Antimicrobial activity of Asteraceae species against bacterial pathogens isolated from postmenopausal women

Purpose Investigation of the antibacterial action of aqueous extracts of Bidens sulphurea, Bidens pilosa, and Tanacetum vulgare, species of Asteraceae family that are popularly used for the treatment of genito-urinary infection. Methods The minimum inhibitory concentration (MIC) and minimal bacterial concentration (MBC) of the extracts against standard strains of Staphylococcus aureus (ATCC25923), Enterococcus faecalis (ATCC29212), Escherichia coli (ATCC25922), and Pseudomonas aeruginosa (ATCC27853) and against bacteria that were isolated from cultures of vaginal secretions and urine from menopausal women with a diagnosis of recurrent urinary tract infections (rUTI) were determined by broth microdilution. Results The MIC values of the three extracts against Gram-positive and Gram-negative standard bacterial strains ranged from 7.81 to 125.00 mg ml-1, and the MBC values ranged from 7.81 to 500.00 mg ml-1. However, B. sulphurea was more efficient. In the urine samples, the three extracts inhibited the growth of coagulase-negative Staphylococcus spp., and the B. pilosa was the most active extract against E. coli compared with the other ones. For the vaginal secretion samples, no significant differences in the inhibition of coagulase-positive Staphylococcus spp. and P. mirabilis were found among the extracts. T. vulgare and B. sulphurea were more effective in inhibiting coagulase-negative Staphylococcus spp. compared with B. pilosa. E. coli was more susceptible to the B. sulphurea extract compared with the B. pilosa and T. vulgare extracts. Conclusion The present results suggested the potential medicinal use of Asteraceae species, especially B. sulphurea, as therapeutic agents against rUTI-related bacteria.

in Brazilian folk medicine for the treatment of bacterial infections, cystitis, and renal infections [8,14].
Despite the popular use of these species for the treatment of bacterial infections and genitourinary tract infections, the therapeutic activity of these species has not yet been investigated. The present study evaluated the potential antimicrobial activity of aqueous extracts that were obtained by infusions of these species, as recommended by popular use, against bacteria that were collected from urine samples and vaginal secretions from postmenopausal women with a diagnosis of rUTI.

Patient recruitment
Prior to the collection of clinical samples, the study received approval from the Ethics Committee on Research Involving Human Beings of UNIPAR (CAEE no. 90949218.2.0000.0109). The participants provided both verbal and written consent for urine and vaginal secretion collection for research purposes. Women in the postmenopausal period (45-70 years old) with a diagnosis of rUTI (three episodes of UTI in the previous 12 months or two episodes in the last 6 months) were included in the study [15]. Women with structural genetic abnormalities in the genital or urinary systems, genital dystopias greater than Pelvic Organ Prolapse Quantification stage 2 [16], genetic or drug-induced immune deficiency, neurological deficiency that affected urinary tract function, or malignant pelvic disease or women who had already undergone pelvic radiotherapy and used antibiotics in the last 4 weeks at the time of sample collection were excluded from the study [17].

Collection of clinical samples
A total of 15 urine samples and 15 samples of vaginal secretions were collected using sterile containers and swabs that contained Aimes medium with activated charcoal (Transystem™, Copan Italia, Brescia, Italy), respectively. The samples were obtained from 15 patients who attended a private clinical routine from May to July 2018. Each participant was given a printed sheet outlining the details of the method to be used for urine collection. It was instructed to wash their hands and clean the genital area with soap and water, discard the first jet and collect the mid-stream urine specimens in a sterile container. Immediately after collection, the samples were sent to the Laboratory of Preventive Veterinary Medicine and Public Health of UNI-PAR, Brazil. The samples were transported under ice-cold conditions.

Identification of clinical samples
Urine samples were seeded on plates that contained Mannitol Salt Agar and MacConkey Agar and incubated at 37˚C for 24 h to isolate Gram-positive and Gram-negative aerobic bacteria. The swabs were first placed in tubes that contained 3 ml of Brain Heart Infusion (BHI) medium and incubated in an oven at 37˚C for 24 h. Afterward, they were seeded on plates according to the procedure for urine samples. Subsequently, macroscopic and microscopic analyses and biochemical tests were performed [18]. Gram-positive, catalase-positive cocci underwent a coagulase assay to classify coagulase-positive and coagulase-negative Staphylococcus. The Enterobacteriaceae family was biochemically identified using a set of biochemical tests in the Enterobacteria Kit (NewProv, Paraná, Brazil) according to the manufacturer's instructions.

