Efficacy of leaf extracts of Psidium guajava (L.) on enteric bacterial isolates from faecally impacted groundwater

Waterborne diseases are the major cause of morbidity and mortality in low- and middle- income countries and antimicrobial agents are essentially important in reducing the global burden of waterborne infectious diseases (Mandal et al. 2011). Most waterborne diseases are often transmitted via the faecal-oral route, and this occurs when human faecal material is ingested through drinking contaminated water or eating contaminated food as a result of inadequate water, sanitation and hygiene.

Plants have been a veritable source of drugs and the scientific exploration of medicinal plants for the benefit of man is intensified because these plants may likely reduce the dependence on orthodox chemotherapeutic agents (Ayodele et al. 2019). Medicinal plants are important with respect to new drug and pharmacological research development and are widely used and accepted as home remedies and raw materials for the pharmaceutical industry (Mule et al. 2013). Development of antibiotics resistance by enteric bacterial pathogens including Salmonella and Shigella, against easily accessible and commonly prescribed drugs has become a major concern throughout the world, particularly in developing countries (Oncho et al. 2021). There is a need to search for new antimicrobial agents among plant extracts because plant-based antimicrobials represent a vast untapped source of medicine.

Guava (Psidium guajava L.) belongs to the family Myrtaceae and is considered to have originated from tropical South America. Guava trees grow in tropical and sub-tropical regions of the world such as Asia, Africa and Hawaii (Biswas et al. 2013). The leaves have long been recognized for their antimicrobial activity. P. guajava Linn is a phytotherapeutic plant used in folk medicine for the management of various disease conditions including enteric bacterial infections and it is also a good source of natural antioxidants (Oncho et al. 2021). Studies on phytochemical components of different guava extracts has revealed numerous bioactive compounds such as tannin, flavonoid, terpenoid, steroids, glycoside, cardiac glycoside, alkaloid, phlobatannin, polyphenol, saponin anthraquinones and phytosteroid (Oncho et al. 2021). Plant-derived bioactive compounds are promising sources of antimicrobials. These compounds act by the inhibition of microbial cell wall development, disruption, and lysis, hampering biofilm formation, repression of DNA replication and transcription, impeding adenosine triphosphate (ATP) production, suppression of bacterial toxins, and the generation of reactive oxygen species (ROS) (Kumar et al. 2021). Guava leaves, owing to the presence of different organic and inorganic antioxidants and anti-inflammatory compounds, have been shown to demonstrate antimicrobial activities against Pseudomonas aeruginosa, Escherichia coli, Streptococcus faecalis, Staphylococcus aureus and Bacillus subtilis (Kumar et al. 2021).

Several studies have reported the antidiarrheal potential of guava leaves. For instance, Mazumdar et al. (2015) observed that a dosage level of extracts of guava leaves at a concentration of 500 and 750 mg/kg had antidiarrheal effect in rats. Similarly, Kumar et al. (2021) reported that guava leaf extracts at doses of 52–410 mg/kg when administrated orally were found to combat diarrhea, and also resulted in reduced intestinal transit and dilatory removal of unwanted gastric products in rodents. In addition, symptoms, such as secretion of interstitial fluid and wetness of faecal droppings in a dose-dependent manner were reduced significantly. In another related study, Dewi et al. (2013) combined aqueous leaf extracts of guava and leaf extracts of green tea at different ratios and reported that all combinations had potent antidiarrheal activities, depicted by enhanced stool weight, stool onset, stool consistency and diarrhea period.

This study set out to assess the antibacterial effects of leaf extracts of guava P. guajava (L.) on enteric bacteria isolated from selected groundwater sources in Akure, Nigeria. The objectives of this study were to determine the level of faecal contamination of the wells; examine the phytochemical components of the leaf extracts of guava P. guajava (L.); and assess the antibacterial effects of the leaf extracts on enteric bacteria from the wells.

Fresh leaves of P. guajava (L.) were collected from guava trees growing at a residential house in Owo, Ondo State, Nigeria. The leaves were collected into plastic bags, stored in a cool box with ice packs and transported to the Department of Crop, Soil and Pest Management, Federal University of Technology, Akure, Nigeria for identification. Thereafter, the leaves were washed thoroughly with running tap water, dried, homogenized to fine powder, stored in an air tight bottle and kept at room temperature until required. One hundred grams (100 g) each of the dried powdered plant leaves was weighed and sequentially extracted with 150 ml of ethanol and hot distilled water by maceration method. The extracts were filtered using Whatman No. 1 filter paper and filtrates were concentrated appropriately, weighed, labelled and stored at 4 °C. Two grams (2 g) of the filtrate were dissolved in 10 ml Dimethyl sulfoxide (DMSO) containing 70% distilled water and 30% DMSO to achieve a 100% concentration.

