Antimicrobial Effects of Aqueous Extract from Calyces of Hibiscus sabdariffa in CD-1 Mice Infected with Multidrug-Resistant Enterohemorrhagic Escherichia coli and S almonella Typhimurium

Abstract

To determinate the antimicrobial effect of chloramphenicol and aqueous extract against multidrug-resistant enterohemorrhagic Escherichia coli (EHEC) and Salmonella enterica serovar Typhimurium in CD-1 mice. Aqueous extract was isolated from Hibiscus sabdariffa calyces. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of chloramphenicol and aqueous extract were determined for EHEC and S. Typhimurium. Nine groups of six mice each were formed. Three groups were inoculated orally with 1 × 10⁴ colony-forming units (CFU) of S. Typhimurium, three groups were inoculated with 1 × 10⁴ CFU of EHEC and the remaining three groups were not inoculated. Six hours postinoculation, the mice of some groups were orally administered solutions of aqueous extract (50 mg/mL), chloramphenicol (82 μg/mL), or isotonic saline. The EHEC and S. Typhimurium concentration in all mice feces was determined. For both pathogens, the MIC and MBC values of aqueous extract were 20 y 50 mg/mL, respectively; for chloramphenicol, they were between 17.5 and 82 μg/mL. EHEC and S. Typhimurium were not detected in the feces of mice that were administered aqueous extract on the 2nd and 3rd days posttreatment. Furthermore, these mice recovered from the infection. In contrast, in mice not treated, or treated with chloramphenicol alone, pathogens were isolated from their feces throughout the study, and some mice died. The H. sabdariffa calyx extracts could be an alternative to control multidrug-resistant bacteria in humans and animals.

INTRODUCTION

Numerous studies have been carried out aimed at understanding the different antimicrobial and phytochemical constituents of medicinal plants and using them for the treatment of microbial infections (both topical and systemic) as possible alternatives to chemically synthetic drugs to which many infectious microorganisms have become resistant.¹ It has been reported that the pace of development of new antimicrobial drugs has slowed down; while the prevalence of resistance (especially multiple resistances) has increased astronomically, the increase in the number of bacteria resistant to antibiotics no longer corresponds to the expansion of the arsenal of agents available to treat infections.²

Literature reports and ethnobotanical records suggest that plants are the sleeping giant of the pharmaceutical industry.¹ They can provide a natural source of antimicrobial drugs that will provide new or leading compounds that can be used to control some infections globally. The medicinal value of plants lies in some chemical substances that produce a defined physiological action on the human body.

Enterohemorrhagic Escherichia coli (EHEC), as a subgroup of Shiga toxin (Stx)-producing E. coli (STEC), are foodborne pathogens responsible for human diseases. In addition to uncomplicated diarrhea, EHEC can cause hemorrhagic colitis and life-threatening complications such as hemolytic-uremic syndrome.³ The recognition of EHEC as an etiological agent of diarrhea with life-threatening complications has made this type of infection a serious public health problem concern.⁴ In various studies, antibiotic multiresistant EHEC has been isolated from some sources such as raw beef and its products^5,6 from shrimp,⁷ from chicken viscera,⁸ and raw vegetables.⁹

Salmonella enterica serovar Typhimurium is the number one cause of food poisoning in Western countries, causing around one million cases of illnesses in the United States every year. The pathogen is shown to be remarkably adaptive, being able to invade a large range of host organisms, and, within the host, has to go through numerous different environments.¹⁰ In various studies, multidrug-resistant Salmonella has been isolated from raw vegetables such as tomatoes¹¹ and carrots.¹² These are widely consumed around the world and, therefore, represent a severe public health risk. Antibiotic-resistant Salmonella has also been isolated from ground meat,¹³ chicken carcasses,¹⁴ shrimp,¹⁵ and buffalo meat.¹⁶

Hibiscus sabdariffa, with an attractive flower, is widely grown in many developing countries. More than 300 species are distributed in tropical and subtropical regions around the world.¹⁷ They are originally native from India to Malaysia,¹⁸ here, it is commonly cultivated and was carried at an early date to Africa. It is also cultivated in Sudan, Egypt, Nigeria, Mexico, Saudi Arabia, Taiwan, West Indies, and Central America.^19,20 Traditionally roselle is cultivated for its stem, leaves, calyces, and seeds as all parts have industrial, medicinal, and other applications.²¹

The extracts from H. sabdariffa calyces have been reported to have antimicrobial effects on different antibiotic-resistant and nonresistant pathogens.^{11,12,22

–31} The pure compounds isolated from the calyces of H. sabdariffa could be an alternative for the control or treatment of infections caused by different types of pathogenic bacteria that are resistant to antibiotics.

