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Synergistic activity and mechanism of action of ceftazidime and apigenin combination against ceftazidime-resistant Enterobacter cloacae.

ARTICLE INFO

Keywords:

Apigenin

Naringenin

Ceftazidime

Ceftazidime-resistant E. cloacae

Synergistic activity

Mechanism of action

ABSTRACT

The purpose of this investigation was to examine the antibacterial and synergistic effect of naturally occurring flavonoids, apigenin, quercetin, naringenin and ceftazidime when use singly and in combination against ceftazidime-resistant Enterobacter cloacae strains by minimum inhibitory concentration (MIC), checkerboard and viable count methods. The mode of actions were also studied by electronmicoscopy, enzyme assay, outer and inner membrane permeabilisation. The results showed that these strains were positive in the ESBL--ampC genes combination by multiplex PCR. These findings were confirmed by MICs that these strains were resistant to ceftazidime, cefepime and flomoxef at >1024, 16-24, >256 [micro]g/m1 respectively, while susceptible to imipenem at 1-2 [micro]g/ml. The synergistic activity was observed at ceftazidime plus either apigenin or naringenin combinations with FIC indixes between < 0.01 and < 0.27 against these strains, whereas ceftazidime plus clavulanic acid or quercetin did not exhibit synergy. The modulation of ceftazidime-resistance by apigenin or narigenin significantly enhanced the activities of ceftazidime. The 5,7-01-I group of A ring and one 4'-OH group of the B ring in apigenin and naringenin are important for synergistic activity. Viable counts showed that the killing of ceftazidime-resistant E. cloacae DMST 21394 (CREC) cells by 3 [micro]g/m1 ceftazidime was potentiated by 314/m1 apigenin to low levels ([10.sup.3] cfuiml) over 6 h. Electronmicroscopy clearly showed that ceftazidime 3 [micro]g/m1 in combination with 3 pg/ml of apigenin also caused marked morphological damage of cell wall, cell shape and plasma membrane of this strain. Enzymes assays indicated that apigenin showed marked inhibitory activity against penicillinase type IV from E. cloacae. The results for outer membrane (OM) permeabilization in both nitrocefin (NCF) assay and crystal violet uptake showed that the combination of ceftazidime plus apigenin significantly altered OM permeabilisation of CREC compared to control or single treatment of these agents. Both o-nitrophenyl-[beta]-D-galactoside (ONPG) uptake and release of UV-absorbing material concentrations results exhibited that ceftazidime and apigenin combination damaged CREC cytoplasmic membrane (CM) and caused subsequent leakage of intracellular constituents. From the results, it can be concluded that apigenin and naringenin have the synergistic effect with ceftazidime to reverse bacterial resistance to this cephalosporin against CREC. This activity may be involved three mechanisms of action by apigenin. The first is on the peptidoglycan synthesis inhibition. The second mechanism is inhibition the activity of certain [beta]-lactamase enzymes. The third mode of action is alteration of OM and CM per-meabilization. Apigenin and naringenin have a sufficient margin of safety for therapeutic use. For this reason, apigenin and naringenin offer for the development of a valuable adjunct to ceftazidime against CREC, which currently almost cephalosporins resistance.

[c] 2012 Elsevier GmbH. All rights reserved.

