Antibacterial activity of the curcumin derivative FM 0817 against Neisseria gonorrhoeae

The objectives were to analyze the anti-bacterial activity of the curcumin derivative FM0817 and its mechanism of action. We first tested 40 strains of Neisseria gonorrhoeae (N. gonorrhoeae) for their susceptibilities to antibiotics by the Kirby-Bauer and agar dilution methods. Total DNA was extracted by the boiling method, and 22 strains of N. gonorrhoeae were randomly selected and used as templates for polymerase chain reaction (PCR) amplification of the antibiotic-resistance genes: TEM, tetM, erm, and mefA. The filter disc diffusion method was used to determine whether FM0817 had anti-bacterial activity. Eight strains, covering four drug-resistant genotypes/phenotypes and five different types of resistance, were selected. The susceptibility tests confirmed multi-drug resistance in N. gonorrhoeae. The percentage of strains possessing TEM and tetM was 77.2 and 77.2%, respectively. The drug-resistance genes, erm and mefA, were not detected in any of the strains studied. FM0817 exhibited potent antibacterial activity in vitro, but the relationship between FM0817 activity and multi-drug resistance and the resistance genes of N. gonorrhoeae remains unclear, therefore, it requires further investigation.


INTRODUCTION
Neisseria gonorrhoeae is a Gram-negative pathogenic bacterium responsible for an array of diseases ranging from urethritis to disseminated gonococcal infection.The incidence rate of gonorrhea, the disease caused by N. gonorrhoeae, is currently high due to the rapid spread of this pathogen.The United States Centers for Disease Control stated that the penicillin and tetracycline were not the first line of drugs because of the high drug-resistance rates (Gonzalez et al., 2009).It is time to sound the alarm.During the past three years, the wily gonococcus has become less susceptible to our last line of antimicrobial defense, threatening our ability to cure gonorrhea and prevent severe sequelae, and it was needful to find the new drugs (Bolan et al., 2012).
Curcumin is a monomeric phenolic compund, belonging to the ginger family (Zingiberaceae), and it has widespread pharmacological activities, including antitumour oxidation-resistance, anti-sudden change activity, anti-inflammatory, and anti-blood platelet accumulation activity (WG, 1997).In recent years, curcumin pharmacological studies focusing, in particular, on the inhibition of tumour cell proliferation and on the induction of apoptosis have gained attention, along with the in vitro anti-bacterial action of curcumin (Na et al., 2011).The water-solubility of ginger flavine is poor, but hydrolysis can be carried out in peroxide solution.These factors have limited its biological application and have caused an industrial production bottleneck.Studies have been per-*Corresponding author.E-mail: zszcl1988@sina.cn.Tel.: 0086-0760-89880353 0086-0760-88841707.2000, Shoba et al., 1998)).
A study comparing FM0817 and ginger flavine has been reported (NW Zhang et al., 2009).The aim of this study was to determine the anti-bacterial activity of the curcumin derivative FM0817 on N. gonorrhoeae, along with its mechanism of action.

Specimen origin and gonococcus isolation
Clinical strains of N. gonorrhoeae were isolated from specimens submitted to the Guangdong Province Zhongshan People's Hospital from January 2009 to August 2010.Clinical specimens received by the hospital were examined according to the "Nation Clinical Checked Operation Regulations".N. gonorrhoeae isolated from specimens were cultured, and were identified by API NH.

Drug sensitive analyses
The antibiotic sensitivity of forty gonococcal isolates were tested by the National Center for Clinical Laboratory (China) using the Kirby-Bauer.The results were interpreted according to the standard reference, CLSI (2008).The gonococcus (Zopf) Trevisan standard strain, ATCC 49226, was used as a positive control.

Extraction of bacterial DNA
Bacterial DNA was extracted according to the previously published boiling method (Tanaka et al., 1998).

The sequence of drug-resistant genes primers
Previously reported primers were used in this study (Li et al., 2005), as shown in Table 1.Primers were synthesized by Boya Biological Corporation.

