One-Step Combinatorial Strategy for Optimization of Antibiotics With Plant Extract Against Drug Resistant Clinical Bacteria

doi:10.21203/rs.3.rs-2102326/v1

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One-Step Combinatorial Strategy for Optimization of Antibiotics With Plant Extract Against Drug Resistant Clinical Bacteria

https://doi.org/10.21203/rs.3.rs-2102326/v1

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Antimicrobial resistance (AMR) is one of the most worrying health problems worldwide, antimicrobial research with reference to medicinal plants is a growing domain, likewise the synergism between plant extract and antibiotics.

The present study seek to propose and develop strategic synthetic means to overcome antibiotic resistance in bacteria using ethnopharmacological approach with conventional antibiotics. Concentrated sulphuric acid and 10% sodium hydroxide were used in combinatorial synthesis of Calotropis procera extract with Amoxicillin and Ampicillin.to validate the positive results obtain in stage one, Piliostigma reticulatum extract was combine with 100 µg/mL of azithromycin and again with 100 µg/ml of ampicillin with 0.1 mL, 0.2 mL and 0.3 mL the acid to validate and a different bacteria strain was also introduced.

Higher inhibitions zone were observed at 16.7 mm for salmonella spp, 16.4 mm shigella spp, 16.8 mm S. aureus, 21.3 mm E.coli and 22.4 mm streptococus at situations where antibiotics inhibitions zones was 0 mm.

The present study results uses a simple, less time consuming, cost-effective, safe and eco-friendly methods, and further shows that acid combinatorial synthesis of antibiotic with plant extract can be made to be effectively active against bacteria isolates which were initially resistant to the antibiotic used in the synthesis as compared to others done before, as they shows that green synthesis of nanoparticles and combination of plant extracts with antibiotics can only have synergistic effects on resistant bacteria or may enhance in overcoming bacteria resistance in a situation where the antibiotics have already shown some degree of antibacterial activity on the tested pathogens, these methods however has little possibility of overcoming resistance at a concentration were the antibiotic does not.

Medicinal Chemistry

Antibiotics

Bacteria

Calotropis procera

drug-resistance

Infections

Combination

Ethnopharmacology

One of the world health most worrying problem is Antimicrobial resistance (AMR), posing serious health threats to animals, humans, food security and development. International borders will be vulnerable to new forms of antibiotic resistance as it can spread remarkably fast due to international trades and travels. (WHO, 2021). Resistance to every available antibiotic is not a basic requirement for bacteria be fungi to be considered dangerous, serious problem can arise by resistance to just one antibiotic, resistance to antibiotic has caused more than 23,000 deaths a year in the United States alone, reports by Center of Disease control (CDC) United State classify group B Streptococcus, non typhoidal Salmonella, Methicillin resistant Staphylococcus aureus and drug resistant Shigella as bacteria of urgent threat( U.S. Centers for Disease Control and Prevention, 2019)

Discovery of new antibiotic is static, almost all clinical isolates of bacteria have slowly develop resistance mechanism, successful treatment of infection and diseases caused by persistent bacteria are hampered due to antimicrobial resistance. (Olga et al, 2020).

Mutation, Gene Transfer, Inappropriate Use and Inadequate Diagnostics are some of the major way resistance in bacteria happens faster. (Diseases, National Institute of Allergy and Infectious, 2011). Plants used in medicines are complex mixtures of active, and inactive substances, herbal medicines are still in use to treat various diseases in most developing country(Heinrich, 2010) few papers published showed the combination of plant extract with antibiotics displaying synergy to combat resistant bacteria better than fewer antibiotic(Eze et al, 2013) (Fadila and Tajelmolk, 2016) (Haq et al, 2019) plant extract were used to synthesize nanoparticle and were combined with antibiotic to fight resistant bacteria(Vazquez-Muñoz et al, 2019) there are inadequate papers with novel or different combinatorial strategy showing how to effectively improve antimicrobial activity higher than the antibiotic used for combination to overcome drug resistant bacteria at almost equal concentration to the antibiotic. Calotropis procera commonly known as milkweed (Barkha et al, 2021) have been used to cure various forms of common infections and diseases (Alafnan et al, 2021). Fresh juice oral administration is employed in treatment of Malaria and intermittent fever, Decoction for Gonorrhoea treatment. (Barkha et al, 2021).