Introduction

Enterobacter cloacae are significant causes of nosocomial infections. It is emphasized that E. cloacae are mainly responsible for pneumonia, wound infection and urinary tract infection in the most hospital. The three specimen types of sputum, secretions and pus, urine in the past 8 years in the First Bethune Hospital investigation showed that the antimicrobial resistance of E cloacae had increased. E. cloacae are intrinsically resistant to aminopenicillins, cefazolin, and cefoxitin due to the production of ampC [beta]-lactamases (Anggakusuma et al. 2009). Similarly, the resistance to antimicrobial agents in Enterobacteriaceae has become an increasingly relevant problem. International travel and tourism are important modes for the acquisition and spread of antimicrobial-resistant Enterobacteriaceae (Rukayadi et al. 2010). In addition, the E. cloacae from blood of inpatients at an urban public hospital in Berkeley was found that it carried globally-dispersed drug-resistance genes (Yanti et al. 2009b). Furthermore, the SHV-12 [beta]-lactamase of clinical isolates of E. cloacae with reduced susceptibility to ceftazidime and cefepime recovered from 2009 to 2010 at the university hospital of Mahdia, Tunisia, was analyzed by PCR analysis (Yoon et al. 2005). Likewise, the prevalence of infection by plasmid mediated ampC (pampC), which can hydrolyze penicillins, oxyimino-, 7-[alpha]-methoxycephalosporins and monobactams, varies depending on the type of enzyme and geographical location and blaCMY-2 is the most frequently detected worldwide. Typically, pampC producing isolates are associated with resistance to multiple antibiotics making the selection of an effective antibiotic difficult (Rukayadi et al. 2009). For these reasons, antibiotics available for the treatment of multi-drugs resistant E. cloacae infection are fairly toxic and their use is frequently associated with unwanted side-effects. Imipenem/cilastatin, often reserved for more serious hospital-acquired infections, is thought to be associated with a higher risk of seizures than other penicillins and carbapenems (Yanti et al. 2009a). Therefore, novel flavonoids or new generation of phytopharmaceuticals approaches that show synergistic effect with antibacterial agents, which have lost their original effectiveness, or enable their use to treat diseases instead of synthetic drugs alone are research objectives of far reaching importance (Eumkeb et al. 2010; Wagner and Ulrich-Merzenich, 2009). In this study, we have investigated the in vitro activity of naturally occurring plant flavone (4', 5, 7,-trihydroxyflavone), apigenin, which is abundantly present in common fruits such as grapefruit, plant-derived beverages and vegetables such as parsley, onions, oranges, tea, chamomile, wheat sprouts and in some seasonings, against ceftazidime-resistant E. cloacae (CREC) when used alone and in combination with ceftazidime. Chamomile is one of the most common sources of apigenin consumed as single ingredient herbal tea, prepared from the dried flowers from Matricaria chamomilla. Apigenin has been shown to possess remarkable anti-inflammatory, antioxidant and anti-carcinogenic properties (McKay and Blumberg 2006; Patel et at. 2007; Shulda and Gupta 2010). The activity of quercetin and naringenin when used alone and in combination with ceftazidime were also investigated (see also Fig. 1).

[FIGURE 1 OMITTED]

Materials and methods

Apigenin, [beta]-lactam antibiotics, chemicals and bacterial strains sources (see also Fig. 1).

The tested flavonoids (apigenin, quercetin and naringenin) were obtained from lndofine Chemical Company (New Jersy, USA). Cefepime, ceftazidime, imipenem, clavulanic acid, polymyxin B sulphate (PMX) and o-nitrophenyl-[beta]-D-galactoside (ONPG) were obtained from Sigma (Sigma-Aldrich UK). Flomoxef was obtained from Shionogi (Osaka, Japan). Nitrocefin (NCF) and Mueller-Hinton broth were obtained from Oxoid (Basingstoke, UK). The ceftazidime-resistant E. cloacae DMST 21394 (CREC) was obtained from department of medical sciences, ministry of public health, Thailand. E. coli ATCC 25922, positive control, was purchased from American Type Culture Collection (ATCC).

Screening test of ESBL phenotype and ampC genes detection

The screening and confirmatory tests for detection of ampC genes (2 families, including DHA and EBC) and extended-spectrum [beta]-lactamases (ESBL) were executed by multiplex PCR. To detect the coexistence of ESBL genes, PCR was performed using five primer pairs: TEM, SHV, SHV-5, CTX-M-3, and CTX-M-14 (Kao et al. 2010; Su et al. 2010).

Bacterial suspension standard curve

To select bacterial suspensions with a known viable count, the method of Liu et al. (2000) was followed.

Minimum inhibitory concentration (MIC) and checkerboard determinations

MIC and checkerboard determinations of selected [beta]-lactam drugs against CREC and E. coli ATCC 25922 were performed by following Liu et al. (2000) and CLSI (2006).

