq and FAPESQ/PB.