Detection of Carbapenemase Producing Enterobacteriaceae from Clinical Samples and their Susceptibility to Conventional Antibiotics and Medicinal Plant Extracts

Introduction

Carbapenemase enzymes are regarded as a serious threat affecting the global healthcare system. These enzymes are diverse and are in rapid circulation throughout the world [1]. Their health burden is coupled with the fact that the antibiotics which it renders inactive, are the ones regarded as the last resort treatment option against all serious bacterial infections caused by both Gram positive and Gram negative organisms [2]. To date, these enzymes are known to be produced by only Gram negative Enterobacteriaceae such as Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, and Acinetobacter baumannii among others [2, 3].

In Northern Nigeria, there are several reports including that of Yusuf T, et al. [4] that the rate of spread of these enzymes is moving at an alarming rate which if nothing is been done, there is the fear that it will continue to spread to far rural areas which have weak health care system. In Kano, Kaduna, Jigawa and other Northwestern states, different classes of Carbapenemase enzymes producing isolates have been reported to be in circulation. These enzymes are responsible for drug and surgical failures, prolong hospitalization, as well a high number of deaths [2].

For many years, the treatment of many infectious diseases using plant concoctions continue to yield promising outcomes. The activity recorded by these plants are mainly due to the reservoirs of bioactive constituents, and these constituents are reported to be in different proportion in different plants. However certain bioactive compounds were reported to be present only among given species of plants. The antibacterial activity of many medicinal plants and plant preparations has not been examined in Nigeria [5, 6]. Ficus ovata known as “Gamji dankwarai” in Hausa and Guiera senegalensis known as “Sabara” in Hausa, are known to be very active in managing different ailments such as stomach pain, wound and infertility in Kano and other part of Hausa land [7, 8, 9].

As newer classes and groups of carbapenemase enzymes continue to be discovered in Nigeria and Kano state in particular, and the high resistance profile coupled with fatal public health consequences exhibited by bacterial harboring Carbapenamases genes, it become very important to test their susceptibility to different classes of other antibiotics as well as Ficus ovata and Guiera senegalensis leaf extracts which were known to be effective in the treatment of various form of bacterial infections (Appendix 1 & 2).

Result and Discussion

Following the reconfirmation of the isolates using morphological appearance, motility, Gram reaction and biochemical test. All the isolates were observed to be Gram negative rod, motile, catalase positive with special ability to produce gas (Table 1). These characteristics as exhibited by all the test isolates (E. coli, Klebsiella pneumoniae and Pseudomonas aeruginosa) are in agreement with the report of Cheesbrough M, et al. [11, 13].

Keys: +:Positive, -:Negative Table 1: Microscopy and Biochemical test.

In all the two plants, the highest yield was observed in N-Butanol having 32.7% in Ficus ovata and 26.3% in Guiera senegalensis. The lowest yield was observed in N-Hexane extract having 5.6% for Ficus ovata and 6.7% in Guiera senegalensis (Table 2). This may be due to the variation in the Phytoconstituents present in each of the plant. More so, the place of sample collection as well as time of sample collection was reported to directly affect the presence and or level of Phytoconstituents presents in plant extracts [1, 14, 15]. As reported by Yusha’u M, et al. [16] the varying extracts colour may be due to the level of abundance of some given Phytoconstituents which gives a distinct colour to each extracts.

Keys: NH: N-Hexane, NB: N-butanol, ET: Ethanol. Table 2: Physical properties of Ficus ovata and Guiera senegalensis plants extracts.

The Modified Hodge Test was carried out for the Detection of carbapenemase production (Table 3 & Figure 2), where the highest occurrence of carbapenemase was observed in E. coli (14.3%), while the least was observed in K. pneumoniae and P. aeruginosa having 3(12%) and 2(6.7%) respectively. These variations in the occurrence of this enzyme may be because not all strains or species of these isolates are capable of producing Carbapenamases. More so, it may be because the test isolates are clinical isolates and not all patients harbored these categories of isolates that produce Carbapenamases [2]. The work of Yusuf T, et al. [4], which report the first cases of the circulation of Carbapenemase isolates in Kano also revealed that carbapenemase-producing isolates are in circulation in different parts of Kano, at varying levels and locations.

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The Antibacterial susceptibility test of the test isolates (E. coli, Klebsiella pneumoniae and Pseudomonas aeruginosa) revealed a decreased sensitivity of the test isolates to some commonly used antibiotics such as Cefotaxime (25 µg}, Cefixime {5µg}, Levofloxacin {5 µg}, Amoxicillin{30µg} and Cefuroxime{30µg} (Table 4). This shows that the resistant isolates exhibit resistance to other different classes of antibiotics which is a clear indication of their public health implications. These isolates have the capability in transferring resistance among the same and or different species making their existence a continuing threat [17, 18].