Side-effect associated with antimicrobials and the development of bacteria resistance has shifted the focus of research toward Ethnopharmacology, this current work seeks to demonstrate a one-step combinatorial strategy which antibiotics may be optimize using aqueous plant extract making it highly effective against resistant clinical isolates of Streptococcus, Salmonella Spp, Staphylococcus aureus, Shigella Spp and E. coli.

2.1 Sampling

Amoxicillin 500 mg and Ampicillin 500 mg 10 capsules gsk Manufactured By: MEDREICH LIMITED, Virgonagar, Bangalore – 560049, INDIA, 500 mg Azithromycin VATROMAX oral capsules Vivax Pharmaceutical Co., LTD. CHINA were purchased in Laboratory and pharmaceutical vendors within Kaduna metropolis along Kano Road Kaduna North Kaduna State Nigeria, there originality were confirmed by sending each drug pin to 38353.

Calotropis procera and Piliostigma reticulatum specimens were collected in Sabon Tasha Kaduna. Calotropis procera voucher number V/N – ABU900086 of the specimen was collected at the Department of Botany Ahmadu Bello University Zaria, Kaduna, Nigeria.

Clinical Isolates of Streptococcus spp (High Vaginal Swab) Salmonella Typhi (Stool), Escherichia Coli (Urine), Shigella spp (Stool) and Staphylococcus Aureus (High Vaginal Swab) were collected at Chemical Pathology, Hematology and Microbiology diagnostic laboratory of Oxford Hospital Makera, Kakuri, RC KD0010980 Kaduna State Nigeria.

Fresh aerial part of Calotropis procera weighing 10 g containing both leaves and flowers were washed with distilled water, chopped with knife, squeezed with 50 mL of distilled water to extract its juice and filtered using a hand sieve. 100 µg/mL of Ampicillin and 100 µg of azithromycin were prepared dissolved with distilled water separately in a 500 mL volumetric flask, 8 test-tube were labelled Amx, AmxA, AmxM, AmxB for amoxicillin and Ap, ApA, ApM, ApB for ampicillin. Concentrated sulphuric acid was used for acidic combinations while 10% Sodium hydroxide (NaOH) were prepared and used for alkaline combination reactions.

The compositions of the 8 test-tubes were detail in order of addition below:

Amx, 2 mL of 1mg/mL amoxicillin

AmxA, 2 mL of 1mg/mL of amoxicillin + 2 mL plant extract + 0.4 mL of Acid

AmxM, 2 mL of 1mg/mL of amoxicillin + 2.4 mL plant extract

AmxB 2 mL of 1mg/mL of amoxicillin + 2 mL plant extract + 0.4 mL of Base

Ap, 2 mL of 1mg/mL of ampicillin + 2.4 mL distilled water

ApA, 2 mL of 1mg/mL of ampicillin + 2 mL plant extract + 0.4 mL of Acid

ApM, 2 mL of 1mg/mL of ampicillin + 2.4 mL plant extract

ApB 2 mL of 1mg/mL of ampicillin + 2 mL plant extract + 0.4 mL of Base

All 6 with the exception of Amx and Ap were heated at 100^oC for 20 minutes in a water bath.

2.2 Screening for the antibiotic resistance among the test bacteria and antimicrobial test of prepared samples

Streptococcus spp (High Vaginal Swab) Salmonella spp (Stool), Escherichia Coli (Urine), Shigella spp (Stool) and Staphylococcus Aureus (High Vaginal Swab) were isolated, characterized and identified. The patterns for antimicrobial susceptibility of confirmed clinical isolates of E. coli, Salmonella spp, Shigella spp and S. aureus strains for selected antibiotics were done by Kirby– Bauer disc diffusion test using Mueller-Hinton Agar (MHA) adopting the method used by (Fadila and Tajelmolk, 2016), purchased High profile positive /negative 10 tipped multiple susceptibility antibiotic discs were placed on the top layer of agar plates, incubated at 37ºC for 24 h, the isolates resistant to antimicrobials were considered as drug-resistant strains according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI).

