RESEARCH ARTICLE Jin et al., Journal of Medical Microbiology 2017;66: DOI /jmm

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1 RESEARCH ARTICLE Jin et al., Journal of Medical Microbiology 2017;66: DOI /jmm Biochanin A partially restores the activity of ofloxacin and ciprofloxacin against topoisomerase IV mutation-associated fluoroquinolone-resistant Ureaplasma species Hong Jin, 1 Chao Qi, 1 Yanping Zou, 1,2 Yingying Kong, 1,3 Zhi Ruan, 1,3 Honghui Ding, 4 Xinyou Xie 1,3, * and Jun Zhang 1,3, * Abstract Purpose. This study aims to investigate the synergistic antimicrobial activity of four phytoalexins in combination with fluoroquinolones against Ureaplasma spp., a genus of cell wall-free bacteria that are intrinsically resistant to many available antibiotics, making treatment inherently difficult. Methodology. A total of urogenital tract specimens were assessed for Ureaplasma spp. identification and antimicrobial susceptibility. From these, 31 epidemiologically unrelated strains were randomly selected for antimicrobial susceptibility testing to determine the minimum inhibitory concentration (MIC) of four fluoroquinolones and the corresponding quinolone resistance-determining regions (QRDRs). Synergistic effects between fluoroquinolones and four phytoalexins (reserpine, piperine, carvacrol and biochanin A) were evaluated by fractional inhibitory concentration indices (FICIs). Results. Analysis of the QRDRs suggested a vital role for the mutation of Ser-83fiLeu in ParC in fluoroquinolone-resistant strains, and the occurrence of mutations in QRDRs showed significant associations with the breakpoint of levofloxacin. Moreover, diverse synergistic effects of the four phytoalexins with ofloxacin or ciprofloxacin were observed and biochanin A was able to enhance the antimicrobial activity of fluoroquinolones significantly. Conclusion. This is the first report of the antimicrobial activity of biochanin A in combination with fluoroquinolones against a pathogenic mycoplasma, and opens up the possibility of using components of biochanin A as a promising therapeutic option for treating antibiotic-resistant Ureaplasma spp. infections. INTRODUCTION Ureaplasma spp., a member of the class Mollicutes, are recognized as one of the smallest-known self-replicating and freeliving organisms. This bacterium has an extremely small genome size, lacks a cell wall, hydrolyzes urea to generate ATP and requires cholesterol. To date, 14 serovars have been identified and reclassified into two species using molecular biology techniques. U. parvum (UPA) comprises serovars 1, 3, 6 and 14, with a relatively small genome size ( Mbp). U. urealyticum (UUR) contains the remaining 10 serovars, with a relatively large genome size ( Mbp) [1]. Ureaplasma spp. are associated with numerous infectious diseases affecting pregnant women, neonates, sexually active individuals and the immunocompromised. [2, 3]. Approximately 40 to 80 % of asymptomatic women are colonized with Ureaplasma spp., and the vertical transmission rate to infants born is reported to range from 18 to 88 % [4]. The absence of a bacterial cell wall renders Ureaplasma spp. intrinsically resistant to all beta-lactam and glycopeptide antibiotics, and therefore treatment of infections is limited to three classes of antibiotics that inhibit DNA replication (i.e. fluoroquinolones) and protein synthesis (i.e. macrolides and tetracyclines). These therapeutic options are further limited in situations involving pregnant females or neonates, for whom macrolides are the only recognized treatment, due to the associated toxicity of the tetracyclines and fluoroquinolones [5]. However, acquired resistance to all of the above-mentioned antibiotics, especially Received 25 June 2017; Accepted 5 September 2017 Author affiliations: 1 Clinical Laboratory, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang , PR China; 2 Clinical Laboratory, The 117th Hospital of PLA, Hangzhou, Zhejiang , PR China; 