The result of the antibacterial activity of Ficus ovata and Guiera senegalensis extracts against some clinical isolate of Enterobacteriaceae (Table 5 and 6), shows that the activity of the extracts is quite dependent on the level of concentration. The activity decreases with a decrease in concentration from 200 mg to 100 mg and then 50 mg. This is in agreement with the work of Yaro MN, et al. [7] and Yusha’u M, et al. 16]. All the extracts of the two plants were observed to have the highest activity against P. aeruginosa, followed by E. coli while the least activity was recorded in K. pneumoniae isolate. Considering the very high resistance profile exhibited by Pseudomonas aeruginosa, as it remains one of the few isolates which have found to contain almost all the resistant genes that has been reported to exist among the diverse groups of Enterobacteriaceae [19, 20, 21]. The extracts activity can serve as a source of hope for the extraction of novel therapeutic agents that can be used to curtail the health problems posed by this bacterium.

Keys: FO: Ficus ovata, NB: N-Butanol, NH: N-Hexane, ET: Ethanol. Table 5: Antibacterial activity of Ficus ovata Extracts against some clinical isolate of Enterobacteriaceae.

Keys: GS: Guiera senegalensis, NB: N-Butanol, NH: N-Hexane, ET: Ethanol Table 6: Antibacterial activity of Guiera senegalensis Extracts against some clinical isolate of Enterobacteriaceae.

The activity recorded by Ficus ovata extracts as recorded in this study is in agreement with the findings of Oyeleke SB, et al. [5] and Kuetea V, et al. [9] who reported the presence of Alkaloids, flavonoids and tannins in the plants with inhibitory activity against E. coli, Shigella and Salmonella spp. The work of Onwuliri FC, et al. [22], Ogbeba J, et al. [6] conducted in Bauchi State and that of [8] conducted in Borno state, (North east) Nigeria report the aqueous and ethanol extracts of Guiera senegalensis to contain Tannins, flavonoids, saponins and alkaloids, with a significant inhibitory activity against both Gram negative (E. coli, Salmonella) and Gram positive (S. aureus) bacterial pathogens. Yaro MN, et al. [7] also report a significant activity by the extracts of Guiera senegalensis.

Conclusion

The highest occurrence of carbapenemase was observed in E. coli (14.3%), while the least was observed in K. pneumoniae and P. aeruginosa having 3(12%) and 2(6.7%) respectively. Of all the three plants extracts, highest yield was observed in N-Butanol extracts having 32.7% in Ficus ovata. The test isolates were observed to have a decreased sensitivity to some commonly used antibiotics. All the extracts of the two plants were observed to have the highest activity against P. aeruginosa, followed by E. coli while the least activity was recorded in K. pneumoniae isolate. The inhibitory activity exhibited by the extracts against the test isolates could justify their use locally in traditional medicine. The extracts have also demonstrated the potentials for the extraction of novel chemotherapeutic agents therefore future research should focus on identifying the individual components of each of the extracts with a view to identifying the specific active compounds. On the spread of CPE, the populace needs to be enlightened on the dangers of over- the-counter drugs, reckless use of antibiotics, and the need for adequate personal and community hygiene and dose completion therapy among others.

Recommendation

• The use of plants locally to treat infections should be backed by scientific evidences, else they can be toxic to humans. This also necessitate the need for toxicity studies on the extracts.
• Carbapenemase producing isolates can be spread easily as such extra care must be taken in ensuring an all-round environmental and personal hygiene as this has been noted as a means of dissemination of infectious agents with CPE inclusive.
• The co-selection of antibiotics, use of over the counter medication and over consumption of carbapenems antibiotics has been identified as the major causes and persistence of carbapenems resistance, therefore extra caution need to be applied when handling antibiotics.

• People with urinary tract infection should be fully diagnosed for confirmation before treatment using carbapenems antibiotics.
• Future research should focus on identifying the individual components of each of the extracts via Gas chromatography Mass spectroscopy (GC-MS), Thin layer chromatography and or phytochemical test with a view to identifying the specific active compounds.
• A larger sample size should be tested to cover a wider range of isolates, and appropriate bodies should take necessary measures to avoid the risk of occurrence of these enzymes of medical importance
• Funding This work received no funding.
• Conflict of Interest The authors declare no conflict of interest and have read and agree to the content of the manuscript.