The identified resistant bacteria strains were cultured and the zone of inhibition (ZOI) in millimeters (mm) for the 8 test-tube composition were recorded.

2.3 Validation of positive results

Washed, dried and pulverized 10 g of Piliostigma reticulatum (Boualam et al, 2021) were extracted by decoction using 100 mL distilled water and filtered using a Whattman filter paper. 100 µg/mL of azithromycin and 100 µg/mL Ampicillin were prepared, 8 test-tube were labelled A, A1, A2, A3 for azithromycin and B, B1, B2, B3 for ampicillin. Concentrated sulphuric acid was used to catalyse the reactions.

The compositions of the 8 test-tubes were detail in order of addition below:

A, 1 mL of 100 µg/mL azithromycin

A1, 2 mL of 100 µg/mL azithromycin + 2 mL plant extract + 0.1 mL of Acid

A2, 2 mL of 100 µg/mL azithromycin + 2 mL plant extract + 0.2 mL of Acid

A3 2 mL of 100 µg/mL azithromycin + 2 mL plant extract + 0.3 mL of Acid

B, 2 mL of 100 µg/mL of ampicillin + 2.mL distilled water

B1, 2 mL of 100 µg/mL of ampicillin + 2 mL plant extract + 0.1 mL of Acid

B2, 2 mL of 100 µg/mL of ampicillin + 2.4 mL plant extract + 0.2 mL of Acid

B3 2 mL of 100 µg/mL of ampicillin + 2 mL plant extract + 0.3 mL of Acid

All 6 with the exception of A1 and B1 were heated at 100^oC for 20 minutes in a water bath.

Purchased high profile positive and negative antibiotic disc containing Amoxicillin, Perfloxacin, Erythromycin, Septrin, Streptomycin, Ciprofloxacin, Rocephin, Zinnacef, Ampiclox, Gentamycin, Spafloxacin, Chloramphenicol, Augmentin and Tarivid each of different concentration were tested on the identified bacteria isolates by disc diffusion method(Fadila and Tajelmolk, 2016) These antibiotics were tested because they seem to be common in Nigerian markets.

Table 1

Susceptibility pattern of antibiotics against different microorganisms.
S/N	Bacteria	(µg)	Streptococcus spp	S.aureus	Salmonella Spp	Shigella Spp	E. coli
1	Amoxicillin	30	+	-	-	-	-
2	Perfloxacin	10	++	+++	-	++	+++
3	Erythromycin	10	++	NT	NT	NT	NT
4	Septrin	30	+++	+++	-	+	+
5	Streptomycin	30	+++	+++	-	+	++
6	Ciprofloxacin	10	+++	+++	-	+++	+++
7	Rocephin	25	-	+++	NT	NT	NT
8	Zinnacef	20	-	NT	NT	NT	NT
9	Ampiclox	20	-	NT	NT	NT	NT
10	Gentamycin	10	++	NT	+	++	+++
11	Spafloxacin	10	NT	++	-	++	++
12	Chloramphenicol	30	NT	++	-	-	+
13	Augmentin	30	NT	-	-	-	+
14	Tarivid	10	NT	-	-	+	+
# Diameter of inhibition zone: NT; Not tested, no inhibition (-); 5–15 mm (+); 16–25 mm (+ +); 26–35 mm (+ + +)

Streptococcus spp, were resistant to rocephine, zinnacef and ampiclox at 20 µg and showed little activity for amoxil. S. aureus showed resistance to 10 µg tarivid, 30 µg of amoxicillin and augmentin. Salmonella spp, were resistant to all tested antibiotic but gentamycin at 10 µg. Shigella spp showed resistance to 30 µg of amoxicillin, chloramphenicol and augmentin. Escherichia Coli was only resistant to amoxiciln at 30 µg.

