Antibacterial Properties of Senecio sandrasicus. on Multidrug-Resistant Stenotrophomonas maltophilia.

Abstract

In this study, the antibacterial activity of Senecio sandrasicus. P.H. Davis (Asteraceae), an endemic species of southwestern Anatolia, was investigated. The antibacterial effects of extracts of chloroform, ethanol, hexane, and ethyl acetate, which were obtained from voucher specimens of the plant, were tested by a disk diffusion method against 10 strains of Stenotrophomonas maltophilia.. The strains are resistant to various antibiotics including some widely used β-lactam antibiotics (penicillin and cephalosporins), β-lactam + β-lactamase inhibitors, monobactams, carbapenems, and aminoglycosides. The results showed that all of the four extracts of S. sandrasicus. had similar potencies against all tested bacteria. The activities were increased dependies on the doses of extracts; 3.6 µg/disk extracts had higher antibacterial activity than 0.4 µg/disk. The strains of S. maltophilia., which are important pathogens, were inhibited by some extracts of S. sandrasicus..

Keywords:

• Antibacterial activity
• multidrug-resistant Stenotrophomonas maltophilia
• Senecio sandrasicus

Introduction

Stenotrophomonas maltophilia. (Xanthomonodaceae) (Palleroni & Bradbury, 1993), previously known as Pseudomonas maltophilia. (Pseudomonadaceae) (Hugh & Ryschenkow, 1961) and subsequently as Xanthomonas maltophilia. (Xanthomonodaceae) (Swings et al., 1983), has received much attention in the past decade because of its role as a pathogenic microorganism in an increasing number of clinical syndromes (Robin & Janda, 1996), such as bacteremia, infections of the respiratory and urinary tracts, skin and soft tissue infections, biliary tract infection, meningitis, serious wound infections, conjunctivitis, endocarditis (Fisher et al., 1981; Denton & Kerr, 1998), cystic fibrosis, and central nervous system infection. S. maltophilia. has also been described to be an important nosocomial pathogen (Denton & Kerr, 1998).

The treatment of infections caused by this microorganism is difficult because S. maltophilia. is frequently resistant to most of the widely used antibiotics (Vartivarian et al., 1994; Liu et al., 1995; Skaehill, 2000; Krueger et al., 2001). This resistance is usually attributed to low cell wall permeability (Yamazaki et al., 1989; Cullmann, 1991) and, in the case of β-lactams, mainly to two β-lactamases: L1, (a metallo-β-lactamase) and L2 (a non-metallo-serine-β-lactamase) (Bush et al., 1995). The metallo-β-lactamase (MBL) hydrolyzes most β-lactam drugs including carbapenem (Bush et al., 1995). None of the commercially available β-lactamase inhibitors are active against these enzymes (Miller et al., 2001). The L2 cephalosporinase is inhibited by clavulanic acid (Bush et al., 1995). L2 appears to be active against the monobactam aztreonam (Babalova et al., 1995). Trimethoprim-sulfamethoxazole (TMP-SMZ) is bacteriostatic for most isolates, hence high doses are usually recommended (Bush et al., 1995). High doses may be especially difficult for elderly patients with poor renal function to tolerate because of severe skin reactions, bone marrow suppression, and thrombocytopenia. Combination therapy may be attempted when the patient is seriously ill, clinical response is poor, or when in vitro. testing demonstrates resistance to all agents tested. For patients allergic to TMP-SMZ, a combination of ticarcillin-clavulanate and ciprofloxacin may be useful. Patients with a documented penicillin allergy may be treated with TMP-SMZ and ciprofloxacin (Poulos et al., 1995). The aminoglycosides show a minimum activity against S. maltophilia., due to both temperature-dependent changes in outer membrane conformation and the presence of aminoglycoside modifying enzymes (Denton & Kerr, 1998). Minocycline, doxycycline, and the newer quinolones (clinafloxacin, sparfloxacin) appear to have reasonably good in vitro. activity and may be considered as an option (Felegie et al., 1979).

The intrinsic broad-spectrum antimicrobial resistance of S. maltophilia. is probably one of the factors responsible for its emergence as an opportunistic pathogen and often leads to therapeutic problems (Lesco-Bornet & Bergogne-Berezin, 1997). Available therapeutic alternatives for S. maltophilia. are limited; hence, there is a continuous need for alternative inhibitors. One of the routine approaches for the search of biologically active substances is the systematic screening of plants, which have been sources of many useful therapeutic agents.