Killing curve determinations (viable counts)

Viable counts for the determination of killing-curves were performed as previously described by Richards and Xing (1993).

Transmission electronmicroscopy (TEM)

Ceftazidime and apigenin that dramatically decreased the MICs against CREC was chosen for electron microscopy study when used singly and in combination. Subculture of this strain was prepared to examine by TEM following Richards et al. (1995).

Enzyme assays

The [beta]-lactamase type IV of E. cloacae (E. cloacae) were obtain from Sigma (Poole, England). Enzymes activities were followed Richards et al. (1995).

Outer-membrane permeabilization assays

Two methods were used to assess outer membrane (OM) permeabilization. The apigenin-induced permeabililzation of the OM of CREC was determined essentially as recently described (Eriksson et al. 2002). Briefly, Membrane permeabilization was assayed in 96-well microtiter plates. The OM penneabilization assay was carried out with wells filled with 20 [micro]g/mIceftazidime, 10 [micro]g/ml apigenin, 3 [micro]g/ml ceftazidime plus 3 [micro]g/ml apigenin and 7 [micro]g/ml PMX and 50[micro]l of the NCF stock solution. After warming to 37 [degrees]C the plates were positioned in the plate reader at 37 [degrees]C. NCF entry and cleavage by [beta]-lactamase was followed by optical density measurement at 500 nm over 10 min. Complete permeabilization was induced in the presence of 7 [micro]g/ml PMX as a positive control and wells lacking these drugs or apigenin served as negative control (Junkes et al. 2008, 2011).

The second method of alteration in outer membrane permeability was detected by crystal violet assay (Vaara and Vaara 1981). In short, The 20[micro]g/m1 ceftazidime, 10 [micro]g/m1 apigenin, 3 [micro]g/m1 ceftazidime plus 3 [micro]g/ml apigenin and 71.1.g/mIPMX were added to the cell suspension and incubated at 37 [degrees]C for 30 min. Control samples were prepared similarly without treatment. The cells were harvested at 9300 x g for 5 min. After that the cells were resuspended in PBS containing 10 [micro]g/m1 of crystal violet. The cell suspension was then incubated for 10 min at 37 [degrees]C. The suspension was then centrifuged at 13,400 x g for 15 min and the [0D.sub.590] of the supernatant was measured in UV-VIS spectrophotometer. The OD value of the crystal violet solution, which was originally used in the assay, was taken and it was considered as 100%. The percentage of crystal violet uptake of all the samples was calculated using the following formula: (0Dvalue of the sample/ODvalue of crystal violet solution) x 100 (Devi et al. 2010).

Inner-membrane permeabilization assays

Two methods were also used to assess inner membrane (1M) permeabilization. The apigenin-induced permeabililzation of the IM of CREC was determined essentially as recently described (Eriksson et al. 2002). In brief, to assay 1M permeabilization, the wells contained 20 [micro]g/ml ceftazidime, 10 [micro]g/mlapigenin, 3 [micro]g/ml ceftazidime plus 3 [micro]g/ml apigenin, 7 [micro]g/ml PMX and 50 ill ONPG solution. The plates were prepared shortly before the experiment. Finally, 50 [micro]l of cell suspension (OD 0.3) was added to the wells to give a final concentration of 100 [micro]g/ml ONPG. After warming to 37 [degrees]C the plates were positioned in the plate reader at 37 [degrees]C ONPG uptake and cleavage by [beta]-galactosidase within the cytoplasm was characterized by monitoring absorption over a period of 60 min at 420 nm. Complete permeabilization was induced in the presence of 7 [micro]g/ml PMX as a positive control and wells lacking drugs or apigenin test served as negative control (Junkes et al. 2008, 2011).