3.1 Antibacterial activity of Amx, AmxA, AmxM, AmxB, Ap, ApA, ApM, and ApB

The antimicrobial susceptibility test in Table 1 shows a zone of inhibition (ZOI) of 0 mm for 1 g/mL of amoxicillin against all four of the bacterial strain, which were increased to a minimum value of 16.7 ± 1.2 mm for Salmonella spp and a maximum of 21.3 ± 1.2 mm for S. aureus due to the acid influence in the combination process.

Table 2

Zone of inhibition (mm) of resistant bacterial strains in response to Amx, AmxA, AmxM, AmxB, Ap, ApA, ApM, and ApB.
S/N	Bacteria	Ap	AmxA	ApA	AmxB	ApB	AmxM	ApM
1	Salmonella spp	5.6 ± 0.3	16.7 ± 1.2	17.4 ± 1	5.8 ± 0.6	0	5.5 ± 0.4	0
2	Shigella spp	5.8 ± 0.5	15.4 ± 0.9	16.6 ± 0.7	0	5.3–0.5	0	5.1 ± 0.3
3	S. aureus	0	16.2 ± 0.8	25.9 ± 1.2	0	0	5.0 ± 0.2	16.8 ± 0.8
4	E. coli	10.8 ± 1.2	21.3 ± 1.2	18.3 ± 0.8	0	0	0	13.4 ± 0.9

Ampicillin at 1 mg/mL shown a ZOI of 0 mm for S. aureus, 5.6 ± 0.3 mm for Salmonella, 5.8 ± 0.5 mm for Shigella and 10.8 ± 1.2 mm for S. aureus but, it acid enhanced combination showed a minimum increase of + 7.5 mm for S. aureus and + 25.9 mm for E. coli. All the acid combinatorial synthesis showed a synergistic effect against all the tested bacteria and successfully overcame resistance presented by the tested strains, this may be due to possible conjugation of some of the functional groups or substituents of either the antibiotic and the different phytochemicals present in the plant extract, Fig. 5 confirms a reaction in the mixture with a colour change from light green to brown. (Reusch, 2013)

No observable changes in ZOI of alkali combinatorial synthesis, this may be due to the conjugation or possible substitution of the active sites of either the antibiotic or the phytochemicals themselves, with a colour change from light green to a dark green cloudy solution.

The heat catalysed mixture of both antibiotics were able to overcome resistance with ZOI increase from 0 to 5.5 ± 0.4 mm for salmonella spp, 0 to 5.0 ± 0.2 E. coli in amoxicillin and a + 16.8 mm increase in S. aureus for ampicillin only, thus confirming the antimicrobial properties exhibited by the plant Calotropis procera extract (Barkha et al, 2021)

3.2 Validation of positive results

The validation of positive results obtained from the first combinatorial synthesis method as shown in Fig. 5 and Table 2 were carried out using a different plant extract of Piliostigma reticulatum (Boualam et al, 2021), with an additional antibiotic ( azithromycin) and an additional bacteria strain ( streptococcus spp )

The acid catalysed combinations were still able to overcome resistance in Streptococcus spp for azithromycin, in streptococcus spp and S. aureus also for ampicillin with a minimum ZOI of 11.3 mm and a maximum ZOI of 22.4 mm, an increased in ZOI from 11.3 mm to 22.4 mm was observed in streptococcus spp. An increase in ZOI with increase in acid volume were observed in Streptococcus spp as shown in Table 3.

Table 3

Zone of inhibition (mm) of 3 resistant bacterial strains in response to A, A1, A2, A3, B, B1, B2 and B3
S/N	Bacteria	A	A1	A2	A3	B1	B2	B3
1	Streptococcus spp	0	11.3 ± 1.5	15.5 ± 1.5	22.4 ± 1.2	13.5 ± 0.8	19.7 ± 1	21.2 ± .9
2	S. aureus	13.40 ± 0.9	21.4 ± 1.2	19.2 ± 0.7	21.1 ± 1.1	12.2 ± 0.9	17.2 ± 1.2	16.6 ± 0.8
3	Salmonella spp	0	0	0	0	0	0	0

No ZOI was observed in salmonella spp, this may be due to the nature of phytochemicals present in Piliostigma reticulatum or may be no fresh leaves were used. Piliostigma reticulatum is commonly used to treat fungal infection while Calotropis is used to treat typhoid fever as practiced in Nigeria. This further confirms the necessity of prerequisite knowledge of the plant ethnopharmacolgical usage and also that of the resistant pathogen before any combinatorial method.