3 Biomedical Research Center, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang , PR China; 4 Yiwu Maternity and Child Care Hospital, Jinhua, Zhejiang , PR China. *Correspondence: Xinyou Xie, scottxie@mail.hz.zj.cn; Jun Zhang, jameszhang2000@163.com Keywords: Ureaplasma spp.; fluoroquinolone resistance; QRDRs; phytoalexin; synergistic effect. Abbreviations: B-A, biochanin A; CAR, carvacrol; CCU, colour-changing units; CIP, ciprofloxacin; CLSI, Clinical and Laboratory Standards Institute; FICI, fractional inhibitory concentration indices; LEV, levofloxacin; MIC, minimum inhibitory concentration; MOX, moxifloxacin; OFL, ofloxacin; PIP, piperine; QRDR, quinolone resistance-determining region; RES, reserpine; UPA, U. parvum; UUR, U. urealyticum ã 2017 The Authors 1545

2 fluoroquinolones, has increasingly been documented in clinical isolates [3, 6 9]. Chromosomal mutations of quinolone resistance-determining regions (QRDRs), including type II topoisomerase DNA gyrase (encoded by gyra and gyrb) and topoisomerase IV (encoded by parc and pare), have been identified as the major molecular mechanism resulting in resistance to fluoroquinolones in Ureaplasma spp. [10 12] In the QRDRs, amino acid substitutions of Asp-82fiAsn and Ser-83fiLeu in the ParC protein, and Gln-100fiArg and Gln-104fiLys in the GyrA protein are commonly described as being responsible for the increasing fluoroquinolone resistance of Ureaplasma spp. [11, 13 17] In our previous study, the resistance rates of Ureaplasma spp. to fluoroquinolones (ofloxacin and ciprofloxacin) were much higher than those for any other antibiotics, and increased rapidly from 1999 to 2013 in China [15, 17]. For these reasons, alternatives are urgently required to prevent antimicrobial resistance in part or completely. Plants produce enormous quantities of low-molecularweight antimicrobials, commonly recognized as phytoalexins, which are related to the mechanisms of plant defence in response to microbial invasion or tissue disruption. These have been confirmed to be a promising natural product with potent antimicrobial activity against some bacterial pathogens, such as Staphylococcus aureus, Sphingomonas paucimobilis and Klebsiella oxytoca [18, 19]. The development of a combination of antibiotics and plant-derived compounds seems to be an attractive strategy to slow down the spread and development of antimicrobial resistance. In this study, we described the prevalence of antibiotic resistance among Ureaplasma spp. isolates in China, in addition to the molecular mechanisms for fluoroquinolone resistance. We also investigated the synergistic antimicrobial activity of four phytoalexins in combination with ofloxacin and ciprofloxacin against fluoroquinolone resistance isolates. METHODS Bacterial strains and clinical specimens A total of Ureaplasma spp. specimens were collected from clinical patients between January 2013 and September 2014 in Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, People s Republic of China. Of these, samples were from female patients and 8370 samples were from male patients. The urine specimens were obtained from the male patients, and the cervical or vaginal swabs were collected from the female patients. Chemicals and antimicrobial agents Four antibiotics (ofloxacin, ciprofloxacin, levofloxacin and moxifloxacin) and four phytoalexins (biochanin A, reserpine, carvacrol and piperine) were purchased from Sigma- Aldrich (St Louis, Missouri, USA). All of the antibiotics were dissolved in deionized water and the three phytoalexins (biochanin A, reserpine and piperine) were dissolved in DMSO (Sigma-Aldrich) to make 128 g l 1 stock solutions. Ureaplasma spp. culture and antibiotic susceptibility testing A commercially available Mycoplasma IST2 kit (bio- Merieux, Marcy l Etoile, France) was used for the isolation of Ureaplasma spp., and we distinguished it from Mycoplasma hominis according to the manufacturer s instructions. Clinical samples collected