Determination of susceptibility to antimicrobials
Antibiotic resistance and susceptibility of the identified organisms were determined using the disc diffusion method [19] with commercially available discs of metronidazole (50 μg/disc), amoxicillin (10 μg/disc), norfloxacin (10 μg/disc), and oxacillin (10 μg/disc). The samples were thawed and added to the culture medium to grow each isolate and incubated. After growth, the bacterial inoculum was padronized on the McFarland 0.5 scale and seeded on Mueller Hinton agar using a sterile swab. After 15 min, the antimicrobial-impregnated disks were incubated at 37˚C for 18-24 h. Antibacterial activity was evaluated by measuring the diameter of the growth inhibition zones (in millimeters; including the 6.5 mm disc diameter) for each of the microorganisms. The inhibition zones were measured in triplicate.

Multiple antibiotic resistance index
The multiple antibiotic resistance (MAR) index of each strain was calculated according to the formula of Krumperman (1983) [20]: a / b, where a is the number of antibiotics to which a particular isolate was resistant, and b is the total number of antibiotics tested.

Plant material and extract preparation
Botanical material (B. sulphurea and B. pilosa) was obtained from the Medicinal Garden of Paranaense University (UNIPAR; S23˚47'55", W53˚18'48"), Umuarama, PR, Brazil. One specimen of each species was registered in the Medicinal Garden of Campus 2 of UNIPAR (no. 131 and 40, respectively). Aerial parts of T. vulgare were collected in the Municipal Nursery of Saudade do Iguaçu, PR, Brazil (S25˚41'30.069", W52˚37'06.207"), and an exsiccate was deposited in the Herbarium of the Federal Technological University of Paraná of Dois Vizinhos, PR, Brazil (no. DVPR006294). All of the species were collected in May 2018. The aqueous extracts of the plants were obtained by infusion as recommended by popular use [12] with minor modifications. The dried and ground vegetable material (100 g, the material was pulverized in a knife mill until granulometry of 850 μm) was subjected to an extraction process by infusing with 1 L of boiling water. Extraction was performed until the extraction medium reached room temperature (24 hours). The residue was separated by filtration, and the supernatant was resuspended in ethanol (1:3 extract/ethanol) for the precipitation of proteins and polysaccharides, obtaining a precipitate and an ethanolic supernatant from the infusion (48 hours of precipitation). After complete removal of the organic solvent by rotavaporation (3 hours/400 mL; 45˚C), the extract was subjected to lyophilization (72 hours; -42˚C). The final yields of the extracts of B. sulphurea, B. pilosa, and T. vulgare were 21.43%, 17.45%, and 23.13%, respectively. The extracts were stored in a freezer until use.

Gas chromatography/mass spectrometry
The chemical constituents of the extract samples were identified using a gas phase chromatograph (Agilent 7890 B) coupled to a mass spectrometer (Agilent 5977 A) equipped with an Agilent HP-5MS UI capillary column (30 m × 0.250 mm × 0.25 μm). For the analysis, an injection volume of 1.0 μl of a solution that was prepared by the dissolution of 20 mg of the B. sulphurea, B. pilosa, and T. vulgare extracts in 1.0 ml of methanol was used. The analytical conditions were the following: 280˚C injector temperature operating in spline mode (1:2), 280˚C transfer line, and 1 ml min -1 carrier gas (helium) flow. The initial column temperature was 80˚C (1 min) with a ramp of 2˚C min -1 until reaching 185˚C. The temperature remained at 185˚C for 1 min, followed by heating at 9˚C min -1 until reaching 275˚C. The temperature remained at 275˚C for 2 min, followed by heating at 25˚C min -1 to 300˚C. The temperature was then held at 300˚C for 1 min. The extracts of B. sulphurea, B. pilosa, and T. vulgare underwent electron impact ionization scanning at 70 eV with a 40-600 mass/charge ratio (m/z). The ionization source temperature and quadrupole temperature were 230˚C and 150˚C, respectively. The compounds were identified by comparing their mass spectra with the NIST 11.0 library and by comparing the retention indices (IRs) that were obtained by the homologous series of n-alkane standards (C7-C28; [21]).