The objective of the present study is to evaluate the antimicrobial effect of the aqueous extract obtained from H. sabdariffa calyces when administered to mice infected with antibiotic-resistant EHEC and S. Typhimurium.

MATERIALS AND METHODS

Preparation of H. sabdariffa extract

A kilogram of dehydrated calyces of H. sabdariffa (“Criolla de Oaxaca” variety) grown in Oaxaca, Mexico, was used in the study. The calyces were stored in a closed polyethylene container at room temperature until use.

The aqueous extract from calyces of H. sabdariffa was obtained exactly as we previously described.³¹ In brief, samples (100 g) of dehydrated calyces were placed in glass flasks and 900 mL of distilled water. It was heated to boiling for 15 min and allowed to cool to room temperature. After, the liquid phase was filtered through filter paper (Whatman Grade 4). The filtered extracts were concentrated in a rotary evaporator (V-800, Vacuum Controller, BÜCHI, Switzerland). The water was completely removed from the rota evaporated concentrate by placing it in an air recirculation oven (Ambi-Hi-Low Chamber, Lab-Line, Jefferson, MO, USA) at 45°C for 24 h.^27,32

Determination of antimicrobial effects in culture medium

Preparation of test solutions

Solutions of Aqueous extract (final concentration of 100 mg/mL) and chloramphenicol (final concentration of 1.5 mg/mL) were prepared. Sterile distilled water was used to prepare the solutions.

Bacterial strains

Two multidrug-resistant bacterial strains isolated from food were used: S. Typhimurium C65 isolated from coriander³⁰ and EHEC A enterohemorrhagic E. coli isolated in our laboratory from raw beef. The two bacterial strains were resistant to 10 antibiotics (kanamycin, neomycin, streptomycin, amikacin, tetracycline, erythromycin, chloramphenicol, ceftriaxone, nalidixic acid, and trimethoprim/sulfamethoxazole) according to the protocol indicated by the Clinical and Laboratory Standards Institute.³³

Preparation of bacterial strains

S. Typhimurium and EHEC multidrug-resistant bacteria were inoculated separately in 3 mL of trypticasein soy broth (TSB; Bioxon, Becton Dickinson, Mexico) and incubated at 35 ± 2°C for 18 h. The cultures were washed twice in sterile isotonic saline solution (ISS, 0.85% NaCl) by centrifugation at 3500 rpm for 20 min, and the pellet was resuspended in ISS at approximately 10⁹ colony-forming units/mL (CFU/mL). Finally, a decimal dilution of these washed cultures was done with ISS to produce a final approximate concentration of 10⁸ CFU/mL.^27,32

The antimicrobial effect in culture medium

The gel diffusion technique with paper discs was used. In brief, separately, 100 μL washed bacterial cultures, from a concentration of 1 × 10⁸ CFU/mL, were inoculated onto trypticasein soy agar plates (TSA; Bioxon, Becton Dickinson) and distributed over the agar by the surface extension method. Sterilized paper disks (Whatman grade 5, 6 mm diameter) were placed on the surface of the inoculated agar. Then, 20 μL aliquots containing a solution of aqueous extract (100 mg/mL, final dose on disk 2 mg) and chloramphenicol (1.5 mg/mL, final dose on disk 30 μg) were placed on the paper discs. Chloramphenicol and ISS were used as controls. Treatments were performed in triplicate.

The plates were incubated for 24 h at 35°C ± 2°C. For each treatment, the diameters (mm) of the resulting inhibition zones were measured and expressed as the average.³² All treatments were done in triplicate.

Minimum inhibitory concentration and minimum bactericidal concentration

The broth macro dilution method³⁴ was used to obtain the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Tubes were prepared with TSB containing aqueous extract or chloramphenicol at different concentrations. The tubes were inoculated with a final suspension of S. Typhimurium or EHEC at 1 × 10⁵ CFU/mL (from the culture washed in ISS at a concentration of 1 × 10⁹ CFU/mL, two decimal dilutions were made in TSB, and from the last dilution, 10 μL was taken and inoculated in a tube containing 990 μL, to give a final concentration of 1 × 10⁵ CFU/mL) and incubated at 37°C for 24 h.