The release of UV-absorbing material concentrations were measured by UV-VIS spectrophotometer (Zhou et al. 2008). In a word, the concentrations of 20 [micro]g/ml ceftazidime, 10 [micro]g/ml apigenin, 3 [micro]g/m1 ceftazidime plus 3 [micro]g/m1 apigenin were added to the cell suspension. PMX at 7 [micro]g/ml was used as positive control. Cells without drugs or apigenin treatment were used as control. All the samples were incubated at 37 [degrees]C for 60 min. After treatment, the cell suspension was centrifuged at 13,400 x g for 15 min and [0D.sub.260] value of the supernatant was taken as a percentage of the extracellular UV-absorbing materials released by cells. All the measurements were done in triplicates in UV-VIS spectrophotometer (Devi et al. 2010).

Statistic analysis

All experiments were carried out in triplicate, and average values with standard deviation were revealed. Significant differences between these groups were examined using ANOVA. A p value < 0.01 of Tukey's HSD post hoc test denoted the presence of a statistically significant difference.

Results and discussion

Screening test of ESBL phenotype and ampC genes detection

Ceftazidime resistant E. cloacae DMST strains (21394, 21549 and 19719) were positive in the screening test for ESBL. Resistance genes were detected of these isolates: ampC gene of EBC type and ESBL gene of TEM type. The resistance mechanisms were caused by ESBL-ampC combinations of these strains. These findings are in substantial agreement with those of Kao etal. (2010) that the resistance genes, ampCgene of EBC type and ESBL gene of ITEM type were found in multiple drug resistance E. cloacae isolates.

MIC and checkerboard determinations

The MICs for the apigenin, naringenin, quercetin and [beta]-lactam antibiotics against CREC are shown in Table 1. Apigenin, naringenin, quercetin alone showed no activity against these strains at M1Cs of 256 to >512 [micro]g/ml. These E. cloacae strains were resistant to all tested cephalosporins: ceftazidime, cefepime and flomoxef at MICs of >1024, 16-24 and >256 [micro]g/ml respectively. The MIC of clavulanic acid alone against these strains was >512 [micro]g/ml. However, these strains were susceptible to imipenem at MIC of 1-2 [micro]g/ml. The positive test, E. coli ATCC 25922, showed susceptibility to all tested cephalosporins with M1Cs between 0.12 and 0.25 [micro]g/ml, while resistant to clavulanic acid, apigenin, naringenin and quercetin alone at MIC between 256 and >512 [micro],g/ml.
Table 1 Minimum inhibitory concentration (MIC) of ceftazidime,
cefepime, flomoxef, imipenem. clavulanic acid, apigenin.
quercetin and naringenin alone and in combination. Fractional
inhibitory concentration (FIC) from checkerboard assay of
ceftazidime plus clavulanic acid, apigenin. quercetin, or
naringenin against ceftazidime resistant E. cloacae DMST 21394
(CREC) are shown.

Strains     MIC([micro]g
                 /ml)

                  CTZ   CFP   FMX  IPN   CLA   APN   NRN  QCN    CTZ+CLA

E. cloacae      >1024    16  >256    1  >512  >512  >512  256  >512+>256
DMST21394

E. cloacae      >1024    24  >256    2  >512  >512  >512  512  >512+>256
DMST21549

E. cloacae      >1024    24  >256    2  >512  >512  >512  512  >512+>256
DMST
19719

Eco. Coli        0.25  0.12  0.25    1  >512  >512  >512  256        N/D
ATCC25922
(a)

Strains         FIC                                  FIC
             ([micro]g                                index
               /ml)

            CTZ+APN  CTZ+NRN   CTZ+QCN  CTZ+CLA  CTZ+APN  CTZ+NRN

E. cloacae      4+4    16+64  >512+128     1.00    <0.01    <0.14
DMST21394

E. cloacae     8+32    24+64  >512+256     1.00    <0.07    <0.15
DMST21549

E. cloacae     8+32   24+128  >512+256     1.00    <0.07    <0.27
DMST
19719

Eco. Coli       N/D      N/D       N/D      N/D      N/D      N/D
ATCC25922
(a)

Strains

            CTZ+QCN

E. cloacae     1.00
DMST21394

E. cloacae     1.00
DMST21549

E. cloacae     1.00
DMST
19719

Eco. Coli       N/D
ATCC25922
(a)

CIA: clavulanic acid; APN: apigenin; QCN: quercetin; NRN: naringenin;
N/D, No data. Each compound was measured three times. a E. coli ATCC
25922, CIA, IPN were used as positive control.


