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Assessment of the antibacterial activity of galangin against 4-quinolone resistant strains of Staphylococcus aureus.

Abstract

The flavonol galangin is present in numerous plants and is a major constituent of Helichrysum aureonitens, a perennial herb used by South African indigenes to treat infection. In the present study, the antibacterial activity of galangin was assessed against 17 strains of 4-quinolone resistant S. aureus using an agar dilution assay. It was determined that the flavonol had a minimum inhibitory concentration (MIC) of approximately 50 [micro]g/ml against 16 of these strains, including those which exhibited 250- and 500-fold increases in norfloxacin resistance. The remaining one strain, which possessed an amino acid alteration in the GrlB subunit of topoisomerase IV, had increased susceptibility to galangin. Control strains of 4-quinolone sensitive S. aureus were also found to have MICs of 50 [micro]g/ml. The topoisomerase IV enzyme may therefore be implicated in the antibacterial mechanism of action of galangin. Clearly however, there is no cross-resistance between galangin and the 4-quinolones, and the flavonol therefore warrants further investigation as an antibacterial agent.

[c] 2005 Elsevier GmbH. All rights reserved.

Keywords: Helichrysum aureonitens; Galangin; Flavonol; Antibacterial activity; 4-quinolone; Topoisomerase; Staphylococcus aureus

Introduction

Staphylococcus aureus is an important human pathogen, which causes life-threatening systemic infections such as pneumonia, septicemia, endocarditis and osteomyelitis (Pan et al., 2002). During the last decade, there has been an increase in the prevalence of methicillin resistant S. aureus (MRSA), and such strains now account for almost 50% of all clinical isolates in the UK. The recent emergence of vancomycin resistance among strains of MRSA is deeply troubling and highlights the urgent need for new classes of antibiotics (Adcock, 2002).

Natural products have traditionally been a rich source of antimicrobial agents (Silver and Bostian, 1990) and flavonoids, a group of heterocyclic organic compounds that occur widely in the plant kingdom (Havsteen, 1983), are increasingly becoming the subject of medical research (Harborne and Williams, 2000). In the last couple of years, the flavonol galangin (3,5,7-trihydroxy-flavone) has been the focus of at least two antibacterial investigations (Denny et al., 2002; Cushnie et al., 2003). This compound is present in numerous plants, including Alpinia officinarum, Populus nigra (Harborne and Baxter, 1999) and Castanea sativa (Basile et al., 2000), and is one of the major constituents of Helichrysum aureonitens, a perennial herb used by South African indigenes to treat infection (Afolayan and Meyer, 1997). Galangin also contributes significantly to the antimicrobial activity of propolis or 'bee glue' (Pepeljnjak et al., 1982; Park et al., 1998; Bosio et al., 2000; Hegazi et al., 2000). In the study by Denny et al. (2002), galangin was shown to inhibit the activity of [beta]-lactamase from Stenotrophomonas maltophilia. This might suggest a possible future role for the phytochemical in combination with [beta]-lactam antibiotics. In the previous study by our group (Cushnie et al., 2003), galangin was shown to have antibacterial activity in its own right, and although the spectrum of activity was found to be quite narrow, inhibitory activity was detected against all six of the [beta]-lactam sensitive and resistant strains of S. aureus tested.

It has been suggested that, if a mechanism of action could be established for galangin, future optimisation may allow the development of a pharmacologically acceptable antibacterial agent (Cushnie et al., 2003). However, the structural similarity between galangin and the 4-quinolones (Fig. 1) raises concerns that these compounds might share similarities in their mechanisms of action targeting DNA replication, and that consequently the flavonol may have decreased activity against strains of S. aureus that are already 4-quinolone resistant. If there was substantial cross-resistance between the 4-quinolones and galangin, then this would immediately make redundant any plans to develop the phytochemical as an antibacterial agent. The present study sought to address these concerns by determining the activity of galangin against a number of 4-quinolone sensitive and resistant strains of S. aureus.

