Antagonistic Activity of Antimicrobial Producing Bacteria Against Multidrug-Resistant Bacteria Isolated From Hotspot Environments

Abstract

Background:

To overcome antibiotic resistance, there is an interest in improving or discovering novel classes of antibiotics from microorganisms that have different mechanisms of action worldwide. In Ethiopia, no significant studies have been conducted to isolate antibiotic-producing bacteria from hotspot environments and evaluate their antagonistic activities against multidrug-resistant bacteria. Therefore, the present study aimed to isolate antibacterial-producing bacteria from wastes and evaluate their antagonistic activities against multidrug-resistant bacteria in Bahir Dar town.

Methods:

A total of 49 bacteria were isolated from 30 waste and soil samples and tested against multidrug-resistant bacteria by primary screening using the single line streak method. Crude extracts of promising isolates were produced using submerged state fermentation methods and tested against multidrug-resistant bacteria using agar well diffusion methods. The minimum inhibitory and bactericidal concentrations of the crude extracts were determined using two-fold serial dilution and streak plate methods.

Results:

The results revealed that 11 (22.5%) bacterial isolates showed antibacterial activity against 1 or more tested multidrug-resistant bacteria. Isolates Ampb17 and Ampb33 isolated from soil and poultry waste showed the highest antagonistic activity against Salmonella spp., with a mean zone of inhibition of 20.3 ± 0.3 mm. The crude extract from isolate Ampb30, isolated from dairy waste, showed the highest antibacterial activity against Salmonella spp., with a mean zone of inhibition of 27.7 ± 0.3 mm. One-way analysis of variance confirmed that most of the crude extracts were statistically significant at a 95% confidence interval. The minimum inhibitory concentration of crude extracts was 0.13 mg/ml against S. aureus, E. coli, Salmonella spp., Pseudomonas spp., and Klebsiella spp., whereas the minimum bactericidal concentration of crude extracts was 0.25 mg/ml against S. aureus, E. coli, Salmonella spp., and Pseudomonas spp.

Conclusion:

The results of the present study revealed that antibiotic-producing bacteria from waste and soil have vast potential sources of antibacterial compounds.

Keywords

antagonistic activity multidrug-resistant bacteria antibiotic-producing bacteria hotspot environments isolation

Introduction

Antimicrobial drugs used for prophylactic or therapeutic purposes in humans, veterinary, and agricultural purposes favor the survival and spread of resistant microorganisms.¹ Some antibiotics, such as penicillin, erythromycin, and methicillin are effective against infectious diseases,² are now less effective because the bacteria are resistant to such antibiotics. Antibiotic resistance is a great challenge to modern medicine, which causes substantial morbidity and mortality, especially among elderly and immunocompromised patients, and requires the discovery of novel antibiotics.³

To overcome this situation, there is an interest in improving or discovering novel classes of antibiotics from microorganisms that have different mechanisms of action worldwide.⁴ Antibiotic-producing microorganisms are abundant in different habitats, where they produce antimicrobial metabolites with specific activities against coexisting microorganisms.⁵ They produce different secondary metabolites, known as antibiotics, to maintain their territory and defend them from other microorganisms. These microbial secondary metabolites are considered the most promising resources for novel antibiotics.^6,7

Some scholars have isolated antimicrobial-producing actinomycetes from different ecosystems. A study by⁸ reported that antimicrobial-producing actinomycetes were isolated from soil samples of the Mount Everest region, and they showed antibacterial activity against at least 1 of the tested bacteria among the 2 gram-positive and 9 gram-negative bacteria. Cwala et al. and Valli et al. also isolated antimicrobial-producing actinomycetes from aquatic and marine environments, respectively, and reported that all isolates were promising against at least 1 tested organism.^9,10 However, there is a need to explore indigenous actinomycetes for their potential to produce effective antibiotics from different sources in the country to combat infectious diseases caused by antimicrobial-resistant microorganisms.