The MIC was the lowest concentration of aqueous extract or chloramphenicol to inhibit bacterial growth without turbidity in the tubes. To determinate the MBC, TSB tubes containing the lowest concentration aqueous extract or chloramphenicol with no turbidity were inoculated into TSA using the pour plate technique and incubated at 35°C for 24–48 h. The MBC was defined as the lowest concentration of aqueous extract and chloramphenicol that showed no colony growth in TSA. All treatments were done in triplicate.

The antimicrobial effect in CD-1 mice infected with EHEC or S. Typhimurium

Rifampicin resistance of bacterial strains for use in mice

Rifampicin-resistant mutant strains (Sigma-Aldrich, Mexico) were obtained from S. Typhimurium and EHEC strains resistant to 10 other different antibiotics. In brief, a suspension of each pathogen was prepared by centrifuging 24 h cultures grown in TSB (Bioxon, Becton Dickinson), discarding the supernatant, and washing twice in saline water (NaCl 0.85%) with peptone (0.1%). The washed cells were finally resuspended in 1 mL of sterile 0.1% peptone. A 0.3 mL volume of this suspension was spread on TSA plates containing 1 mL of rifampicin (100 mg) per 1000 mL of TSA (Rif-TSA plates). The inoculated Rif-TSA plates were incubated at 35°C ± 2°C for 24 h, and then, 3–5 colonies that developed on the culture medium were selected.

The rifampicin-resistant (R+) mutants were streaked in slanted TSA tubes, incubated for 24 h at 35°C, and subsequently refrigerated until use.³⁵

Preparation of inoculum

The EHEC R+ and S. Typhimurium R+ strains were inoculated in TSB and incubated at 35°C ± 2°C for 24 h. Each of the cultures was diluted in decimal series in peptone diluent (0.1%) until a cell concentration of 1 × 10⁵ CFU/mL was obtained.

Experimental animals

Sixty-six healthy male mice of the CD-1 strain, aged 8 weeks old, were used. The mice were obtained from the certified Bioterium of the Universidad Autónoma del Estado de Hidalgo (UAEH) and were randomly split into groups of six. Each group was kept in an individual cage, and each cage was kept in the same room under temperature conditions of 22°C ± 2°C, with a 12 h/12 h light-dark cycle. The mice were allowed to adapt to their new environment for a week before inoculation with the pathogenic strains. Food and water were provided by the animal house (standard rodent food) and were administered ad libitum. It is important to note that the experimental protocol involving mice was analyzed and approved byInstitutional Review Board (IRB) named as the UAEH Ethics Committee for the Care and Use of Laboratory Animals (registration number: UAEH2019-A1-S-8288).

Preparation of test solutions for administration to mice

For inoculation into mice, the MBC against the R+ pathogens were used. Therefore, the concentrations of test solutions used were 50 mg/mL and 82 μg/mL for aqueous extract and chloramphenicol, respectively.

Oral inoculation of EHEC R+ and S. Typhimurium R+ and administration of the aqueous extract or chloramphenicol in CD-1 mice

The 54 mice were divided into 9 groups of 6 mice each (I to IX). All groups were maintained for 1 week of adaptation, providing them with standard food and water ad libitum. After this adaptation time, the mice were inoculated with EHEC R+ and S. Typhimurium R+ orally. The mice were held firmly by the scruff of the neck in a vertical position then inoculated with the R+ pathogen in suspension, antimicrobial solution, or saline solution using an esophageal cannula adapted to a sterile needleless syringe.

Mouse Group I was not infected with the R+ pathogenic strains and no treatment was administered (blank, only ISS was administered orally). Groups II and III were not infected with the R+ pathogenic strains, but they were administered chloramphenicol and aqueous extract, respectively (uninfected and treated controls). Groups IV and V were inoculated orally (0.1 mL) with ∼1 × 10⁴ CFU of S. Typhimurium R+ or EHEC R+, respectively, then 6 h after infection, 0.5 mL of ISS was administered orally (infected and untreated controls). Groups VI and VIII were inoculated orally with ∼1 × 10⁴ CFU of S. Typhimurium R+, in a volume of 0.1 mL, then 6 h after infection, received 0.5 mL of chloramphenicol or aqueous extract, respectively (infected and treated groups).