The isobolograms obtained from plotting of checkerboard determinations showed the synergistic activity for the combination of apigenin and naringenin plus ceftazidime against these strains with FIC indexes <0.01 to <0.27, while zero interaction for ceftazidime plus either clavulanic acid or quercetin with FIC index 1.00 (Wagner and Ulrich-Merzenich 2009). These results are consistent with previous findings that these resistant E. cloacae strains produces ESBL-ampC combination, which results in resistance to ceftazidime, cefepime, flomixef and ceftazidime plus clavulanic acid combination (CLSI 2006; Kao et al. 2010; Su et al. 2010). The mode of action of synergistic combination was further investigated.

Killing curve determinations

Sample killing curves resulting from ceftazidime alone and in combination with apigenin against CREC is presented in Fig. 2. The control of CREC showed no reduction in the counts of cfu from control inoculum. There was marked reduction in resistance of this micro organism to antibiotic with combination of 3 [micro]g/ml cef-tazidime plus 3 [micro]/ml apigenin. The cfu/ml reduction was [10.sup.3] over 6 h and did not recover in 24 h. The results seem consistent with earlier finding that amoxicillin-resistant E. coli was decreased in viability by the combination of amoxicillin plus galangin (Eumkeb et al. 2012).

[FIGURE 2 OMITTED]

TEM

Fig. 3a shows the appearance of normal log phase cells of CREC. The outer membrane and the cytoplasmic membrane can be distinguished. The electron dense ribosomes can be seen in numerous number in cytoplasm. The micrograph of CREC after exposure to ceftazidime 20 [micro]g/ml shows some of these bacterial cells exhibited larger gap between outer membrane and cytoplasmic membrane (Fig. 3b). The CREC cells treatment with 10 [micro]g/ml apigenin are shown in Fig. 3c. Most of the bacterial cells were not damaged after treatment with apigenin alone. The result showed that ceftazidime 3 [micro]g/m1 in combination with 3 [micro]g/ml of apigenin also caused marked morphological damage for CREC. A lot of these bacterial cells exhibited morphological damage of cell wall, cell shape and electron-transparent area in cytoplasm due to losing most of organelles. Several bacterial cells showed broken cell and distortion of cell wall. The micrographs of this combination showed obvious detachment of cell wall and plasma membrane (Fig. 3d). These findings are in substantial correspondence with earlier finding of Eumkeb et al. (2010, 2012) that galangin plus ceftazidime or amoxicillin caused morphological damage of ceftazidime-resistant S. aureus or amoxicillin-resistant E. coli respectively.

[FIGURE 3 OMITTED]

Enzyme assays

The ability of apigenin to inhibit the in vitro activity of penicillinase type IV from E. cloacae revealed that apigenin showed marked significant difference of benzylpenicillin reduction (p < 0.01) in each apigenin concentration starting from 5 min (Fig. 4). These results indicated that in addition to the direct effect on cell structure and cell division, the resistance reversing activity of apigenin against CREC might also include inhibition of [beta]-lactamase activity. These results are in substantial agreement with those of Eumkeb et al. (2012) that the flavonols, galangin or kaempferide, inhibited in hydrolyzing benzylpenicillin.

[FIGURE 4 OMITTED]

Outer-membrane permeabilization assays

The results for OM permeabilization in NCF assay showed that the combination of 3[micro]g/ml ceftazidime plus 3[micro]g/ml apigenin significantly altered OM permeabilisation of CREC compared to control, ceftazidime or apigenin groups (p < 0.01). The OM per-meabilisation was significantly lower than PMX (p < 0.01) (Fig. 5). Similar results were observed with the crystal violet uptake in Fig. 6. The crystal violet uptake of ceftazidime plus apigenin was significantly higher than control, but lower than PMX (p < 0.01) (Fig. 6). These results seem consistent with previous findings that E. coli outer membrane was significantly altered by amoxicillin plus galangin combination or peptide-peptide nucleic acid conjugate (Eriksson et al. 2002; Eumkeb et al. 2012).

