Multi-drug resistance of bacterial infections is a public health menace in the 21st century. This is aggravated by the   continuous  misuse  of   antibiotics   in  humans  and animals. Systemic bacterial infections cause urinary tract infections, meningitis, septicemia, pneumonia, peritonitis and  gastritis.  Community and hospital acquired systemic infections are mostly caused by Enterobacteriaceae, spread easily among humans though contaminated water and food. Plasmids and transposons are mostly responsible for the transfer of the multi-drug resistant genetic material among the Enterobacteriaceae (Nordmann et al., 2011).

Until recently carbapenems such as meropenem, ertapenem, imipenem and doripenem were the drug of choice for the successful treatment of most systemic infections. Carbapenems are also valuable in the treatment of extended-spectrum beta-lactamases (ESBLs) producing bacteria (Pitout and Laupland, 2008) (Vardakas et al., 2012). It has been established in Accra that ESBL producers were increasingly becoming a public health nuisance and carbapenems were the best treatment option for the ESBL infections (Hackman et al., 2013).

The emergence of carbapenemase-resistant Enterobacteriaeae (CRE) is a threat to the effective treatment of systemic infections. The carbapenemese enzymes produced by Enterobacteriaceae are capable of inactivating carbapenems. Some of the emerging CRE responsible for systemic infections include Klebsiella pneumoniae, Escherichia coli, Providencia rettgeri, Proteus mirabili, Pantoea species, Citrobacter koseri and Acinetobacter baumanii (Hackman et al., 2017).

Several carbapenemeses have been characterized (Queenan and Bush, 2007) after Nordmann reported carbapenemase-producing Enterobacteriaceae in 1993 (Naas and Nordmann, 1994). There is limited antibiotic treatment option for these multidrug-resistant systemic infections (Falagas et al., 2013). Natural products remain the important source of therapeutic agents for systemic infections and other conditions (Korosecchviz et al., 1992). The phytochemical constituents and pharmacological potential of a significant number of higher plants remain unknown (Saraswathy et al., 2011). This calls for consistent and deliberate studies of plant species to discover alternative treatment options.

Psidium guajava (guava) is known for its fruit and medicinal purposes in the tropical countries. Guava is indicated for the treatment of gastroenteritis, caries, diarrhea, dysentery, diabetes, hypertension and pain relief (Naseer et al., 2018). It contains high content of organic and inorganic compounds such as antimicrobials, antioxidants, polyphenols and anti-inflammatory compounds (Naseer et al., 2018).

In this study, the antimicrobial activity of ethanolic extract of the guava leaves was tested against carbapenem-resistant K. pneumoniae. The study hypothesized that P. guajava possess phytochemical compounds with antimicrobial efficacy against carbapenem-resistant   K.  pneumoniae   through  in  vitro analysis.

The carbapenem-resistant Enterobacteriaeae used in the study was K. pneumoniae.

Subculturing of carbapenem-resistant K. pneumonia

In order to obtain pure culture of K. pneumoniae, the isolates were sub-cultured on Mueller Hinton agar plate and incubated for 24 h. Using an inoculation needle, at least four colonies from the pure culture were transferred into sterile peptone water and adjusted to turbidity equivalent to 0.5 McFarland Standard.

Antibacterial susceptibility testing using agar well-diffusion method

The agar-well diffusion method was used to assess the antibacterial activity of the guava leaves extracts. A 3 ml inoculum with 0.5 McFarland was uniformly spread on the Muellar Hinton agar plate using the glass spreader. Cork borer was used to create wells (6mm diameter) in the plate. Twenty microlitres (20 µl) of the guava leaves extract concentrations (200, 100, 50, 25 and 12.5 mg/ml) were transferred into the wells. Amikacin (30 µg/ml) and colistin (10 µg/ml) disks were used as positive controls and 5% v/v DMSO and sterile distilled water (SDW) were used as the negative controls. The guava leaves extract and controls were allowed to diffuse at room temperature for 30 min and the plates were then incubated at 37°C for 24 h. The experiments were carried out in triplicates. After incubation, the diameter zone of inhibition (DZI) was measured and recorded in millimeters. According to Junior and Zanil (2000), DZI < 7mm was considered inactive; 7-10 mm partially active; 11-16 active and > 16 very active.