Finally, Groups VII and IX were inoculated orally with ∼1 × 10⁴ CFU of EHEC R+, in a volume of 0.1 mL, then 6 h after infection, received 0.5 mL of chloramphenicol or aqueous extract, respectively (infected and treated groups). Each of the treatments with the test solutions and the ISS were administered to the mice every 12 h for 7 days.

Enumeration of EHEC R+ and S. Typhimurium R+ in mouse feces

Under aseptic conditions, feces were collected from each cage bed every 8 h and stored under refrigeration. The feces of the test animals were taken directly from the sawdust found in the base of each of the cages, containing each group of mice. The feces were taken with sterilized forceps and placed in plastic bags with hermetic closure. Every 24 h, the bags containing the feces of each group of rodents were transported to the laboratory in refrigeration and under aseptic conditions. In the laboratory, the feces of each 24 h period were mixed and numbered for R+ pathogenic bacteria. The bacterial counts were determined for each of the nine study groups.³⁶

The sawdust from each of the nine cages was changed and sterilized daily during the collection of the stool samples to avoid cross-contamination. To prepare for enumeration of EHEC R+ and S. Typhimurium R+ in each stool sample, 9 mL of sterile peptone diluent (0.1%) was placed in the plastic bag containing 1.0 g of stool, then the feces were homogenized manually by rubbing from outside the bag for 1 min. The enumeration of the pathogenic R+ bacteria was performed by the pour plate technique using TSA supplemented with Rifampicin (100 mg/L), and incubating at 35°C ± 2°C. Each dilution was inoculated in triplicate.

To confirm the presence of the R+ mutant strain in the TSA-Rif-plates, the colonies from these plates were taken and streaked onto Eosin and Methylene Blue (EMB) agar or Brilliant Green Agar (BGA), both containing rifampicin (100 mg/L) for EHEC R + or S. Typhimurium R+, respectively.^36,37

Mouse mortality rate and pathological manifestations

The mortality rate of the mice in the different groups was calculated as the number of mice that died during the experiment, about all the mice used in each group.³⁸ Throughout the study, the consistency of the fecal matter of each rodent was registered. Animals were also observed daily for any physiological and pathological abnormalities (weight loss, loss of appetite, weakness/slow movement, and mortality) during the period of the experiment. At the end of the study (7 days after inoculation and treatment), all infected and uninfected mice were sacrificed by cervical dislocation, and incinerated, to prevent the spread of infection by EHEC R+ and S. Typhimurium R+ in the environment.

RESULTS AND DISCUSSION

Antimicrobial effects of solutions in culture medium

The results of the effects of the three solutions examined are found in Table 1. It can be seen in Table 1 that the aqueous extract presented radial inhibition zones of 10.1 and 9.2 mm for S. Typhimurium and EHEC, respectively. Chloramphenicol presented zones of inhibition of 14.3 and 10.7 mm for S. Typhimurium and EHEC, respectively. According to the Clinical and Laboratory Standards Institute (CLSI),³³ these results indicate that EHEC presents resistance to chloramphenicol (zone of inhibition ≤12 mm), while S. Typhimurium presents intermediate resistance to this antibiotic (13–17 mm zone of inhibition).

Table 1.

Diameters of Inhibition Halos of Aqueous Extract and Chloramphenicol Against Multidrug-Resistant S. Typhimurium and Enterohemorrhagic E. coli

Bacteria	Treatments
Bacteria	Aqueous extract	Chloramphenicol
S. Typhimurium	10.1 ± 0.5^a	14.3 ± 0.6
EHEC	9.2 ± 0.4	10.7 ± 0.5

Mean ± standard deviation of three replicas of zone of inhibition diameter (mm).

EHEC, enterohemorrhagic Escherichia coli.