[FIGURE 1 OMITTED]

Materials and methods

Chemicals

Galangin was purchased from Aldrich (Gillingham, UK) and ammonia (0.88 specific gravity) was obtained from Fisons Scientific Apparatus (Loughborough, UK). Norfloxacin and sodium hydroxide were supplied by Sigma (Poole, UK) and sparfloxacin was kindly provided by Prof. Mark Fisher (Pan et al., 2002). Iso-sensitest nutrient agar and broth was from Oxoid (Basingstoke, UK).

Bacterial strains

Fifteen methicillin sensitive clinical isolates of S. aureus were generously supplied by Prof. Glen Kaatz. One of these was also 4-quinolone sensitive with wildtype gyrase and topoisomerase IV genes. The remaining 14 possessed various GyrA, GrlA and GrlB alterations (see Table 1) and exhibited different degrees of 4-quinolone resistance (Kaatz and Seo, 1998; Gootz et al., 1999). Prof. Laura Piddock provided S. aureus F265, a second step 4-quinolone resistant mutant carrying grlA and gyrB mutations (Griggs et al., 2003; Table 2). Unfortunately, the first step grlA mutant from which this had been derived was no longer viable so the original antibiotic sensitive parent strain, S. aureus NCTC 8532, was selected for comparative analysis instead. Two 4-quinolone resistant S. aureus strains (2SSA4 and 2SSA6), each possessing a 15 base pair deletion in the gyrB gene, together with their parent strain (1SSA1; generated from antibiotic sensitive S. aureus RN4220) were also donated by Prof. Mark Fisher (Pan et al., 2002; Table 2). Bacteria were subcultured and maintained on agar. To obtain bacteria in exponential growth phase, three to five well isolated colonies of the same morphological type (NCCLS, 2000) were lifted from these plates and inoculated into 100ml of broth. With the exception of strains 2SSA4 and 2SSA6, all bacteria were then incubated statically at 37[degrees]C for 18 h (Mark II proportional temperature controller; LEEC Ltd., Nottingham, UK). S. aureus strains 2SSA4 and 2SSA6 required 26 and 24h incubation at 37[degrees]C, respectively.

Determination of minimum inhibitory concentrations (MICs) for 4-quinolones and galangin against the 4-quinolone sensitive and resistant strains of S. aureus

MICs were determined using the agar dilution assay described previously (Cushnie et al., 2003). Six strains of S. aureus were analysed at a time, and 4-quinolone(s) and galangin were tested concurrently to allow the detection of any wild-type reversions. Stock solutions of 400 [micro]g/ml galangin and 1000 [micro]g/ml norfloxacin were prepared in 0.1% (v/v) ammonia solution and 6.93 mM sodium hydroxide, respectively. A 2.56 mg/ml sparfloxacin stock solution was made in 0.1 M sodium hydroxide (Pan, 2003).

Results

The MICs determined for norfloxacin and galangin against the 15 clinical isolates of S. aureus are shown in Table 1. Data from restriction fragment length polymorphism (RFLP) analysis (Kaatz and Seo, 1998) and DNA sequence analysis (Gootz et al., 1999) is included for ease of reference. The MICs determined for sparfloxacin, norfloxacin and galangin against the wild type and gyrB mutant strains of S. aureus are presented in Table 2, along with data from DNA sequence analysis (Griggs et al., 2003; Pan et al., 2002), again for the purpose of reference.

Discussion

The pattern of susceptibility exhibited by the 15 clinical isolates to norfloxacin (Table 1) is very interesting and, in some cases, the correlation between specific mutations and resistance is more straightforward than previously published data suggests (Kaatz and Seo, 1998). Importantly, this indicates that unidentified variables affecting norfloxacin resistance are minimal. When determining MIC values in the present study, instead of adjusting the turbidity of all bacterial cell suspensions to the same standard value (NCCLS, 2000), enumeration graphs [optical density at 500 nm vs. viable count (cfu/ml)] were prepared and employed in the testing of each individual isolate. Therefore, despite performing the MIC analyses in complete accordance with the NCCLS guidelines, the values obtained by Kaatz and Seo may be less accurate than those determined in the present study. Importantly though, the results in Table 1 are in overall agreement with these findings, with norfloxacin resistance increasing as the number of mutations in the type II topoisomerase genes increase. It can be seen that there is an enormous (500-fold) difference in the susceptibilities of the wild-type S. aureus 1199 strain and the heavily mutated S. aureus 1628 strain. When Gootz et al. (1999) examined these norfloxacin resistant clinical isolates, it was shown that the strains also have increased resistance to trovafloxacin, ciprofloxacin, levofloxacin, sparfloxacin and pefloxacin. Encouragingly though, the results presented in Table 1 indicate that mutations in the quinolone resistance determining region (QRDR) of gyrA, grlA and grlB genes of S. aureus, do not cause an increase in galangin resistance.