In Ethiopia, some researchers have demonstrated the presence of antibiotic-producing actinomycetes in different ecosystems. A study conducted by Atsede and Fassil reported that antimicrobial-producing actinomycetes were isolated from the southern part of the Ethiopian Rift Valley alkaline lakes.¹¹ Bizuye et al. and Gebreyohannes et al. also isolated antimicrobial-producing actinomycetes from soils in Gondar town water and sediments of Lake Tana, Ethiopia, respectively.^12,13

However, in Ethiopia, no significant studies have been conducted to isolate antimicrobial-producing bacteria from different waste samples and evaluate their antagonistic activities against multidrug-resistant (MDR) bacteria. Therefore, the present study aimed to isolating antimicrobial-producing bacteria from different wastes and evaluate their antagonistic activities against MDR bacteria in Bahir Dar town, Northwestern Ethiopia.

Materials and Methods

Sample Collection and Preparation

A total of 30 waste samples were collected from poultry waste, municipal wastewater, medical wastewater, beef waste and wastewater, dairy waste and wastewater, and soil. The samples were transported to Bahir Dar University Postgraduate Microbiology Laboratory, Bahir Dar town, from October 2020 to June 2021, using sterile containers. One gram of a solid sample or 1 ml of a liquid sample was measured and transferred to 9 ml of sterile physiological saline and homogenized using a vortex mixer. Serial dilutions (10⁻¹,10⁻², 10⁻³, 10⁻⁴ . . . 10⁻⁹) were prepared by adding 1 ml of a homogenized sample to a sterile test tube containing 9 ml of sterile peptone water and mixing properly.

Isolation and Identification of Antibiotic Producing Bacteria

From the serial dilution of 10⁻³ and 10⁻⁵, 0.1 ml sample was spread plated on the Starch Casein Agar (SCA) plate (composition: soluble starch: 10 g, K₂HPO₄:2 g, KNO₃:2 g, casein (vitamin free: 0.3 g, MgSO₄.7H₂O: 0.05 g, CaCO₃: 0.02 g, FeSO₄.7H₂O: 0.01 g, agar: 18 g, and distilled water: 1000 ml and pH: 7.0 ± 0.1) for actinomycetes and nutrient agar (NA) for other bacteria in triplicate. SCA and NA plates were incubated for 7 days at 28°C and 24 h at 37°C, respectively. Colonies that appeared to have clear zones around them were carefully isolated into pure cultures.¹⁴ Repeated subculture was performed by streaking the SCA and NA to obtain a pure culture. Bacterial identification was performed by colony morphology, staining, microscopy, and biochemical tests¹⁵ and comparing the characterization of the isolates with Bergey’s Manual of Determinative Bacteriology.¹⁶

Primary Screening of Isolates for Antagonistic Activity

The isolated bacteria were subjected to antagonistic effects against MDR bacteria using the single-line streak method. In this method, isolates were inoculated in a single streak down the middle of a plate of MHA (Mueller-Hinton agar) medium (HiMedia) in triplicate. The plates were incubated at 28°C for 4 days and at 37°C for 24 h for actinomycetes and other bacteria, respectively. The MDR bacterial isolates were transferred into the fresh nutrient broth and incubated at 37°C for 24 h until visible turbidity was observed. The test organisms were streaked perpendicular to the isolate after adjusting the turbidity to 0.5 McFarland with a cell count of 1.5 × 10⁸ CFU/ml. A single streak of each test organism (MDR bacteria) was streaked perpendicular to the isolated antimicrobial-producing bacteria. The plates were further incubated at 37°C for 24 h, the zone of inhibition of the test organisms was measured using a caliper and the antimicrobial activity was recorded.¹⁵

Production of Crude Extract Using Submerged-State Fermentation

Based on the inhibitory zone size produced in primary screening, isolates with greater inhibitory capacity were selected for submerged fermentation to produce crude extracts that can be used in secondary screening.

In this method, 1000 ml of starch casein broth was prepared, in which 100 ml was dispensed into a 500 ml Erlenmeyer flask, sterilized, and cooled. At room temperature, the broth was inoculated with a 2 ml suspension of isolates and incubated for 14 days in a rotary shaker at 200 rpm. The culture broth was harvested by centrifugation at 4000 rpm for 15 minutes. The supernatant was collected, and equal volumes of ethyl acetate (1:1 v/v) were added and shaken vigorously for 1 h twice. Then, the ethyl acetate containing the active metabolite was separated from the solid residue using Whatman No. 1 filter paper. The solvent phase was separated from the aqueous phase using a separating funnel and subjected to a rotary vacuum evaporator at a water bath temperature of 60°C at 100 rpm to remove the solvent and to obtain crude extracts.¹⁷ The crude extracts obtained from each isolate were dissolved in 10% dimethyl sulfoxide (DMSO; 10 mg/ml) and used as stock concentrations for the determination of antimicrobial activity against tested MDR bacteria using DMSO as a negative control.