We have recently reported the in vitro antimicrobial effect of another extract from H. sabdariffa against the same pathogenic strains used in this study.³⁹

Determination of MIC and MBC of aqueous extract and chloramphenicol

The values obtained for the MIC and MBC of aqueous extract and chloramphenicol against S. Typhimurium and EHEC are reported in Table 2. It should be noted that the MIC values that are obtained with aqueous extract were 20 mg/mL for S. Typhimurium and EHEC, while, for chloramphenicol, it was 17.5 μg/mL for both pathogenic strains. Concerning to MBC, for aqueous extract and chloramphenicol, the values were 50 mg/mL and 82 μg/mL for both pathogenic bacteria, respectively. According to CLSI, members of Enterobacterales are sensitive to chloramphenicol if MIC equal to or below 8 μg/mL.³³ As one would expect, both S. Typhimurium and EHEC strains had a MIC of 17.5 μg/mL, which confirms that both strains were resistant to chloramphenicol.

Table 2.

Minimum Inhibitory Concentration, Minimum Bactericidal Concentration, and MBC/MIC Ratio of Aqueous Extract and Chloramphenicol on Multidrug-Resistant S. Typhimurium and Enterohemorrhagic E. coli

	Aqueous extract (mg/mL)			Chloramphenicol (μg/mL)
Bacteria	MIC	MBC	MBC/MIC	MIC	MBC	MBC/MIC
S. Typhimurium	20	50	2.5	17.5	82	4.7
EHEC	20	50	2.5	17.5	82	4.7

MBC, minimum bactericidal concentration; MIC, minimum inhibitory concentration.

Farooqui et al. ⁴⁰ reported that the MIC of chloramphenicol for the S. Typhimurium AmpCSxtTNa strain was 512 μg/mL. These values are much higher than those obtained in the present study. In another study, Obinna et al. ⁴¹ obtained MIC values of 1–256 μg/mL for chloramphenicol with different pathogenic bacteria. Similarly, Andrews⁴² reported MIC values of 2–4 mg/mL of chloramphenicol for E. coli strains; values are very high compared to most of the reported studies. However, the differences in MIC values may be due to the intrinsic resistance of each of the pathogenic strains to this antibiotic.

By comparing the MIC and MBC values, it was also determined whether the antimicrobial effects of aqueous extract and chloramphenicol were bactericidal or bacteriostatic. A compound is considered bactericidal when the ratio of MBC/MIC ≤4, and considered bacteriostatic when the ratio of MBC/MIC >4.⁴³ As shown in Table 2, the aqueous extract showed bactericidal activity for both pathogenic strains, while chloramphenicol showed bacteriostatic activity. We have previously reported the bactericidal effect of another extract of H. sabdariffa.³⁹

Effects of aqueous extract and chloramphenicol in mice infected with EHEC or S. Typhimurium

The results of the antibacterial activity of aqueous extract and chloramphenicol in CD-1 mice infected with S. Typhimurium R+ or EHEC R+ are reported in Table 3. As observed in Table 3, on the day the pathogenic bacteria were administered orally to the rodents, which we call “day 0,” neither of the pathogenic bacteria strains was detected in the feces of any of the nine groups of rodents. However, 24 h after inoculation, both S. Typhimurium R+ and EHEC R+ were detected and quantified in the fecal matter of the mice that were inoculated (Groups IV–IX, Table 3).

Table 3.

Effect of Treatments in CD-1 Mice on the Fecal Excretion of S. Typhimurium R+ and Enterohemorrhagic E. coli R+

Groups	Treatments	No. of R+ bacteria excreted in the feces of mice on each day throughout the study (CFU/g)
Groups	Treatments	Day 0	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6	Day 7
I (Mouse)	ISS	0	0	0	0	0	0	0	0
II (Mouse)	Chloramphenicol	0	0	0	0	0	0	0	0
III (Mouse)	Aqueous extract	0	0	0	0	0	0	0	0
IV (Mouse + S. Typhimurium R+)	ISS	0	^*3.7 × 10²	1 × 10³	7 × 10³	1 × 10⁴	6 × 10⁴	1 × 10⁵	8 × 10⁵
V (Mouse + EHEC R+)	ISS	0	6 × 10¹	5 × 10²	1.3 × 10⁴	5 × 10⁴	1 × 10⁵	9 × 10⁵	1 × 10⁷
VI (Mouse + S. Typhimurium R+)	Chloramphenicol	0	3 × 10¹	1.6 × 10²	6 × 10³	7 × 10³	1 × 10⁴	5 × 10⁴	1 × 10⁵
VII (Mouse + EHEC R+)	Chloramphenicol	0	1 × 10²	5.8 × 10²	6 × 10³	8 × 10³	1.1 × 10⁴	1 × 10⁵	1 × 10⁶
VIII (Mouse + S. Typhimurium R+)	Aqueous extract	0	4.5 × 10³	1.3 × 10⁴	0	0	0	0	0
IX (Mouse + EHEC R+)	Aqueous extract	0	3.9 × 10³	0	0	0	0	0	0

(CFU/g).