Since S. aureus 2SSA4 and 2SSA6 were generated from the parent strain 1SSA1, it is almost certain that the observed increases in sparfloxacin and norfloxacin MICs (Table 2) are a direct result of the alterations to the GyrB subunit. It is worth noting that the sparfloxacin MICs determined in the present study correspond well with those obtained previously (Pan et al., 2002). Although the parent strain of S. aureus F265 was not available for study, by contrasting the results obtained with F265 and its 'grandparent' strain (S. aureus NCTC 8532), it can be seen that alterations in the GyrB and/or GrlA subunits are the likely cause of an increase in sparfloxacin and norfloxacin MIC values (Table 2). S. aureus F265 has also been reported to have increased resistance to nalidixic acid, ciprofloxacin, moxifloxacin, trovafloxacin, grepafloxacin, levofloxacin and ofloxacin, compared to S. aureus NCTC 8532 (Griggs et al., 2003). Importantly however, alterations in the GyrB topoisomerase subunit of S. aureus which appear to cause an increase in 4-quinolone resistance, do not cause an increase in resistance to galangin (Table 2).

Of the 20 strains of S. aureus tested (Tables 1 and 2), all but one (1066) exhibited a relatively uniform level of susceptibility to galangin. Interestingly, this strain possessed an amino acid substitution (serine to proline) at position 410 of the GrlB subunit. Fournier and Hooper (1998) have reported a grlB mutation in S. aureus (asparagine replaced by aspartic acid at position 470 of the GrlB subunit), which is responsible for both quinolone resistance and coumarin hypersus-ceptibility. It is possible that the grlB mutation in S. aureus 1066 has similar pleiotropic effects. Although this mutation did not cause an increase in norfloxacin resistance (Table 1), it is associated with a 2-fold increase in resistance to ciprofloxacin and sparfloxacin (Gootz et al., 1999), and a 4- to 8-fold increase in susceptibility to galangin (Table 1). The type II topoisomerase enzymes may therefore be implicated in galangin's antibacterial mechanism of action. Ohemeng and colleagues previously detected no inhibitory activity from galangin against Escherichia coli DNA gyrase (Ohemeng et al., 1993; Hilliard et al., 1995), but the flavonol may have activity against S. aureus DNA gyrase or it may primarily target the topoisomerase IV enzyme. It is also possible, however, that S. aureus 1066 has an additional uncharacterised alteration, which is responsible for the strain's increased susceptibility to galangin, and is wholly unrelated to the topoisomerase enzymes. This could be investigated by further screening of S. aureus topoisomerase mutants, including those generated from exposure to coumarins (e.g. novobiocin) or cyclothialidine.

In summary, the results of the present study indicate that there is no cross-resistance between galangin and the 4-quinolones. Clearly therefore, the flavonol's antibacterial activity warrants further attention. The connection between the amino acid substitution at position 410 of the GrlB subunit and increased galangin susceptibility is unknown but it is possible that, as is the case with the coumarins, the type II topoisomerase enzymes are involved in galangin's antibacterial mechanism of action.

Acknowledgements

The authors are deeply indebted to Dr. Glen Kaatz, Prof. Mark Fisher and Prof. Laura Piddock for supplying the 4-quinolone resistant strains of S. aureus, and wish to thank Dr. Xiao-Su Pan and Dr. Maggie Johnson for their advice and assistance in subculturing these strains. Thanks are extended to Dr. Derek Chapman and Miss. Vivienne Hamilton for their continued support and encouragement.

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Kaatz, G.W., Seo, S.M., 1998. Topoisomerase mutations in fluoroquinolone-resistant and methicillin-susceptible and-resistant clinical isolates of Staphylococcus aureus. Antimicrob. Agents Chemother. 42 (1), 197-198.