from patients were placed in R1 medium. Then, this mixture was added to R2 medium and vortexed until the pellet dissolved. A Mycoplasma IST2 strip, inoculated with the rehydrated R2 growth medium, and the remainder of the broth were incubated at 37 C. After 24 h of incubation, the colour of broth changed and the strips were able to provide information about Ureaplasma spp. identification, semi-quantification of the concentration of bacterium and the antibiotic susceptibility for nine antibiotics (ofloxacin, ciprofloxacin, pristinamycin, josamycin, azithromycin, doxycycline, erythromycin, clarithromycin and tetracycline). DNA extraction and the identification of Ureaplasma species To prepare the template for PCR, a total of 0.5 ml of Ureaplasma spp. broth culture for each strain was used to isolate genome DNA as described in our previous study [20]. The genome sequences of 14 ATCC Ureaplasma spp. serovars were retrieved from the NCBI GenBank database, as follows: ABES (UPA1), ABFL (UUR2), CP (UPA3), AAYO (UUR4), AAZR (UUR5), AAZQ (UPA6), AAYP (UUR7), AAYN (UUR8), AAYQ (UUR9), CP (UUR10) AAZS (UUR11), AAZT (UUR12), ABEV (UUR13) and ABER (UPA14). A total of 47 UPA-specific and 45 UUR-specific genes were found using the BLASTN program. Of these, UU295 and UUR10_0588 showed the greatest conservation property in each species and tested suitably for the identification of Ureaplasma spp. by using 14 reference strains. The primers to distinguish UPA from UUR were: UU295-F (5 -GCCAA- GAAAACATTTAATCGCT-3 ) and UU295-R (5 -CTGA- TATTGTCCGCTGCTCATT-3 ) for UPA, and UUR10_0588 F (5 -AAAGTTAAAGAGTCTTGGTGGA-3 ) and UUR10_0588 R (5 -AATAGGTAATAGCCTCTTTGAT- 3 ) for UUR. Determination of QRDRs and comparison of amino acid alterations Thirty fluoroquinolone-resistant strains and one fluoroquinolone-susceptible clinical Ureaplasma spp. strain were randomly selected to analyse the amino acid substitutions in QRDRs. The PCR primers for amplification of the QRDRs were: primers gyra-f (5 -TTGCTGCTTTCGAAAACGG-3 ) and gyra-r (5 -CTGATGGTAAAACACTTGG-3 ) were used for gyra, primers gyrb-f (5 -CCTGGTAAATTAGCT- GACTG-3 ) and gyrb-r (5 -TTCGAATATGACTGCCATC- 3 ) were used for gyrb, primers parc-f (5 -ACGCAATGAGT- GAATTAGG-3 ) and parc-r (5 -CACTATCAT- CAAAGTTTGGAC-3 ) were used for parc, and primers 1546

3 pare-f (5 -ATGGGCGGAAAATTAACGC-3 ) and pare-r (5 -CTTGGATGTGACTACCATCG-3 ) were used for pare [21]. The amplifications were carried out according to the Taq DNA polymerases (Takara, Japan) protocol, as per the manufacturer s instructions. The PCR products were purified and then sequenced by the ABI 3730xl DNA analyzer as described previously [20]. The amino acid changes derived from mutations in the QRDRs from GyrA, GyrB, ParC and ParE proteins found in the 31 clinical Ureaplasma spp. strains were compared with 14 serovars of the ATCC reference strains. The sequences of gyra, gyrb, parc and pare for the ATCC reference strains were obtained from the NCBI GenBank database. Determination of MIC values and synergy testing The accurate MICs of four fluoroquinolones and phytoalexins were determined by a broth microdilution method with 96-well microtitre plates. Briefly, all of the antibiotics and phytoalexins were twofold serial diluted in 10B broth. The antibiotic gradient ranged from 256 to mg l 1 for ciprofloxacin; 128 to mg l 1 for ofloxacin, levofloxacin and moxifloxacin; and 8192 to 4 mg l 1 for the four phytoalexins. Twenty µl of overnight-cultured clinical Ureaplasma spp. strains of 10 4 colour-changing units (CCU) was added to each well of the microtitre plates. After incubation at 37 C, the MIC was defined as the lowest concentration of antibiotics or phytoalexins that inhibited a colour change after 24 h incubation. The breakpoints were designated according to the Clinical and Laboratory Standards Institute (CLSI) and Cumitech 34 guidelines. The synergistic antimicrobial activity between the two fluoroquinolones (ofloxacin and