Secondary Screening Using the Agar Diffusion Method

A suspension of MDR bacteria isolates was compared with a standard 0.5 McFarland solution, which was equivalent to 10⁶–10⁸ CFU/ml. A cotton swab was used to make the carpet culture of the MDR bacterial suspension on the MHA plate in triplicate. The inoculated plates were allowed to diffuse at room temperature. A sterile 6 mm diameter cork borer was used to make the well on the agar, and 60 µl crude extract was carefully dispensed into each well. The plate was allowed to diffuse for 1 h and then incubated at 37°C for 24 h. After the proper incubation period, the zone of inhibition was measured with a caliper and recorded.¹⁸

Determination of MIC and MBC Values of Antibiotic-Producing Isolates

MIC values were determined using a two-fold serial dilution method. A series of 6 assay tubes for each organism were taken, and crude extract solutions (1 mg/ml) were serially diluted with nutrient broth as 1:2, 1:4, 1:8, 1:16, 1:32, and 1:64 to bring 0.5, 0.25, 0.125, 0.0625, and 0.03 mg/ml concentrations respectively, and 100 μl of a standard suspension of MDR bacteria were added to each concentration of the extract. Extra test tubes containing nutrient broth and nutrient broth with MDR bacteria were prepared to serve as negative and positive controls, respectively. The tubes were incubated at 37°C for 24 h, and the MIC value of antibiotics produced by the isolates was taken as the lowest concentration that showed no visible growth or turbidity in the test tube.¹⁸

All tubes that showed no visible signs of growth or turbidity were used to determine the MBC value of antibiotic-producing isolates. A loop full of inoculum was inoculated onto sterile nutrient agar plates using the streak plate method and incubated at 37°C for 24 h. The lowest concentration that showed no visible growth after incubation was considered the minimum bactericidal concentration of the tested antibiotic produced by the isolates against MDR bacteria.¹⁸

Data Analysis

The collected and recorded data were coded and analyzed using the SPSS Version 23 software. The mean inhibition zone of measurements, MIC, and MBC of antibacterial producing isolates against tested MDR bacteria were compared by one-way ANOVA followed by Duncan’s multiple comparison tests and presented in tables. A P-value <.05 was used to determine statistical significance.

Results

Sampling and Isolation of Antibiotic Producing Bacteria

From a total of 30 waste and soil samples, 49 different bacterial isolates belonged to Streptomyces spp, Bacillus spp, Pseudomonas spp, Micrococcus spp, and some unknown isolates were obtained using conventional methods. Of these 49 isolates, 8 (16.3%) were isolated from municipal wastewater, 10 (20.4%) from poultry waste, 7 (14.3%) from medical wastewater, 3 (6.1%) from beef waste and wastewater, 9 (18.4%) from dairy waste and wastewater, and 12 (24.5%) isolates were isolated from the soil.

Screening Isolated Bacteria for Their Antagonistic Activities

Primary Screening

The primary screening revealed that 11 (22.5%) bacterial isolates showed antagonistic activity against 1 or more tested MDR bacteria. Of these 11 isolates, 5 (Ampb5, Ampb29, Ampb31, Ampb32, and Ampb33), which accounted for 45.5%, were isolated from poultry waste, and 4 (Ampb8, Ampb17, Ampb18, and Ampb28), which accounted for 36.4% of the soil, and 2 (Ampb20 and Ampb30), which accounted for 18.2% of dairy waste and wastewater. As shown in Table 1, among the 11 isolates, all showed antagonistic activity against MDR Klebsiella spp., 10 (90.9%) against MDR S. aureus, Salmonella spp., and Citrobacter spp., 9 (81.8%) against E. coli, and 8 (72.7%) against Proteus spp. However, only 5 (45.5%) isolates showed antagonistic activity against MDR Pseudomonas spp. (Table 1).

Table 1.

Zone of Inhibition (mm) of the Antibiotic Producing Bacteria Isolates Against Multidrug-Resistant Bacterial Isolates Using Perpendicular Streak Method.