The genus Senecio. is represented by 39 species in Anatolia. Senecio sandrasicus. P.H. Davis (Asteraceae) is an endemic species found on one of the most important mountains for plant reserves in southwestern Anatolia (Davis, 1975). The lack of records of previous chemical and biological studies of the plant stimulated us to initiate them through the current work. Despite the absence of reports of this plant as an antimicrobial agent, the fact that several species of Senecio. exhibit antimicrobial activity (Perez et al., 1999; Denny et al., 2002; Garcia et al., 2003; Rojas et al., 2003) led us to check S. sandrasicus. on S. maltophilia., which is typically resistant to most antimicrobial drugs commonly used in in vitro. screening studies.

In this paper, we examine the efficacy of extracts made from S. sandrasicus. in terms of their ability to inhibit the growth of multiresistant S. maltophilia. under in vitro. conditions.

Materials and Methods

Plant material

S. sandrasicus. was collected during the flowering stage in July 2003 from Sandras Mountain at 1500–1600 m, Köyceğiz, Muğla, Turkey. Voucher specimens of the plants were collected and taxonomically identified by Dr. Ömer Varol, and the voucher specimens (herbarium no. 4463) have been deposited at the Herbarium, Department of Biology, Faculty of Science, the University of Mugla, Mugla, Turkey.

Preparation of the organic extracts

Powdered aerial parts of the plant (107 g) were repeatedly extracted with solvents of increasing polarity beginning with n.-hexane (C₆H₁₂) (S₁), chloroform (CHCl₃) (S₂), ethanol (C₂H₅OH) (S₃), and ethyl acetate (EtOAc) (S₄) (107 g/1000 ml). The extracts were evaporated to dryness under reduced pressure, and the residues obtained yielding 1.36 g (S₁), 1.49 g (S₂), 8.25 g (S₃), and 1.31 g (S₄) were stored at +4°C. Each residue was dissolved at 20 mg/ml and 180 mg/ml with n.-hexane, chloroform, ethanol, and ethyl acetate, respectively.

Antibacterial activity

Bacteria and condition for cultivation

Ten bacterial strains of S. maltophilia., which have been resistant to various antibiotics, were used to assess the antibacterial properties of the test samples. The sensitivity of the strains to the standard antibiotics was determined by disk diffusion method. The inhibition zone diameter (mm) was assessed by using the NCCLS standard (NCCLS, 1999). S. maltophilia. MU 64 is resistant to all the antibiotics. S. maltophilia. MU 25, MU 52, MU 63, and MU 137 are resistant to all the antibiotics except trimethoprim + sulfamethoxazole. S. maltophilia. MU 69 and MU 99 are resistant to all the antibiotics except ciprofloxacin. S. maltophilia. MU 23, MU 94, and MU 136 are resistant to all the antibiotics except ciprofloxacin and trimethoprim + sulfamethoxazole. The sensitivity of the strains to antibiotics is given in Table 1. All the strains were provided by the Mugla University Culture Collection. The cultures of bacteria were maintained in nutrient agar (NA) (Difco) slants in the dark at 4°C during the study. The bacteria were cultured in nutrient broth (NB) (Difco) at 30 ± 0.1°C.

Table 1 Antibiotic resistance patterns of S. maltophilia.

Stenotrophomonas maltophilia. strains	Resι.stance Patterns^a.
MU 23	MEZ, TIM, CAZ, FEP, CRO, CTX, KF, IPM, P, AK, TOB, NET, CN, TE, NOR, C, TVA, AM, PRL, ATM, SAM, AMC
MU 25	MEZ, TIM, CAZ, FEP, CRO, CTX, KF, IPM, P, AK, TOB, NET, CN, TE, CIP, NOR, C, TVA, AM, PRL, ATM, SAM, AMC
MU 52	MEZ, TIM, CAZ, FEP, CRO, CTX, KF, IPM, P, AK, TOB, NET, CN, TE, CIP, NOR, C, TVA, AM, PRL, ATM, SAM, AMC
MU 63	MEZ, TIM, CAZ, FEP, CRO, CTX, KF, IPM, P, AK, TOB, NET, CN, TE, CIP, NOR, C, TVA, AM, PRL, ATM, SAM, AMC
MU 64	MEZ, TIM, CAZ, FEP, CRO, CTX, KF, IPM, P, AK, TOB, NET, CN, TE, CIP, NOR, C, SXT, TVA, AM, PRL, ATM, SAM, AMC
MU 69	MEZ, TIM, CAZ, FEP, CRO, CTX, KF, IPM, P, AK, TOB, NET, CN, TE, NOR, C, SXT, TVA, AM, PRL, ATM, SAM, AMC
MU 94	MEZ, TIM, CAZ, FEP, CRO, CTX, KF, IPM, P, AK, TOB, NET, CN, TE, NOR, C, TVA, AM, PRL, ATM, SAM, AMC
MU 99	MEZ, TIM, CAZ, FEP, CRO, CTX, KF, IPM, P, AK, TOB, NET, CN, TE, NOR, C, SXT, TVA, AM, PRL, ATM, SAM, AMC
MU 136	MEZ, TIM, CAZ, FEP, CRO, CTX, KF, IPM, P, AK, TOB, NET, CN, TE, NOR, C, TVA, AM, PRL, ATM, SAM, AMC
MU 137	MEZ, TIM, CAZ, FEP, CRO, CTX, KF, IPM, P, AK, TOB, NET, CN, TE, CIP, NOR, C, TVA, AM, PRL, ATM, SAM, AMC