The study area was New Castle Hostel along Deeper Life Camp Ground, FUTA North gate, Akure, Ondo State, Nigeria. Both wells (W1 and W2) were situated at approximately 6 m and 5 m from a septic tank, respectively, and were also in close proximity to a landfill (approximately 10 m from the wells). W1 and W2 were within coordinates 5° 8° 30°E and 7° 18° 30°N as shown in Figure 1. Water samples were collected in duplicates using 500 ml sterile bottles labelled A and B over a period of 16 weeks (March to June, 2019), totaling 32 water samples from each well. The samples were transported in a cool box with ice packs to the laboratory for analyses within one hour.

Membrane faecal coliform agar (m-FC), membrane lauryl sulphate agar (MLSA), Salmonella Shigella agar (SSA) and Eosin Methylene Blue (EMB) agar were used as the selective media. The concentrations of faecal coliforms, E. coli, Salmonella, and Shigella in the water samples were determined using the membrane filtration technique. Membrane filters (0.45 μm) were placed on prepared media aseptically and agar plates were incubated at 44 °C for 24 hours (m-FC), 37 °C for 24 hours (MLSA, EMB, SSA). Colonies were counted, calculated and expressed as colony forming units (CFU) per 100 ml.

Bacterial colonies from 24 hours overnight culture were used to make a suspension. Four or five isolated colonies of the organism to be tested were picked with a sterile inoculating loop and suspended in 2 ml of sterile saline. The saline tube was mixed thoroughly using a vortex and the turbidity of the suspension was adjusted to a 0.5 McFarland standard. More organism was added to the suspension when it was too light and diluted with sterile saline when the suspension was too heavy to achieve a 0.5 McFarland standard. The suspension was used within 15 minutes of preparation.

The agar well diffusion method was used for the assessment of antibacterial activity of leaf extracts of P. guajava (L.). Mueller Hilton agar was prepared according to manufacturer's specification and a standardized inoculum was swabbed on the agar. Four wells were made on each plate using a sterile cork borer of 6 mm diameter. The wells were filled with 0.1 ml of different diluted concentrations (200 mg/ml, 100 mg/ml, 50 mg/ml and 25 mg/ml respectively) of the extract with the aid of a filter syringe. The plates were allowed to stand for 15 minutes to allow pre-diffusion of the extracts, and thereafter incubated at 37 °C for 24 hours. Diameters of zones of inhibition were measured using a transparent plastic ruler and recorded appropriately.

The leaf extracts of P. guajava (L.) were tested qualitatively for the presence of saponin, tannin, phlobatannin, flavonoid, steroids, terpenoids and alkaloids using standard methods (Poongothai et al. 2011). In addition, the leaf extracts of P. guajava (L.) were tested quantitatively for the presence of saponin, tannin, flavonoid, steroids and terpenoids using standard methods (Sofowora 2008).

About 600 μl of bacterial culture was added to a 1.5 ml microcentrifuge tube, and centrifuged for 30 seconds at 14,000 rpm. The supernatant was discarded and 100 μl of 7X lysis buffer (blue) 1 was added and mixed. About 350 μl of cold neutralization buffer (yellow) was added to the solution, mixed thoroughly and centrifuged at 11,000–16,000 rpm for 2–4 minutes. The supernatant was transferred into Zymo-Spin™ IIN column and placed into a collection tube, then centrifuged for 15 seconds. Other buffers (Endo-wash, Zyppy™ wash, Zyppy™ elution) were added appropriately to obtain the plasmid DNA. Plasmid curing was performed by inoculating 100 ml of culture grown in broth containing 10% of Sodium Dodecyl Sulfate (SDS). After inoculation, the cultures were incubated at 37 °C for 24 h. After 24 h incubations, the above procedure was used to purify the plasmid. Antibiotics sensitivity testing of isolates was carried out. Briefly, a sterile swab was dipped into the broth suspension containing the isolate and excess moisture was removed. The surface of a plate containing Mueller-Hinton agar was inoculated with the isolate by streaking and was allowed to dry for about 5 minutes before placing the antibiotic disks on the agar. Thereafter, the plates were inverted and placed in an incubator at 37 °C for 24 h. Diameters of zones of inhibition were measured using a transparent plastic ruler (Kirby et al. 1996).