ISS, isotonic saline solution; R+, rifampicin-resistant.

Groups IV and V were infected with S. Typhimurium R+ or EHEC R+, respectively, and only received ISS every 24 h. In these groups, pathogenic bacteria were detected in the feces at concentrations of 3.7 × 10² and 6 × 10¹ CFU/g, respectively, 24 h after inoculation. By day 7, the detected bacterial concentration reached 8 × 10⁵ and 1 × 10⁷ CFU/g. It should be noted that Group V mice infected with EHEC R+ also developed signs of disease, and some died (Table 4). Groups VI and VII were infected with the pathogenic bacteria and administered with chloramphenicol (82 μg/mL per dose) every 24 h. In these groups, both pathogenic bacteria were also detected at high levels in the feces of rodents during the 7 days of study. Also, in this case, the EHEC R+-infected mice developed signs of disease and some died (Table 4).

Table 4.

Clinical Signs and Mortality Observed in Groups of Mice Infected and Not Infected with S. Typhimurium R+ and Enterohemorrhagic E. coli R+ During the Course of the Experiment

Groups	Treatments	Mortality rate/clinical symptoms
Groups	Treatments	Loss of weight	Loss of appetite	Body weakness/Slow movement	Mortality rate
I (Mouse)	ISS	0/6 (0)^a	0/6 (0)	0/6 (0)	0/6 (0)
II (Mouse)	Chloramphenicol	0/6 (0)	0/6 (0)	0/6 (0)	0/6 (0)
III (Mouse)	Aqueous extract	0/6 (0)	0/6 (0)	0/6 (0)	0/6 (0)
IV (Mouse + S. Typhimurium R+)	ISS	0/6 (0)	0/6 (0)	0/6 (0)	0/6 (0)
V (Mouse + EHEC R+)	ISS	5/6 (83)	5/6 (83)	5/6 (83)	2/6 (33.3)
VI (Mouse + S. Typhimurium R+)	Chloramphenicol	0/6 (0)	0/6 (0)	0/6 (0)	0/6 (0)
VII (Mouse + EHEC R+)	Chloramphenicol	5/6 (83)	5/6 (83)	5/6 (83)	2/6 (33.3)
VIII (Mouse + S. Typhimurium R+)	Aqueous extract	1/6 (17)	1/6 (17)	1/6 (17)	0/6 (0)
IX (Mouse + EHEC R+)	Aqueous extract	1/6 (17)	1/6 (17)	1/6 (17)	0/6 (0)

R+, rifampicin-resistant.

Number of affected mice/total number of mice in each group. The values in parentheses represent the percentage of affected mice in each group during the experiment.

The results show that chloramphenicol had no antibiotic effect on any of the pathogenic bacteria (Table 3). It should be remembered that EHEC R+ and S. Typhimurium R+ had shown resistance to chloramphenicol (Table 2), according to the CLSI.³³ In contrast, in the groups of rodents infected with the R+ pathogens (Groups VIII and IX) that received treatment every 12 h with an aqueous extract (50 mg/mL per dose), no R+ pathogenic bacteria were detected in the feces of the rodents by day 3 (Group VIII) or day 2 (Groups IX, Table 3). Besides, for the mice in these groups, although at the beginning, some developed signs of disease; after 48 h, the signs of disease disappeared and none of the rodents died (Table 4).

Finally, the rodents in the control groups (Groups I, II, and III) did not develop signs of disease and the pathogenic bacteria R+ were not detected in the feces throughout the study.

The results clearly show that aqueous extract could be an alternative therapy for the treatment of infections caused by both antibiotic-resistant, and nonresistant, bacteria.