Determination of minimum inhibition concentration (MIC)

Hundred microlitres (100 µl) of ethanolic extract of the guava leaves were added to 100 µl of 5% DMSO in the first well of the 96-well microplate. Hundred microlitres (100 µl) of the mixture was transferred to the next well in the column. The two-fold serial dilution was continued from 10¹ to 10¹⁰ with the aid of a micropipette. The final volume in each well was 100 μl. Amikacin (30 µg/ml) and colistin (10 µg/ml) as positive controls were also serially diluted in different columns of the microplate. Five microlitres of 0.5 McFarland microbial suspension was dispensed into the wells and incubated at 37°C for 24 h.

Forty microlitres of 0.2 mg/ml INT (p-Iodonitrotetrazolium violet) dissolved in sterile distilled water was added to each of the wells after incubation. After additional 30 to 120 min of incubation, the microplates were examined to determine bacterial growth. A red colour of INT reduced to the formazan indicated bacterial growth. The MIC was determined based on the lowest concentration at which a decrease in the red colour is apparent compared to the next dilution.

Determination of minimum bactericidal concentration (MBC)

A small sample from the wells with lowest concentration was transferred to fresh nutrient agar plates. The plates were incubated overnight at 37°C and examined for the growth of living organisms. The wells with lowest concentrations at which no growth took place after culture for 24 h of incubation were regarded as the minimum bactericidal concentrations.

Phytochemical screening

The    phytochemical    constituents   in   the   guava   extracts were screened using standards methods as described by Fong et al. (1977) and Ciulei (1982). This was used to determine the presence of saponins, reducing compounds, polyuronides, phenolic compounds, alkaloids, triterpenes, phytosterols, flavonoids and anthracenosides.

Antimicrobial susceptibility of carbapenem-resistant K. pneumoniae against ethanolic extract of P. guajava

The zones of inhibition of the various concentrations of the ethanolic extract of guava leaves are shown in Table 1.

Minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC)

The minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) shown in Table 2 were determined using the 96 well microplate.

Phytochemical constituents

The phytochemical constituents of the guava leaves extract were saponins, phenolic compounds, reducing sugars, polyuronoids and flavonoids as shown in Table 3. Previous work by Arima and Danno (2002) isolated the antimicrobial compounds in guava and elucidated their chemical structure.

The varied concentrations (12.5- 200 mg/ml) of the guava leaves extracts, which were used against the CRE strain employing the agar diffusion method, revealed significant antibacterial activity against the test organism as shown in Table 1. Furthermore, ethanolic extracts of guava leaves had MIC and MBC of 6.25 mg/ml against CREs.

This may be because guava leaves possess phytochemicals such as saponins, phenolic compounds, reducing sugars, polyuronoids and flavonoids. Flavonoids have antibacterial effect. According to Arima and Danno (2002), four flavonoid compounds: morin-3-O-alpha-L-lyxopyranoside, morin-3-O-alpha-L-arabopyranoside, guaijavarin and quercetin were isolated from guava leaves extract. It could be that the plant extract used in this study may contain these four flavonoids as shown in Table 1. It is plausible that other phytochemicals that were not determined in this study may also contribute to the antimicrobial efficacy of the guava leaves.

The activity of the positive controls (amikacin and colistin) used in this study correspond with the results of Hackman et al. (2017) although amikacin (30 µg/ml) used in this study shows a higher diameter zone of inhibition. According  to  Junior  and  Zanil  (2000), diameter zone of inhibition of 11-16 is considered active and >16 is considered very active. From the results, guava leaves extract concentrations of 50, 100 and 200 mg/ml indicated active diameter zone of inhibition. From Table 2, P. guajava had an MIC of 6.25 mg/ml, MBC of 6.25 mg/ml with the percentage inhibition index being 13.33. This showed that the appropriate concentration of the ethanolic extract of guava leaves is efficacious  against  the  growth of the carbapenem-resistant K. pneumoniae.

The hypothesis for this study proved positive since the plant under study showed activity against CREs. Ethanolic extracts of guava leaves have been proved to be efficacious against multi-drug resistant  K. pneumoniae,  which  is a major cause of systemic infections. Phytochemical screening of the leaves showed that it possesses flavonoids, which is responsible for the antibacterial efficacy of the guava leaves extracts. This offers hope for the development of effective antibiotics for the treatment of systemic infections caused by carbapenem-resistant Enterobacteriaceae. This requires the determination of the toxicological effect  of  the  structure  of  the active antibacterialcompounds isolated in the ethanolic leaves extracts of P. guajava.

The authors have not declared any conflict of interests.

The authors appreciate the logistical support by Centre for Research into Plant Medicine.