Polymerase chain reaction (PCR) amplification of drugresistance genes
PCR reactions (total volume of 25 μl) comprised TaqE reaction buffer, 12.5 μl of dNTPs, 1 μl each of forward (F) and reverse (R) primers, and 1 μl of DNA template.The reaction conditions involved DNA amplification by 30 cycles of 94°C for 30 s, 55°C for 45 s, 72°C for 1 min, with a final extension step of 72°C for 7 min.After 2% agarose gel electrophoresis, the DNA bands were visualized and recorded via a computer image analyzer.The TEM gene from Escherichia coli and the tetM gene from Staphylococcus aureus were used as positive control genes.

Anti-bacterial activity of the curcumin derivative FM0817
The disc diffusion method was used to determine the anti-bacterial activity of FM0817.Among the clinical N. gonorrhoeae isolates obtained, 8 strains, covering four drug-resistant genotypes and five drug-resistance phenotypes, were selected for this experiment, as shown in Table 2.
The inculum was prepared in normal saline to turbidity equivalent to a 0.5 McFarland standard (ca.1.5 x 10 8 colony-forming units (CFU)/ml) and was swabbed on Mueller-Hinton agar.Every strain was inoculated in three Mueller-Hinton agar, respectively, Three kind of discs were used in each Mueller-Hinton agar, the bank, curcumin and curcumin derivatives FM0817 filter disc.The experiment was repeated three times.
Qualitative filters made of 6 mm disks, soaked in sterile water, were applied, along with FM0817 curcumin, to the centre of three Petri dishes.The dishes were then incubated in a 5-10% thermostatic CO2 cultivation cabinet, for 24 h at 20-35°C.After removing the disk, bacteriostatic measurements were made and the average values were recorded.

Statistical analysis
The inhibition zones among different drug-resistance gene mutations were analyzed using the SPSS software.

Drug sensitivity of the N. gonorrhoeae strains
All 40 of the N. gonorrhoeae strains tested were resistant to penicillin.All strains were suscesptible to spectinomycin and cefuroxime axetil.The levofloxacin, ciprofloxacin, and tetracycline-resistance rates of the strains were 95.0, 92.5, and 62.5%, respectively, as shown in Table 3. N. gonorrhoeae strains, representing eight antibiotic-resistance phenotypes, were divided into five  & the number of antibiotics to which a given strain showed resistance groups based on drug-resistance antibiotic species, as shown in Table 3.

The prevalence of drug-resistance genes among N. gonorrhoeae strains
The four drug-resistance genes of twenty-two strains were detected in this study (Figures 1-3).The genemutation rates of TEM and tetM were both 77.2%, and the erm and mefA genes were not detected.These data showed the enzymes were responsible to the penicillin resistance.

Anti-bacterial activity of the curcumin derivative FM0817
The curcumin derivative, FM0817, was tested in vitro for its activity against N. gonorrhoeae.For eight of the N. gonorrhoeae strains, bacteriostatic annulus diameters of between 33-37 mm were observed, but the blank disc and the curcumin disc had no inhibition zone.It indicated that the FM0817 had the activity of inhibiting N. gonorrhoeae.However, no significant differences were detected about the inhibition zones among different drugresistance gene mutations.Taken together these data suggest there was no relation between the inhibition activity and the mutation kinds.