ciprofloxacin) and phytoalexins was determined in this study using the checkerboard method, which is commonly used for evaluating interactive inhibition. Initially, twofold serial dilutions of antibiotics were tested in combination with twofold serial dilutions of phytoalexins. Then, the most promising combinations were chosen for further testing, where twofold serial dilutions of antibiotics were combined with constant concentrations of phytoalexins (64 and 128 mg l 1 for carvacrol, 256 and 512 mg l 1 for reserpine, biochanin A and piperine). The fractional inhibitory concentration index (FICI) was calculated for each combination using the following formulae: FICA=MIC of drug A in combination/mic of drug A alone; FICB=MIC of drug B in combination/mic of drug B alone; FICI=FICA+ FICB. The results were interpreted as follows: synergy (FICI 0.5); no interaction (FICI >0.5 but4); and antagonism (FICI >4) [22]. Apoptosis analysis Apoptosis was detected by flow cytometric analysis using an Annexin V-PE/7-AAD apoptosis detection kit (MultiSciences Biotech Co. Ltd, Hangzhou, China) according to the protocol provided. Briefly, the human ovarian surface epithelial cells (HOSEpiC) were seeded (310 5 cells well in a six-well plate. After culturing for 48 h, the treated cells were harvested, incubated with Annexin V-PE and 7-AAD for 15 min at room temperature in the dark, and immediately analysed by flow cytometry (FACSCalibur flow cytometer, BD, CA, USA). The group of cells treated with DMSO formed the control group and the data are expressed as the means±sd of at least three independent experiments. The extent of apoptosis is expressed as the sum total percentages of annexin-positive populations. Ethical approval All methods of this study were carried out in accordance with the relevant guidelines. All experimental protocols were approved by the Ethics Committee of the Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University. All study participants provided written informed consent prior to initiation of the study. RESULTS Prevalence and antibiotic susceptibility of Ureaplasma spp. Of the urogenital tracts specimens assessed, 8186 (35.7 %) were positive for Ureaplasma spp. Of these, 5978 (41.0 %) and 2208 (26.4 %) specimens were positive for female patients ( samples) and male patients (8370 samples), respectively. The prevalence of Ureaplasma spp. was significantly greater in female patients compared to male patients in the test period (P<0.05). The antibiotic resistance rate of Ureaplasma spp. is shown in Fig. 1. There were significant differences in the levels of susceptibility to antibiotic agents. The resistance rates to ofloxacin and ciprofloxacin were considerable higher than for the other antibiotic agents, with rates of % for ciprofloxacin and % for ofloxacin. However, Ureaplasma spp. showed extremely low resistance rates (below 5 %) to the remaining seven antibiotics, especially pristinamycin and josamycin, for which it showed an almost zeroresistance state. Fig. 1. Antibiotic resistance rate of Ureaplasma spp. to nine antibiotic agents over the study period. The number of resistant samples is indicated at the tip of each column. 1547

4 MICs of fluoroquinolones and molecular characterization of QRDRs Thirty-one clinical Ureaplasma spp. strains were selected randomly to determine the MICs of four fluoroquinolones (ofloxacin, ciprofloxacin, levofloxacin and moxifloxacin) and analyse the QRDRs of the gyra, gyrb, parc and pare genes (Table 1). Among these, ofloxacin and ciprofloxacin were two older fluoroquinolones included in Mycoplasma- IST2, and levofloxacin and moxifloxacin were recognized as two newer effective fluoroquinolones and had recognized CLSI standardized breakpoints (resistance 4 µg ml 1 for both). Ofloxacin and ciprofloxacin had relatively high MIC values, with MICs ranging from 0.5 to 128 mg l 1 for ofloxacin, and 1 to 256 mg l 1 for ciprofloxacin. According to the CLSI breakpoint, the