Data obtained were transformed to log₁₀ using Microsoft Excel and examined using Statistical Package for Social Sciences (SPSS) Version 23.0. All experiments were performed in triplicates and data derived from the study were subjected to 2-way analysis of variance (ANOVA) and level of significance was documented at P < 0.05.

The mean concentrations of the faecal coliforms, E. coli, Salmonella and Shigella in water samples from the wells (W1 and W2) over the study period are shown in Table 1. The mean concentrations of faecal coliforms in the water samples from W1 and W2 were 4.1 and 4.4 log₁₀ CFU/100 ml, respectively, whereas those of E. coli in the water samples from W1 and W2 were 3.2 and 3.1 log₁₀ CFU/100 ml, respectively. Similarly, the mean concentrations of Salmonella in the water samples from W1 and W2 were 3.8 and 3.9 log₁₀ CFU/100 ml, respectively, whereas those of Shigella in in the water samples from W1 and W2 were 3.6 and 3.7 log₁₀ CFU/100 ml, respectively.

The antibacterial activity of ethanol and hot distilled water leaf extracts of P. guajava (L.) measured at varying concentrations (200, 100, 150 and 25 mg/ml) against Salmonella and Shigella revealed that ethanol leaf extracts of P. guajava (L.) had higher antibacterial effect compared to hot distilled water leaf extracts of P. guajava (L.). Salmonella was observed to be resistant to both ethanol and hot distilled water leaf extracts of P. guajava (L.), whereas Shigella was susceptible to ethanol leaf extracts of P. guajava (L.) with the highest zone of inhibition (22.0 mm) at 200 mg/ml and the least zone of inhibition (6.7 mm) at 25 mg/ml (Table 2).

The qualitative phytochemical components of ethanol leaf extracts of P. guajava (L.) revealed the presence of saponin, tannin, flavonoid, steroid, terpenoid, whereas those present in hot distilled water leaf extracts of P. guajava (L.) include saponin, tannin, flavonoid, terpenoid. However, phlobatannin and alkaloid were absent in both ethanol and hot distilled water leaf extracts of P. guajava (L.) (Table 3).

The quantitative phytochemical components of ethanol leaf extracts of P. guajava (L.) showed that terpenoid (25.80 mg/g) had the highest quantity, while steroid (3.71 mg/g) had the least. On the other hand, the quantitative phytochemical components of hot distilled water leaf extracts of P. guajava (L.) showed that saponin (59.82± 0.21 mg/g) had the highest quantity while steroid (0.00 mg/g) had the least (Table 4).

The gel image of profiling and curing of Salmonella revealed that the resistance of the isolates was plasmid based and the DNA plasmid molecules were lost after curing, as shown in Figure 2. Septrin and ciprofloxacin exhibited the highest zone of inhibition (25.67 mm), while gentamycin exhibited the least zone of inhibition (20.67 mm) on Salmonella (Table 5).

The antibacterial effects of leaf extracts of guava P. guajava (L.) on enteric bacteria isolated from selected groundwater sources in Akure, Nigeria were assessed. The occurrence of faecal coliforms in the water samples from the wells suggest faecal pollution and degraded groundwater quality. Studies have shown that consumption of faecally contaminated water may lead to the occurrence of gastrointestinal diseases (Folorunso et al. 2014; Onyango et al. 2018). In this study, the concentration of E. coli in water samples from well 1 and well 2 were higher than those observed by Olalemi et al. (2021) in water from a well situated in Apatapiti, Akure, Nigeria and this may likely be as a result of the landfill and septic tanks sited around the wells. Onyango et al. (2018) had reported that the presence of E. coli in higher counts in groundwater indicate contamination with faecal matter and other pathogens that may compromise the safety of the water source. The level of faecal pollution in the wells (W1 and W2) were ‘critical’ based on E. coli concentration. The results also revealed the presence of Salmonella and Shigella in the water samples from the wells. This observation is similar to the findings of Folorunso et al. (2014) where the authors reported the presence of the pathogens in the water samples obtained from plastic containers used in storing well water and suggested that the bacteria thrive well in polluted and untreated water source. It also in agreement with Mahagamage et al. (2020), where the authors observed high counts of Salmonella and Shigella in groundwater sources in Kelani River Basin in Sri Lanka.