A decrease in colour intensity and smell were observed with successive addition of acid from 0.1 to 0.3 ml as shown in Fig. 6, at 0.3 mL no smell were detected in the case of ampicillin, implying at 0.1 and 0.2 ml of acid added the antibiotic still retains some of the functional groups or substituents responsible for it characteristic smell, but on addition of another 0.1 ml of acid the smell disappeared, indicating that particular functional groups or substituents were definitely involved in conjugation or might be substituted.

Table 4

Comparison of acid enhanced combination with other methods for resistant bacteria inhibition
Bacteria strain	Antibiotic	Antibiotic MIC	Mixture MIC	Ref
P. chlororaphis P. monteilii	Cefotaxime Rifampicin	Nil Nil	12.5µg/ml 0.195 µg/ml	Haq et al (2019)
E. coli S. enteritidis	Ticarcilin 5 µg/ml	Nil Nil	16.00 mm 18.33 mm	Fadila and Tajelmol (2016)
E. coli S. aureus	Chloramphenicol30 µg Tetracycline 30 µg	Nil Nil	11.00 mm 12.00 mm	Eze et al (2013)

The former combinatorial method as shown in Table 4 is a trial based on probability approach where the combination may or may not inhibit bacterial growth with the effect being either additive, antagonistic or synergistic even with the right medicinal plants, but in acid catalyze methods at lower concentration with the right medicinal plants there is higher possibility that all combinations will be synergistic

In this work, antibiotic resistance were overcome using acid catalyze combinatorial synthesis of calotropis procera extract with three different antibiotics (amoxicillin, ampicillin and azithromycin), higher zones of inhibitions were observed at 16.7 mm salmonella spp, 16.4 mm shigella spp, 16.8 mm S. aureus, 21.3 mm E.coli and 22.4 mm streptococus in situations where antibiotics ZOI was 0 mm. The results of this present research further proposed a cost effective methods which antibiotics can be enhance to overcome resistance in bacteria as compared to others done before, which shows that green synthesis of nanoparticles and combination of plant extracts with antibiotics can only have synergistic effects on bacteria or may enhance in overcoming bacteria resistance in a situation where the antibiotics have already shown some degree of antibacterial activity on the tested pathogens, these methods however has little possibility of overcoming resistance at a concentration were the antibiotic does not. This research there by brings or suggest a novel pathway which antibiotic resistance in bacteria can be effectively minimize.

Acknowledgements

The authors would like to acknowledge the following support: Mr. Samuel Oxford hospital Kaduna state for his assistance in antimicrobial analysis, Professor Zakari Ladan Kaduna State University for being my supervisory guide, Mr. Emmanuel Duniya and Mamman Abakeyah James Kaduna State University my friends for assisting the synthetic process and little financial support.

Legal Ethics

Ethical permit was obtained from the ministry of health Kaduna State, Nigeria and were strictly adhere to from the sampling of the clinical isolates to antimicrobial test.

Conflicts Of Interest

No conflicts of interest is associated to this work.

Funding

This research work is self-funded.

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One-Step Combinatorial Strategy for Optimization of Antibiotics With Plant Extract Against Drug Resistant Clinical Bacteria

Status:

Journal Publication

Version 1

Abstract

Figures

1. Introduction

2.0 Methods

2.1 Sampling

2.2 Screening for the antibiotic resistance among the test bacteria and antimicrobial test of prepared samples

2.3 Validation of positive results

3.0 Result And Discussion

3.1 Antibacterial activity of Amx, AmxA, AmxM, AmxB, Ap, ApA, ApM, and ApB

3.2 Validation of positive results

4.0 Conclusions

Declarations

References

Status:

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