resistance rate to levofloxacin was 71.0 %, with MICs ranging from 0.5 to 64 mg l 1. Showing the best activity against Ureaplasma spp. strains, moxifloxacin (MIC range, to 32 mg l 1 ; resistance rate, 22.6 %) was 2- to 8-fold more active than levofloxacin, 4- to 16-fold more active than ofloxacin and 8- to 64-fold more active than ciprofloxacin. Of the 30 fluoroquinolone-resistant isolates, the mutation of Gln-100fiLeu in GyrA, or Ser-83fiLeu or Arg-121fiGly in ParC was observed in 22 strains. No amino acid mutation Table 1. Amino acid changes in QRDRs and MICs of the four fluoroquinolones The amino acids in bold and italics are not species-specific or serovar-specific differences and may contribute to decreased susceptibility to fluoroquinolones. OFL, ofloxacin; CIP, ciprofloxacin; LEV, levofloxacin; MOX, moxifloxacin. /, not tested in this study., identical to the 14 ATCC Ureaplasma spp. serovar type strains. Strain Species Mutations in QRDR MICs (mg l GyrA ParC OFL CIP LEV MOX Serovar 1, 3, 6, 14 UPA Gln Ser Arg / / / / Serovar 2, 4, 5, 7 13 UUR Gln Ser Arg / / / / UU 1 UPA Leu UU 2 UPA Leu UU 3 UPA Leu UU 4 UPA+UUR Leu UU 5 UPA Leu UU 6 UPA Leu UU 7 UPA Leu UU 8 UPA Leu UU 9 UUR Leu Leu UU 10 UPA+UUR Leu UU 11 UPA Leu UU 12 UPA+UUR Leu UU 13 UPA Leu UU 14 UPA Leu Gly UU 15 UPA Leu UU 16 UPA Leu UU 17 UPA Leu UU 18 UUR Leu UU 19 UPA Leu UU 20 UPA Leu UU 21 UUR Leu UU 22 UPA Leu UU 23 UPA UU 24 UPA UU 25 UUR UU 26 UPA+UUR UU 27 UPA UU 28 UPA UU 29 UUR UU 30 UUR UU 31 UPA

5 Table 2. Synergism between ofloxacin and four phytoalexins against 31 clinical Ureaplasma spp. isolates FICI 0.5, synergy (boldface); FICI >0.5 but 4, no interaction; FICI >4, antagonism. UPA, Ureaplasma parvum; UUR, Ureaplasma urealyticum; RES, reserpine; B-A, biochanin A; CAR, carvacrol; PIP, piperine. 1549

6 Table 3. Synergism between ciprofloxacin and four phytoalexins against 31 clinical Ureaplasma spp. isolates FICI 0.5, synergy (boldface); FICI >0.5 but 4, no interaction; FICI >4, antagonism. UPA, Ureaplasma parvum; UUR, Ureaplasma urealyticum; RES, reserpine; B-A, biochanin A; CAR, carvacrol; PIP, piperine. 1550

7 was detected in GyrB or ParE. Moreover, double substitutions, Gln-100fiLeu in GyrA along with Ser-83fiLeu or Ser-83fiLeu along with Arg-121fiGly in ParC, were found in two clinical strains. The mutation of Ser-83fiLeu in ParC was detected in UU8, which represents non-resistance for both of levofloxacin and moxifloxacin. However, no mutation was discovered in UU30, which represents levofloxacin-resistant and moxifloxacin-susceptible. It was noteworthy that the MICs of moxifloxacin for 15 isolates that harboured mutations in the QRDRs were below the breakpoint (4 mg l. Synergistic effects of ofloxacin and ciprofloxacin with phytoalexins All of the 31 clinical Ureaplasma spp. strains were used to evaluate the combined effects of ofloxacin and ciprofloxacin with four phytoalexins (reserpine, biochanin A, carvacrol and piperine). The susceptibilities of Ureaplasma spp. strains to the antibiotics selected, either alone or in combination with phytoalexins, and the synergism are displayed in Table 2, 3 and 4. All four phytoalexins alone showed only a weak inhibition of Ureaplasma spp. (MICs ranging from 256 to >8192 mg l 1 for reserpine and biochanin A, 256 to 8192 mg l 1 for piperine and 256 to 1024 mg l 1 for carvacrol), and high MICs were observed for ofloxacin and ciprofloxacin. The synergistic effects of phytoalexins plus ofloxacin and ciprofloxacin were observed for the clinical Ureaplasma spp. strains when they were assessed by FICIs. The combination of biochanin A with ofloxacin or ciprofloxacin had the strongest synergistic interaction against Ureaplasma spp. The ofloxacin/biochanin A combination showed synergistic interaction against 16 (51.61 %) Ureaplasma spp. strains with 256 mg l 1 biochanin A, and against 19 (61.29 %) strains with 512 mg l 1 biochanin A. For ciprofloxacin/biochanin A, synergism were observed against 