Minimum inhibitory concentration and minimum bactericidal concentration of extracts
The minimum inhibitory concentration (MIC) and minimal bacterial concentration (MBC) of the extracts against standard strains and bacteria that were isolated from cultures of vaginal secretions and urine were determined by the broth microdilution method using Mueller Hinton Broth according to the CLSI [19] with modifications. The vegetal extract was dissolved in Tween 80 (2%) and diluted in culture medium to an initial concentration of 500 mg mL -1 . Then, serial decimal dilutions (1:2) were prepared by adding culture medium to achieve concentrations ranging to 0.97 mg mL -1 . Thus, a final volume of 100 μL (culture medium plus extract) was distributed in 96-well plates, as well as controls of culture medium and culture medium with extract and Tween. Bacteria were standardized on the McFarland 0.5 scale and the inoculum adjusted to~10 5 CFU/mL. The tests were performed in triplicate and the plates incubated at 37˚C for 24 hours. Readings were performed after the addition of 10 μl of 10% diluted 2,3,5-triphenyltetrazolium chloride, followed by incubation at 37˚C for 30 min. Bacterial growth was considered when the wells presented any pink tone after incubation [22]. The MIC was the lowest concentration of the extract that inhibited bacterial growth. The MBC was determinate by subculturing 10 μL from the culture of each negative well on Mueller Hinton Agar plates as described above [23].

Statistical analysis
Differences between groups were assessed using analysis of variance (ANOVA), followed by Tukey's post hoc test. Values of p < 0.05 were considered statistically significant. The results are expressed as mean ± standard error of the mean (SEM). The statistical analyses were performed using Statistica 13.3 software.

Effect of antibiotics on bacterial pathogens and their MAR index
Thirty-two bacterial samples were isolated from 15 postmenopausal women who were diagnosed with rUTI, predominantly from urine (10; 31.25%) and vaginal secretions (22; 68.75%). Gram-positive (Staphylococcus spp.) and Gram-negative (Escherichia coli and Proteus mirabilis) bacteria were identified in urine and vaginal secretion samples.
The antibiotic resistance of the identified organisms was determined by the disc diffusion method using antibiotics that are routinely used for the treatment of UTIs. Table 1 shows the percentage of urine samples and vaginal secretions that were antibiotic-resistant. Overall, we observed multidrug-resistant isolates, with a majority from vaginal secretions (48; 57.83%) and urine (18; 51.42%).
The MAR index results indicated that all of the tested genitourinary tract isolates of Staphylococcus spp. (19 isolates), E. coli (11 isolates), and P. mirabilis (3 isolates) had a very high MAR index (> 0.2; Table 2), indicating that the samples were classified as high risk.  Table 1

Antibiotics
Urine

Antibacterial activity
The MIC values of the B. pilosa, T. vulgare, and B. sulphurea extracts (  Table 4 shows the results of the in vitro screening of the antibacterial activity of the aqueous extracts against bacteria that were isolated from urine and vaginal secretion samples from postmenopausal women. In urine samples for coagulase-negative Staphylococcus spp., no significant difference was found between the extracts (F 2,12 = 1.72, p = 0.21). The one-way ANOVA indicated a significant difference between the extracts against E. coli (F 2,9 = 4.21, p < 0.05) and P. mirabilis (F 2,6 = 12.00, p <0.01). Tukey's post hoc test showed that the extract of B. pilosa Table 4. Antibacterial activities of Gram-positive and Gram-negative bacterial strains, express as minimal inhibitory concentration (MIC, mg mL -1 ) and minimal bactericidal concentration (MBC, mg mL -1 ). (58.59 mg ml -1 ) was the most active against E. coli (p < 0.05) compared with T. vulgare (125.00 mg ml -1 ) and B. sulphurea (78.12 mg ml -1 ). B. sulphurea (5.85 mg ml -1 ) promoted better inhibition (p < 0.05) of P. mirabilis compared with the extracts of T. vulgare (125.00 mg ml -1 ) and B. pilosa (166.66 mg ml -1 ).
With regard to inhibiting E. coli, the one-way ANOVA showed a significant difference between the extracts (F 2,18 = 7.80, p < 0.05). E. coli was more susceptible to the extract of B. sulphurea (62.5 mg ml -1 ) compared with the extracts of B. pilosa (102.67 mg ml -1 ) and T. vulgare (116.07 mg ml -1 ). The MBC values for the clinical samples ranged from 7.81 to >250.00 mg ml -1 .