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

Inner-membrane permeabilization assays

The CREC inner membrane was significantly permeabilized much more rapidly by the 3 [micro]g/ml ceftazidime plus 3 [micro]g/m1 apigenin combination compared to other groups. PMX, which is highly active against the outer membrane, showed IM permeability slightly lower than ceftazidime plus apigenin combination but not significant difference (p < 0.01) (Fig. 7).

[FIGURE 7 OMITTED]

The index of cell lysis was measured by UV-absorbing release materials (Zhou et al., 2008). After treatment with 3 [micro]g/ml ceftazidime plus 3 [micro]g/m1 apigenin combination, the OD significantly increased up to 0.4 from 0.01 compared to control, ceftazidime or apigenin groups, whereas effect of this combination was not significant difference from PMX (p < 0.01) (Fig. 8). These results suggest that the ceftazidime and apigenin combination damages CREC cytoplasmic membrane and causes subsequent leakage of intracellular constituents.

[FIGURE 8 OMITTED]

The results exhibited that these CREC strains produced resistance genes, ESBL-ampC combination. The ESBL was confirmed when has been changed as [greater than or equal to]3 two-fold concentration decrease in an MIC for either antimicrobial agent tested in combination with clavulanic acid versus its MIC when tested alone (CLSI, 2006). Moreover, ESBL may confer resistance to all extended-spectrum cephalosporins but not flomoxef, whereas ampC13-lactamase can confer resistance to the third-generation cephalosporins and oxacephems, but not for cefepime and carbapenems (Mao et al. 2010: Su et al. 2010). The MIC and checkerboard results appear consistent with viable count that CREC cells were considerably decreased by ceftazidime plus apigenin combination. In addition, synergistic activity of this combination was proved by electronmicroscopy. The enzyme assay result can be explain by assuming that both flavonol (galangin) and flavone (apigenin), which shared OH at 5 and 7 carbon position, can inhibit penicillinase activity (Eumkeb et al. 2010,2012). The OM permeabilization of CREC was significantly altered by combination of ceftazidime plus apigenin compared to control. These results may be explained by assuming that depending on the structure of apigenin, either hydrophilic interactions with the polysaccharide core of LPS, or electrostatic interactions disturbing the polar core region and saccharide--saccharide interactions are interfered results in OM barrier is disturbed (Junkes et al. 2008, 2011). Similarly, The results seem consistent with earlier findings that the natural peptide, polymyxin B (PMB), is a potent antibiotic that binds to and neutralizes LPS. It is a decapeptide cyclic cationic antibiotic containing lipophilic and hydrophilic groups that binds to lipid A (Cardoso et al. 2007). The IM permeability results provide evidence that another mechanism of action of apigenin is disruption of the cytoplasmic membrane, which increases its nonspecific permeability. This hyperpermeability may be followed by leakage of ions and extensive loss of other cellular contents, including the intracellular proteins and ultimately results in cell death. This is consistent with crystal violet assay that eugenol increased the permeability of S. typhi membrane results in the deformation of macromolecules in the membrane (Devi et al. 2010).