26 (83.87 %) and 24 (77.42 %) strains, with 256 mg l 1 and 512 mg l 1 biochanin A, respectively. Similarly, the combination of ofloxacin/reserpine and ciprofloxacin/reserpine exerted synergistic activity against 12 (38.71 %) and 14 (45.16 %) strains. Synergistic effects were found for piperine with ofloxacin and ciprofloxacin against two (6.45 %) and seven (22.58 %) clinical strains. However, the ofloxacin/carvacrol combination only displayed synergistic effects against one (3.22 %) strain with carvacrol at a concentration of 256 mg l 1, while ciprofloxacin/carvacrol showed synergism against two (6.45 %) strains with 512 mg l 1 carvacrol. To assess the suitability of the tested compounds for antimicrobial therapy, a cytotoxicity test revealed that there was no significant difference between the group treated with reserpine or biochanin A and the control group for the cell viability and apoptosis of HOSEpiC cells (Fig. 2). DISCUSSION Ureaplasma spp. are frequently isolated from the urogenital tracts of adults and are associated with numerous clinical sequelae affecting pregnant women, neonates and immunocompromised patients. In the present study, Ureaplasma spp. was isolated more frequently from the female population (41.0 %) than the male population (26.4 %), which is indicative of unequal prevalence in the two populations. This result was in line with our previous studies [15, 20]. In contrast to our data, Ye et al. isolated 53.1 % positive urogenital tract specimens from female patients in China [6]. Francesco et al. found a much lower Ureaplasma spp. prevalence rate (18.4 % for 9956 specimens obtained from adults aged between 18 and 43 years) in Italy [8]. Hunjak et al. found a lower Ureaplasma spp. prevalence rate (34.3 % from 1370 tested women) in Croatia [3]. These differences concerning prevalence might be due to different socioeconomic conditions and living standards. Acquired resistance to the limited number of available antibiotics has been reported in Ureaplasma spp. and appears to be increasing. In particular, high resistance rates have been recorded against fluoroquinolones in most countries [23]. In this study, the resistance rates to ofloxacin and ciprofloxacin were higher than those for the other antibiotic agents, with % for ciprofloxacin and % for ofloxacin, which is in line with the data from our previous epidemiological surveillance study [17]. Fluoroquinolones are commonly used for the treatment of a wide variety of bacterial infections in China, and clinical Ureaplasma spp. strains show an extremely high rate of fluoroquinolone resistance, so treatment with fluoroquinolones only may not be the best choice. Fluoroquinolone antibiotics are potent, broad-spectrum agents that are commonly used to treat a range of infections. Resistance to these agents is multifactorial and can be mediated by chromosomal mutations in the QRDRs encoding the targets gyrase (gyra and gyrb) and topoisomerase IV (parc and pare), by increased levels of the multidrug efflux pump AcrAB, and by the presence of the plasmid-borne Table 4. The rates of synergism between fluoroquinolones and phytoalexins against 31 clinical Ureaplasma spp. isolates Antibiotics* No. of Ureaplasma spp. isolates (n=31) RES (256 mg l RES (512 mg l B-A (256 mg l B-A (512 mg l CAR (64 mg l CAR (128 mg l PIP (256 mg l PIP (512 mg l OFL 10 (32.26 %) 11 (35.48 %) 16 (51.61 %) 19 (61.29 %) 1 (3.23 %) 0 (0) 2 (6.45 %) 2 (6.45 %) CIP 11 (35.48 %) 12 (38.71 %) 26 (83.87 %) 24 (77.42 %) 0 (0) 2 (6.45 %) 4 (12.90 %) 7 (22.58 %) *B-A, biochanin A; CIP, ciprofloxacin; CAR, carvacrol; OFL, ofloxacin; PIP, piperine; RES: reserpine. 1551