Discussion
Infections that affect the genito-urinary tract are caused by Gram-positive and Gram-negative bacteria and are common in both young and old women. Estrogen deficiency plays an important role in the development of bacteriuria [24]. These infections are a serious public health problem because they are recurrent in many patients and can lead to severe sequelae, such as sepsis, pyelonephritis, kidney damage, and premature delivery, and multiresistant strains [2,25]. Urinary tract infections also often result in chronic recurrence, resulting in the frequent use of antibiotics or long-term antimicrobial prophylaxis that exposes patients to the consequences of chronic use of these drugs and long-term changes in normal microbiota of the vagina and gastrointestinal tract [26]. Although rUTIs usually are not life-threatening, the high incidence significantly increases healthcare costs and can negatively impact patients' quality of life [2].
Based on this alarming growth of uropathogens that are resistant to existing drugs and the side effects of antibiotics, new therapeutic agents that are less expensive and have fewer adverse effects need to be developed [24]. Preventing recurrent genito-urinary tract infections and Table 5 improving patients' quality of life have been the goals of many research groups [2]. Herbal treatment may be a viable solution for the effective treatment of diseases that are caused by bacteria [27].

Bidens pilosa
Medicinal plants comprise a large variety of small molecules with antibiotic properties, especially terpenoids, glycosides, flavonoids, and polyphenols. Most of these small molecules have poor activity compared with the actions of common antibiotics that are produced by bacteria and fungi. However, despite the less potent effects of vegetal derivatives, many plants can successfully combat infections because of synergistic effects of their different pharmacologically active compounds [28]. In the present study, such synergistic antimicrobial effects of the crude extracts were observed.
Oral infusion preparations of B. pilosa, B. sulphurea, and T. vulgare are popularly used or by seat baths [7,12,14]. The present study evaluated the effects of extracts of these plants, prepared by infusion, against bacterial strains that were isolated from urine and vaginal secretion samples from menopausal women with a diagnosis of UTIs, with the goal of validating their popular use. Such scientific validation is beneficial for patients because the use of infusion preparations of these plants in the form of a seat bath to treat UTIs may be associated with fewer systemic side effects compared with the current antibiotics that are used clinically. Importantly, medicinal plants are considered low-cost options [29], which would facilitate patients' access to such treatment alternatives for UTIs. Such infections are usually recurrent and present high levels of drug resistance.
In the present study, an elevated MAR index was observed for Staphylococcus spp. (0.25 to 1.00), E. coli (0.33 to 1.00), and P. mirabilis (0.33) that were isolated from urine and vaginal secretion samples. According to Krumperman [20], a MAR index � 0.2 is observed when isolates are exposed to high-risk sources of human or animal contamination. Interestingly, despite the relatively high resistance indices of the studied samples, the extracts effectively inhibited the growth of coagulase-negative Staphylococcus spp. in urine samples and inhibited the growth of both coagulase-positive and -negative Staphylococcus spp. in vaginal secretion samples from menopausal women who were diagnosed with UTIs. This effect was more evident for the extract of B. sulphurea, with intermediate action of T. vulgare. Coagulase-negative Staphylococcus is considered a commensal bacteria in humans, and its role as an etiological agent in various infectious processes has been recognized, especially in urinary infections [30,31].
In addition to the involvement of Staphylococcus spp. in the development of UTIs, enterobacteria are one of the main causes of rUTIs [32]. Bactericidal and bacteriostatic effects of B. sulphurea species were also observed against bacteria, especially E. coli and P. mirabilis, that were isolated from urine and vaginal secretion samples from patients with rUTIs. Such an effect is important because UTIs have a tendency to present recurrence or chronicity, and more than 85% of these infections are caused by uropathogenic E. coli [33].
In addition to exerting antibacterial activity against Gram-negative (P. aeruginosa and E. coli) standard strains, a bacteriostatic effect of B. sulphurea was also observed and with lower intensity in T. vulgare and B. pilosa extracts. Bacteriostatic drugs inhibit the growth of bacteria in the environment, and actions of the immune system are necessary to eliminate them [34]. In addition to exerting bacteriostatic effects, the previously reported antioxidant effects of these medicinal plants may also modulate the immune response by increasing interleukin-2, lymphocytes, and T-cells and decreasing lipid peroxidation and prostaglandin synthesis [35].

Conclusion
The ethnomedicinal form of Bidens pilosa, B. sulphurea and Tanacetum vulgare preparation presented antibacterial activity against standard strains and bacteria that were isolated from cultures of vaginal secretions and urine from menopausal women with a diagnosis of recurrent urinary tract infections. However, B. sulphurea was more efficient. Thus, these results suggest the potential medicinal use of an Asteraceae species, B. sulphurea, as a therapeutic agent against bacteria that cause rUTIs.