Many studies have been carried out to try and understand the different antimicrobial and phytochemical components of medicinal plants. The aim is to be able to use them for the topical or systemic treatment of microbial infections and provide possible alternatives to chemically synthetic antibiotics, to which many infectious microorganisms have become resistant.¹ The development of drug resistance in human pathogens against commonly used antibiotics has required a search for new antimicrobial substances from other sources, including plants, which can combat pathogens as antimicrobial and as antidiarrheal agents.⁴⁴

Recently, Itelima and Agina³⁶ reported the antimicrobial activity of five species of plants (Allium sativum, Mangifera indica, Psidium guajava, Vernonia amygdalina, and Zingiber officinale) in albino rats infected with E. coli O157: H7 and administered with extracts of the plants for 14 days. This study revealed that the ethanolic extracts of the plant species used, not only prevented the development of diarrhea in infected rats but also inhibited the growth of E. coli O157: H7 in them.

Compared with our study, they used a higher concentration bacterial suspension (1 × 10⁹ CFU/mL of E. coli O157: H7), and also administered a single dose (simultaneously with the bacterial inoculum) of each extract at a concentration of 3.0 mg/kg of the bodyweight of the rat. Using this protocol, at days 5, 7, 8, 9, and 10, the microbial load in the feces was reduced to 0 CFU/g with the ethanolic extracts of P. guajava, A. sativum, Z. officinale, V. amygdalina, and M. indica, respectively. These results show that, despite reducing the microbial load to 0 CFU/g, they did so in a longer time than was achieved in our study, as seen in Table 3.

Similarly, Tala et al. ⁴⁵ evaluated the effect of aqueous extract of Euphorbia prostrata Aiton on S. Typhimurium in Wistar rats. The rats were inoculated orally with 1 × 10⁸ CFU of Salmonella and were administered gradual doses (26.34, 44.00, 73.48, 122.71 mg/kg) of the aqueous extract of E. prostrata. Administration of this extract was found to markedly reduce the concentration of S. Typhimurium in feces. S. Typhimurium was no longer detected in the feces of the animals between the 8th and 10th days of treatment.

In another study, Jang-Gi et al.,⁴⁶ determined the in vivo effect of an ethanolic extract of the Punica granatum husk on Balb/c mice infected with S. Typhimurium. Mice were infected with 1 × 10⁵ CFU/mL of S. Typhimurium and then treated with P. granatum extract. In this study, the mice were divided into the following groups: uninfected control (CON), infected with Salmonella but not treated (SI), and infected with Salmonella and administered extract of P. granatum (SIPG). One hour after infection, animals in the SIPG group were administered 5 mg of P. granatum extract orally, and every 24 h thereafter.

On day 1 postinfection, S. Typhimurium was isolated from the feces of rodents in the SI and SIPG groups at concentrations of 2 × 10² to 2 × 10⁵ CFU/g. However, on day 6 of the study, the pathogen was no longer detected in the feces of rodents of the SIPG group, while the pathogen was still isolated in high numbers from the feces of mice of the SI group, and some mice of that group had already died.

The results published by different researchers and also ours shown in this document show that plants can be an alternative for use against bacteria resistant to antibiotics. However, further studies are needed.

CONCLUSIONS

Many authors have recommended a significant decrease in the use of antibiotics and an increase in the use of compounds obtained from plants. This is because several plant compounds show strong antimicrobial activity against a wide range of gram-positive and gram-negative bacteria, without exhibiting toxic effects. ⁴⁷ Therefore, there is a growing interest in the use of plants for the treatment of various diseases in humans and animals.

In our study, the effect of aqueous extract on the control of infection in mice by EHEC R+ and S. Typhimurium R+ is clear, in addition to being rifampicin-resistant, the R+ strains used in this study were also resistant to 10 other antibiotics, including chloramphenicol, making our results on the antimicrobial properties of aqueous extract even more striking. The results of the present study suggest that aqueous extract obtained from H. sabdariffa calyces has the potential to be used as an antimicrobial in human and veterinary medicine.

ETHICAL CONSIDERATIONS

The authors indicate that the procedures followed were in accordance with the ethical standards of the responsible committee on animal experimentation (institutional and national).

Footnotes

AUTHOR DISCLOSURE STATEMENT

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the article, or in the decision to publish the results.

FUNDING INFORMATION

The work was supported by National Council for Science and Technology (CONACyT) for financial support to the project number A1-S-8288 “Antimicrobials from Jamaica flower calyxes alone and in combination with antibiotics: determination of the mechanisms of action on resistant and nonresistant pathogenic bacteria to antibiotics, the antimicrobial effect in vivo, and adverse reactions in animals”

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