8 Fig. 2. The effect of reserpine (RES) and biochanin A (BA) on the cell viability and apoptosis of HOSEpiC cells. The cells were treated with RES (256 mg l, BA (256 mg l or DMSO (vehicle for RES and BA) for 24 h. (a) The cell viability analysed using the MTT assay. (b, c) The percentage quantification of the apoptosis populations of the flow cytometric analysis. There is no significant difference between the group treated with RES or BA and the control group for the cell viability and apoptosis of HOSEpiC cells. fluoroquinolone resistance genes qnra, qnrb, qnrs, aac(6 )- lb and qepa [24 27]. To the best of our knowledge, Ureaplasma spp. do not have plasmids, and the accumulation of chromosomal mutations within the QRDRs is thought to be responsible for the fluoroquinolone resistance described for them thus far [11, 12, 23] In this study, 21/22 levofloxacinresistant strains had an amino acid mutation of Ser- 83fiLeu in ParC, which is homologous to the mutations identified in other fluoroquinolone-resistant bacteria, such as Escherichia coli, Staphylococcus aureus and Streptococcus pneumoniae [16, 28, 29]. Interestingly, 15/24 (62.5 %) moxifloxacin-susceptibility clinical strains harboured a mutation in ParC, which suggests that the spatial structure of moxifloxacin keeps it out of these mutations and allows it to retain antibacterial effectiveness. Several studies have shown that phytoalexins are able to act against bacterial resistance mechanisms in combination with antibiotics [19, 30 33]. As previously reported, biochanin A and reserpine might play an important role in repressing bacterial growth and enhancing fluoroquinolone activity by inhibiting the MDR efflux pump against S. aureus [30, 33, 34]. Reserpine was also reported to reduce the resistance of S. aureus to ciprofloxacin, moxifloxacin and sparfloxacin [30]. Piperine was highly effective in combination with ciprofloxacin and mupirocin against S. aureus [31, 32]. In the present study, four phytoalexins (reserpine, biochanin A, carvacrol and piperine) were selected for an analysis of their synergistic effects with the most commonly used fluoroquinolones, ofloxacin and ciprofloxacin, against Ureaplasma spp. In the synergy testing against fluoroquinolone-resistant strains, a significant reduction was observed in the MICs of fluoroquinolone when combined with biochanin A, while a moderate effect of fluoroquinolone/reserpine was also observed. However, in our study, carvacrol, which has the ability to increase membrane permeability and was also reported to be effective in reducing the MICs of 18 antibiotics against K. oxytoca and S. paucimobilis [19], displayed extremely low activity in combination with fluoroquinolone against Ureaplasma spp. In conclusion, we have successfully demonstrated the synergistic antimicrobial activity of natural phytoalexins, especially biochanin A, which were able to potentiate the antibacterial effects of fluoroquinolones against Ureaplasma spp. and seem to be a promising choice for treating fluoroquinolone-resistant ureaplasmal infections. The underlying mechanisms by which phytoalexins exert antimicrobial activity in this atypical bacterial pathogen, which is of increasing clinical significance, warrant further investigation. Funding information This study was supported by grants from the National Natural Science Foundation of China (grant nos and ), Zhejiang Provincial Health Bureau Foundation (grant nos 2013KYA216, 2014PYA011, 2015DTA008 and 2017KY406), Zhejiang Provincial Traditional Chinese Medicine Foundation (grant no. 2015ZB034) and Zhejiang Provincial Innovative Medical Discipline (11-CX18). Conflicts of interest The authors declare that there are no conflicts of interest. Ethical statement All participants were informed of the study aims, and written informed consent was received from each participant before sample collection. The study protocol was approved by the Ethics Committee of the Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University. Informed consent was obtained from all individual participants included in the study. References 1. Glass JI, Lefkowitz EJ, Glass JS, Heiner CR, Chen EY et al. The complete sequence of the mucosal pathogen Ureaplasma urealyticum. 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