AK, amikacin (30 µg) [≤14 mm]; TOB, tobramycin (10 µg) [≤12 mm]; NET, netilmicin (30 µg) [≤12 mm]; CN, gentamicin (10 µg) [≤12 mm]; TE, tetracycline (30 µg) [≤14 mm]; SXT, trimethoprim + sulfamethoxazole (1.25 + 23.75 µg) [≤10 mm]; TVA, trovafloksasin (10 µg) [≤15 mm]; AM, ampicillin (10 µg) [≤13 mm]; MEZ, mezlocillin (75 µg) [≤15 mm]; TIM, ticarcillin + clavulanic acid (75 + 10 µg) [≤14 mm]; CAZ, ceftazidime (30 µg) [≤14 mm]; FEP, cephepim (30 µg) [≤14 mm]; CRO, ceftriaxone (30 µg) [≤13 mm]; CTX, cefotaxime (30 µg) [≤14 mm]; KF, cephalothin (30 µg) [≤14 mm]; IPM, imipenem (10 µg) [≤13 mm]; CIP, ciprofloxacin (5 µg) [≤15 mm]; NOR, norfloxacin (10 µg) [≤12 mm]; C: chloramphenicol (30 µg) [≤12 mm]; PRL, piperacillin (100 µg) [≤17 mm]; ATM, aztreonam (30 µg) [≤15 mm]; SAM, sulbactam + Ampicillin (10 + 10 µg) [≤11 mm]; AMC, amoxicillin + Clavulanic acid (20 + 10 µg) [≤19 mm].

^a.The NCCLS numerial values for the inhibition zones (in mm) of the bacteria to the antibiotics are given in the footnote above in square brackets (NCCLS, 1999). If the inhibition zones determined in this study are the same or smaller than the inhibition zones cited, then the strains are considered reistant to the antibiotic tested.

Antimicrobial assays

The antimicrobial activities of the various extracts of S. sandrasicus. were assayed by the disk diffusion method, also known as the Kirby-Bauer method (Bauer et al., 1966; Collins et al., 1995; NCCLS, 1999). The inoculum size of the bacteria was prepared by using a no. 0.5 McFarland tube to give concentration of 1 × 10⁸bacteria per milliliter. From the bacterial culture, 0.1 ml was aseptically inoculated into 15 ml of Mueller-Hinton agar (MHA) (Difco) tubes that had been warmed to 48–50°C. These mixtures were transferred into sterile Petri dishes and distributed homogeneously. The plates were held for 15–20 min at room temperature. Each extract (100 µl) was applied under suction to the sterile 6 mm disks (Schleichert & Schuell) using the multipoint inoculator. Prepared disks were added aseptically onto the agar with a cooled flamed pliers. The plates containing bacteria were incubated at 30°C for 24 h. At the end of the incubation periods, diameters of no-growth zones around the disks were measured to the nearest 0.1 mm using vernier calipers. Studies were performed in triplicate. Hexane, chloroform, ethyl acetate, and ethanol used as organic solvents for extractions were also used as controls.

Results

Antibiotic resistance patterns of S. maltophilia., used in this study, are given in Table 1. In the current study, the antibacterial activities of two different doses of S. sandrasicus. extracts against the bacteria were examined and potency quatitatively assessed by the presence or absence of inhibition zones and zone diameter. The results are given in Tables 2 and 3. As shown in Table 1, the standard antibacterial agents, antibiotics used against these bacteria, had no effect on the growth of these bacteria. Especially, antibiotics such as TMP-SMZ, ciprofloxacin, sulbactam + ampicillin, and amoxicillin + clavulanic acid, frequently recommended for the treatment of serious S. maltophilia. infections, had no effect on some strains. On the other hand, all four extracts of S. sandrasicus. inhibited the growth of these bacteria. Furthermore, the doses of these extracts (0.4 µg/disk and 3.6 µg/disk) were much lower than the doses of the antibiotics (Table 1) used.