Ethanol leaf extracts of P. guajava (L.), which showed higher antibacterial activity on Shigella compared to hot distilled water leaf extracts of P. guajava (L.), may likely be attributed to better solubility of the bioactive components of leaf extracts of P. guajava (L.) in ethanol than hot distilled water. This is in agreement with the findings of Nwanneka et al. (2013) where the authors investigated the antimicrobial activity of leaf extracts of P. guajava and observed that ethanol leaf extracts of guava exhibited higher inhibition on tested bacteria than aqueous leaf extracts of guava. The resistance of Salmonella to ethanol leaf extracts of P. guajava (L.) may be due to the presence of resistance genes present in the Gram negative organism. This is in contrast to the findings of Chanda & Kaneria (2011) and Oncho et al. (2021), where the authors reported an inhibitory effect of guava extracts on Salmonella.

Studies have shown that phenolic compounds, particularly flavonoids, are responsible for antibacterial properties of guava (Farhana 2017). Quercetin is one of the most predominant flavonoids of guava leaves with high pharmacological activity (Hirudkar et al. 2020). The presence of other components such as terpenoids, glycosides and saponins in guava extracts has been demonstrated to correlate positively with antibacterial activity (Johnson et al. 2017; Kumar et al. 2021). Similarly, water-soluble tannins present in guava leaves may act as bacteriostatic agents, with mechanism of actions such as withholding substratum, hampering oxidative phosphorylation, and extracellular enzyme inhibition (Das & Goswami 2019; Kumar et al. 2021). The quantitative phytochemical components of ethanol and hot distilled water leaf extracts of P. guajava (L.) in this study revealed differences for the same plant species. Studies have shown that the results of phytochemical analysis may differ because of various factors such as biochemical reaction within species, plant genotypes, developmental stages and geographical locations. Furthermore, variations in extraction methods are usually found in the length of the extraction period, pH, temperature, particle size, and the solvent-to-sample ratio (Taura et al. 2014; Oncho et al. 2021).

A key factor that has led to the rise and global dissemination of multidrug-resistant (MDR) bacteria is mobile antimicrobial resistance genes (ARGs). These are frequently located on plasmids, which are pieces of usually circular, self-replicating DNA that can code for a variety of different functional gene groups (Michelle et al. 2018). Antimicrobial resistance genes that pose the most significant threat to human medicine are typically found in Gram-negative bacteria (Michelle et al. 2018). Previous reports have shown that drug resistance of Salmonella is an emerging global challenge, especially in low- and middle-income countries where misuse and abuse of antimicrobial agents is on the increase (Ocean et al. 2015). Furthermore, the emergence and spread of plasmid-borne resistance genes that undergo horizontal gene transfer are threatening many last-line antibiotic therapies (Crofts et al. 2017). In this present study, Salmonella was observed to be resistant to leaf extracts of P. guajava (L.) and this resistance may likely be plasmid-mediated because the antibiotics sensitivity pattern of Salmonella after curing suggest that the plasmids responsible for resistance had been removed. This was evident in the marked sensitivity of Salmonella to antibiotics such as septrin, chloramphenicol, augmentin, gentamycin and streptomycin.

Antibacterial activity of ethanol and hot distilled water extract of leaf of P. guajava (L.) against Shigella and Salmonella isolated from water samples from the selected wells revealed that the ethanol leaf extracts of P. guajava (L.) had inhibitory effect on Shigella but not on Salmonella. The hot distilled water leaf extracts of P. guajava (L.) had no inhibitory effect on the isolates. Qualitative and quantitative phytochemical components of leaf extracts of P. guajava (L.) showed the presence of saponin, flavonoids, glycoside, terpenoid, steroid and tannin that contributed to the antibacterial effects of the leaf extracts of P. guajava (L.) on Shigella. This study further provides more evidence supporting the hypothesis for the use of leaf extracts of P. guajava (L.) in the treatment of gastrointestinal infections caused by Shigella. Ethanol leaf extracts of P. guajava (L.) may be used for treatment in combination with commercially available antibiotics against gastrointestinal infections. The potential antibacterial effects of P. guajava could be enhanced by extracting with ethanol instead of water as applied in the traditional practice.

The financial support of Mr and Mrs F. M. Sule is highly appreciated for the conduct of this study. The technical support of the staff of Crop, Soil and Pest management and Microbiology departments of the Federal University of Technology Akure, (FUTA) are well appreciated in this study.

All relevant data are included in the paper or its Supplementary Information.