DISCUSSION
N. gonorrhoeae has always readily developed resistance to antimicrobial agents: it became resistant to sulfanilamide in the 1940s, penicillins and tetracyclines in the 1980s, and fluoroquinolones by 2007 (WHO report, 2007).When the prevalence of antimicrobial resistance in the gonococcal isolate surveillance project (GISP) exceeds 5%, national treatment recommendations are changed to focus on other effective drugs.
However, the treatment options recommended by the Centers for Disease Control and Prevention (CDC) are now limited to third-generation cephalosporins.But susceptibility to cephalosporins decreasing rapidly (Katz et al., 2012).In the search for new compounds with biological activity curcumin and derivatives therefore    All strains were negative for the mefA and erm genes have attracted the interest of scientists.
To explore the activity of curcumin on E. coli in a sepsis mice protection model, Hou et al. (2008) randomly divided Kunming mice into control, model and curcumintreated groups.The results were proposed that curcumin could reduce TNF-, NO, and oxygen free radicals in mice with bacterial sepsis, reducing the viscera pathological damage.
There were several researchers who investigated the anti-microbial activity of curcumin and its derivatives in vitro (Na et al., 2011;Zhong et al., 2008).The results show that curcumin may be an alternative antimicrobial agent against fetal bacterial infections.
Based on the molecular structure of curcumin, containing two carbonyl groups, they used aniline, 4-methyl aniline, and benzene nitrofication as hydrazines, and 2,4diflooronitrohenzene hydrazine curcumin as the raw material to synthesize four kinds of Schiff base curcumin stability derivatives.The analysis of in vitro antimicrobial activity revealed that four curcumin derivatives, including Schiff bases, had good antibacterial activity (Zhang et al., 2009).
In the present study, 40 strains of penicillin-resistant N. gonorrhoeae were analyzed.Cefuroxime axetil and spectinomycin-resistant strains that have high drugresistance to ofloxacin, ciprofloxacin, and tetracycline are not known to be prevalent in China.The detection rates for penicillin and tetracycline-resistance genes recorded in this study were significantly higher than those reported by Li et al. (2012); likely due to the widespread application of antibiotics and the rapid spread of drugresistance genes.
The erythromycin-resistance-related methylation enzyme, encoded by the erm gene and the mefA gene, were not detected in our study.Analysis of the prevalence of drug resistance among N. gonorrhoeae strains N. gonorrhoeae infection induces inflammatory cytokines tumor necrosis factor (TNF)-, interleukin (IL)-6, and IL-8 expression, and curcumin can inhibit IKKalpha, IKKbeta and NF-κB, showing that its powerful induced kinase from NF-κB mediates cytokine release and induces innate immune responses (Wessler et al., 2005).Except in the case of N. gonorrhoeae inflam-mation, the study also showed that curcumin at a late stage of infection may fully inhibit bacteria adhered to cells, a huge potential side effect of non-toxic antimi-crobial drugs.
In our experiments with curcumin using eight strains of N. gonorrhoeae, bacteriostatic annulus diameters of 6 mm were recorded (the diameter of the disc), indicating no bacteriostatic activity.However, this result was different to the result obtained from the cells (Wessler et al., 2005).On testing FM0817 derivatives of curcumin against eight strains of N. gonorrhoeae, the bacteriostatic annulus diameters showed no obvious differences in size (33-37 mm on average).The mechanism of action of FM0817 and the N. gonorrhoeae drug-resistance genotypes/phenotypes may not be related, and, furthermore, our analysis of drug-resistance strains and the genotypes/phenotypes of drug resistance may not be comprehensive.Therefore, further analysis of different resistance genotypes and drug-resistance strains of different phenotypes with respect to FM0817 antimicrobial susceptibility is required.However, the FM0817 derivative of curcumin showed larger bacteriostatic annulus diameters (average 35 mm) with eight strains of N. gonorrhoeae, showing antibacterial activity in vitro.Further research is now required to fully elucidate the antimicrobial properties and potential applications of the FM0817 curcumin derivative.

Table 1 .
Primers used to amplify Neisseria gonorrhoeae drug-resistant genes.

Table Kirby -
Bauer anti-microbial susceptibility testing of N. gonorrhoeae strains isolated from January 2009 to August 2010 in Zhongshan district (n = 29).

Table 3 .
Resistance to eight antibiotic species among the Neisseria gonorrhoeae strains isolated from January 2009 to August 2010 in Zhongshan district (n=40).

Table 4 .
Prevalence of drug-resistant genes in 22 strains of Neisseria gonorrhoeae.

Table 5 .
FM0817 in vitro experiments with selected strains of N. gonorrhoeae.