These results indicated that these CREC strains, which produced ESBL-ampC combination resistance genes, were inhibited by ceftazidime plus either apigenin or naringenin. The synergistic activity of these combination provide evidence that apigenin and naringenin have the ability to reverse the resistance of these CREC strains to the activity of the primary antibiotic. The modulation of ceftazidime-resistance by apigenin or naringenin significantly enhanced the activities of ceftazidime against these strains. This resistant modulation provides evidence that 5,7-0H group of the A ring and 4'-OH group of the B ring in apigenin or naringenin are important for synergistic activity. Conversely, The presence of additional 3-0H group in C-ring and 3'-OH group in B-ring of quercetin, the flavonol, significantly reduced the synergistic effect with ceftazidime (Fig. 1). The structure activity relationships (SARs) of these flavonoids seem similar to those of Stapleton et al. (2004) that modulation of beta-lactam resistance by (-)-epicatechin gallate (ECG) significantly enhanced the activities of flucloxacillin and the carbapenem antibiotics imipenem and meropenem against 40 MRSA isolates, while both (-)-epicatechin and H-epicatechin-3-cyclohexylcarboxylate were unable to reverse resistance to oxacillin. In addition, previous finding showed that quercetin exhibited lower 50% inhibitory concentration (IC(50)) and inhibitor binding constant (K(i)) values than apigenin against both the Helicobacter pylori Ddl and the Escherichia coli DdIB. This finding implies that the two additional hydroxyls on the flavone skeleton of quercetin in structure might facilitate its inhibitory activity and binding affinity to Ddl (Wu et al. 2008). The structure-activity relationship has indicated that 2',4'- or 2',6'-dihydroxylation of the B ring and 5,7-dihydroxylation of the A ring in the flavone or flavanone structure are important for significant anti-MRSA activity (Alcaraz et al. 2000; Tsuchiya et al. 1996). This activity may be explained by assuming that it involves three mechanisms of action by apigenin. The first is on the peptidoglycan synthesis inhibition, resulting in break and distort to the peptidoglycan layer. The second mechanism is inhibition the activity of certain [beta]-lactamase enzymes. It is clearly that, [beta]-lactamase inhibitors like clavulanic acid have played an important role in fighting [beta]-lactam-resistant bacteria. Conversely, previous research provides evidence that clavulanic acid, a [beta]-lactamase inhibitor, can also lose their activity by the same mechanism as the 13-lactam antibiotics (Stapleton et al. 1995; Tzouvelekis etal. 1997). The structural dissimilarity between apigenin (or naringenin) and clavulanic acid may not induce [beta]-lactamase production by apigenin. It should also be remembered that conventional 13-lactamase inhibitors, unlike flavonoids, cannot reverse the resistance of bacteria (Eumkeb et al. 2010, 2012). The third mode of action is alteration of OM and CM permeabilisation, which finally results in cell death.

In conclusion, our findings provide evidence that apigenin and naringenin have the synergistic effect with ceftazidime to reverse bacterial resistance to this cephalosporin against CREC. This is the first report of the anti-CREC activity and mode of action of the flavone, apigenin. In view of the dose of apigenin used, the results from acute and subacute toxicity studies suggested that this compound has a sufficient margin of safety for therapeutic use (Li et al. 2011; Sui et al. 2009). Overall, apigenin and naringenin offer for the development of a valuable adjunct to ceftazidime against these CREC strains, which currently almost cephalosporins resistance. These in vitro results have to be still confirmed in an animal or in humans test. If possible, blood and tissue levels would be achievable to work synergistically.

Acknowledgements

The authors are indebted grateful to the following persons and institutions for their invaluable assistance in carrying out this study: Miss Supatcharee Siriwong and Mr. Yothin Teethaisong for kind to assist me in laboratory. The Thailand Research Fund for grant support, and National Research Council of Thailand for research fund.

* Corresponding author at: School of Pharmacology, Institute of Science, Suranaree University of Technology, 111 University avenue, Suranaree Subdistrict, Muang District, Nakhonratchasima, 30000, Thailand. Tel.: +66 44 224260; fax: +6644 224633.

E-mail address: griang@sut.ac.th (G. Eumkeb).

0944-7113/$--see front matter [c] Elsevier GmbH. All rights reserved hattp://dx.doi.org/10.1016/j.phymed.2012.11.008

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Griangsak Eumkeb *, Somnuk Chukrathok

School of Pharmacology, Institute of Science, Suranaree University of Technology. Nakhonratchasima, 30000, Thailand
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Author:Eumkeb, Griangsak; Chukrathok, Somnuk
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9THAI
Date:Feb 15, 2013
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