Table 2 Antimicrobial activity of 0.4 µg extracts

	Diameter of the inhibition zone (mm)
Stenotrophomonas maltophilia. strains	Hexane extracts (0.4 µg/disk)	Ethanol extracts (0.4 µg/disk)	Chloroform extracts (0.4 µg/disk)	Ethyl acetate extracts (0.4 µg/disk)
MU 23	10	13	10	10
MU 25	10	10	9	8
MU 52	—	10	8	12
MU 63	12	10	11	10
MU 64	9	11	10	12
MU 69	—	9	9	11
MU 94	9	10	9	10
MU 99	9	11	9	12
MU 136	10	10	8	11
MU 137	10	11	10	11

Table 3 Antimicrobial activity of 3.6 µg extracts

	Diameter of the inhibition zone (mm)
Stenotrophomonas maltophilia. strains	Hexane extracts (3.6 µg/disk)	Ethanol extracts (3.6 µg/disk)	Chloroform extracts (3.6 µg/disk)	Ethyl acetate extracts (3.6 µg/disk)
MU 23	16	19	16	16
MU 25	15	16	14	13
MU 52	—	15	14	17
MU 63	18	16	16	15
MU 64	15	17	15	19
MU 69	—	15	16	18
MU 94	15	17	14	16
MU 99	14	17	13	19
MU 136	16	15	14	17
MU 137	15	17	16	18

Both doses of extracts of ethanol, chloroform, and ethyl acetate displayed antibacterial activities on all the bacteria. The extracts of hexane had an antibacterial activity on all bacteria except S. maltophilia. MU 52 and MU 69. All of the samples showed different antibacterial activities, but the differences were not excessive. However, the highest activity was obtained from the ethanol extract on S. maltophilia. MU 23. The chloroform extract had the lowest activity against all bacteria. In addition, different doses of extracts showed different antibacterial effects on these strains.

Discussion

The obtained results indicate that the two different doses of all extracts except for hexane extracts have an inhibitory effect on these bacteria. None of the doses of hexane extracts exhibited inhibitory effect on S. maltophilia. MU 52 and MU 69. The inhibition zones for bacterial strains that were sensitive to the 0.4 and 3.6 µg extracts of S. sandrasicus. were in the range of 8–13 and 13–19 mm, respectively (Tables 1 and 2). As can be seen from Tables 2 and 3, when the doses of extracts were increased nine fold, the antimicrobial activities of extracts were also increased. However, the increase in antimicrobial activities was not as much as the increase in doses of extracts. In addition, increasing of doses of extracts did not inhibit the growth of S. maltophilia. MU 52 and MU 69. When all the results were taken into consideration, in general, it was found that the ethyl acetate extracts of S. sandrasicus. displayed the highest antibacterial activity among other extracts. Chloroform extracts showed the lowest activity among all extracts.

The findings in this study supported the observations of some other researches that other species of the Senecio. genus have antibacterial properties (Perez et al., 1999; El-Shazly et al., 2002; Rojas et al., 2003; Uzun et al., 2004). However, no studies on antimicrobial activities of essential oils or extracts of S. sandrasicus. have been found in the literature.

In conclusion, various extracts of S. sandrasicus. have shown strong activity against S. maltophilia., which is resistant to various antibiotics. There is an alternative therapy for these strains of S. maltophilia. especially those resistant to all of the antibiotics such as S. maltophilia. MU 64. Therapies of this type are very important because of the types of organisms that have emerged as most problematic for patients within the ICU, including MRSA, VRE, ESBL, stably derepressed AmpC enzyme producers among Enterobacteriaceae, and MDR nonfermentative Gram-negative bacilli, principally P. aeruginosa., Acinetobacter. spp., and S. maltophilia. (Fluit et al., 2001; Fridkin et al., 2002). Furthermore, the dosages of extracts we used were much lower than the dosages of antibiotics. This in vitro. study provides evidence that the plant is potentially a rich source of antibacterial agents against multiresistant S. maltophilia.. Hence, the extracts of S. sandrasicus. may be useful as an alternative antimicrobial agent for S. maltophilia..