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Ohemeng, K.A., Schwender, C.F., Fu, K.P., Barrett, J.F., 1993. DNA gyrase inhibitory and antibacterial activity of some flavones (1). Bioorg. Med. Chem. Lett. 3 (2), 225-230.

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Park, Y.K., Koo, M.H., Abreu, J.A.S., Ikegaki, M., Cury, J.A., Rosalen, P.L., 1998. Antimicrobial activity of propolis on oral microorganisms. Curr. Microbiol. 36 (1), 24-28.

Pepeljnjak, S., Jalsenjak, I., Maysinger, D., 1982. Growth inhibition of Bacillus subtilis and composition of various propolis extracts. Pharmazie 37 (12), 864-865.

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T.P.T. Cushnie, A.J. Lamb*

School of Pharmacy, The Robert Gordon University, Schoolhill, Aberdeen, Scotland AB10 1FR, UK

Received 27 February 2004; accepted 4 July 2004

*Corresponding author. Tel.: +441224262526; fax: +441224262555.

E-mail address: a.lamb@rgu.ac.uk (A.J. Lamb).
Table 1. The susceptibilities of S. aureus clinical isolates carrying
topoisomerase mutations to norfloxacin and galangin

 Results of RFLP Amino acid substitutions predicted from DNA
Strain analysis (a) sequence analysis of QRDR
no. gyrA grlA GyrA GyrB GrlA GrlB

1199 None None ND ND ND ND
1081 None 79/80 None None None Glu 422 to Asp
1134 None 79/80 None None Ser 80 to Tyr None
1039 None 79/80 None None Ser 80 to Phe None
1035 None 79/80 None None Ser 80 to Phe None
1249 None 79/80 None None Ser 80 to Phe None
1642 None 79/80 None None Ser 80 to Phe None
1305 None 84 None None Glu 84 to Lys None
 872 None 84 None None Glu 84 to Lys Glu 422 to Asp
1066 None 84 None None Glu 84 to Lys Ser 410 to Pro
 & Glu 422 to
 Asp
1639 83/84 79/80 ND ND ND ND
1640 83/84 79/80 Ser 84 to None Ser 80 to Tyr None
 Leu
1275 83/84 79/80 Ser 84 to None Ser 80 to Phe His 478 to Tyr
 Leu
1637 83/84 79/80 Ser 84 to None Ser 80 to Phe His 478 to Tyr
 Leu
1628 83/84 79/80 Ser 84 to None Ser 80 to Phe Glu 422 to Asp
 Leu

Strain MIC ([micro]g/ml)
no. Norfloxacin Galangin

1199 0.7813 50
1081 3.125 50
1134 12.5 50
1039 25 50
1035 25 50
1249 25 50
1642 25 50
1305 25 25 to 50
 872 25 50
1066 25 6.25 to 12.5
1639 50 50
1640 50 50
1275 100 50
1637 200 50
1628 400 50

RFLP, restriction fragment length polymorphism; QRDR, quinolone
resistance determining region; ND, not determined.
(a) Codon(s) in which a mutation(s) resulting in loss or acquisition of
recognition sites was found.

Table 2. The susceptibilities of wild type and gyrB mutant strains of S.
aureus to sparfloxacin, norfloxacin and galangin

 Amino acid substitutions/deletions predicted from DNA
 sequence analysis of QRDR
Strain no. GyrA GyrB GrlA GrlB

NCTC 8532 None None None None
F265 None Asp 437 to His Ser 80 to Phe None
1SSA1 None None None None
2SSA4 None Deletion from None None
 405 to 409
2SSA6 None Deletion from None None
 405 to 409

 MIC ([micro]g/ml)
Strain no. Sparfloxacin Norfloxacin Galangin

NCTC 8532 0.125 1.563 50
F265 1.0 50 50
1SSA1 0.5 3.125 50
2SSA4 4.0 6.25 50
2SSA6 4.0 6.25 50

QRDR, quinolone resistance determining region.
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Author:Cushnie, T.P.T.; Lamb, A.J.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
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Date:Feb 1, 2006
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