Isolates	Mean zone of inhibition (mm)
Isolates	S. aureus	E. coli	Salmonella spp.	Pseudomonas spp.	Klebsiella spp.	Citrobacter spp.	Proteus spp.	P value
Ampb5	0 ± 0.0	12.1 ± 0.4	13.3 ± 0.3	0 ± 0.0	15.3 ± 0.8	14.7 ± 0.3	9.3 ± 0.7	<.0001
Ampb8	11.7 ± 0.5	14.3 ± 0.4	0 ± 0.0	14 ± 1.3	16.7 ± 0.7	15.3 ± 0.3	13.3 ± 0.3	.002
Ampb17	13.2 ± 0.4	16.5 ± 0.3	20.3 ± 0.3	0 ± 0.0	11.7 ± 0.6	15 ± 0.6	10.7 ± 0.3	<.0001
Ampb18	13.7 ± 0.4	11 ± 0.3	18.3 ± 0.3	16.7 ± 0.3	18.3 ± 0.6	9 ± 0.6	11 ± 0.6	<.0001
Ampb20	11.8 ± 0.6	0 ± 0.0	17.3 ± 0.3	0 ± 0.0	16 ± 0.6	13 ± 0.6	0 ± 0.0	<.0001
Ampb28	14.7 ± 0.3	13 ± 0.4	9 ± 0.6	11.6 ± 0.3	17.3 ± 0.7	16.3 ± 0.3	12.3 ± 0.3	<.0001
Ampb29	14.7 ± 0.5	12.3 ± 0.3	16.3 ± 0.3	10.9 ± 0.5	14.3 ± 0.8	20.3 ± 0.3	18.7 ± 0.3	<.0001
Ampb30	11.3 ± 0.7	0 ± 0.0	15.3 ± 0.7	16.3 ± 0	14.3 ± 0.3	0 ± 0.0	12.7 ± 0.3	<.0001
Ampb31	14 ± 0.5	15 ± 0.2	12.7 ± 0.3	0 ± 0.0	16.7 ± 0.6	16.7 ± 0.7	11 ± 0.6	<.0001
Ampb32	12.2 ± 0.5	17.3 ± 0	18.3 ± 0.3	0 ± 0.0	19.3 ± 0.6	13.7 ± 0.3	0 ± 0.0	<.0001
Ampb33	13.8 ± 0.2	19 ± 0	20.3 ± 0.3	0 ± 0.0	17.3 ± 0.7	14.7 ± 0.3	0 ± 0.0	<.0001
Gen	23 ± 0.0	6.8 ± 0.0	9 ± 0.0	0 ± 0.0	0 ± 0.0	21 ± 0.0	8 ± 0.0	<.0001
P value	.001	<.0001	<.0001	<.0001	.001	.001	<.0001

Values are expressed as the mean of 3 replicates ± S.E.M (Kruskal-Wallis, P < .05).

As shown in Table 1, the most promising isolates against S. aureus were the Ampb28 and Ampb29 isolates, with a zone of inhibition of 14.7 ± 0.5 mm. Isolate Ampb33 (19 ± 0.0) mm against E. coli, isolate Ampb17 and Ampb33 (20.3 ± 0.3) mm against Salmonella spp., and isolate Ampb30 (16.3 ± 0.0) mm against Pseudomonas spp. also showed the highest antagonistic activity when compared to other isolates. Moreover, isolate Ampb32 (19.3 ± 0.6 mm), Ampb29 (20.3 ± 0.3 mm), and Ampb29 (18.7 ± 0.3 mm) showed the highest antagonistic activity against Klebsiella spp., Citrobacter spp., and Proteus spp. respectively.

However, isolate Ampb5 against S. aureus, isolate Ampb20 and Ampb30 against E. coli, Ampb8 against Salmonella spp., and Ampb5, Ampb17, Ampb20, Ampb31, Ampb32, and Ampb33 against Pseudomonas spp. did not show any antibacterial activity. Isolate Ampb30 against Citrobacter spp. and isolates Ampb20, Ampb32, and Ampb33 against Proteus spp. also did not show any antimicrobial activity (Table 1). The present results showed that there was a highly significant difference (P < .05) in the antagonistic activity of the isolates against each tested MDR bacterial isolate (Table 1).

Secondary Screening

The crude extracts obtained from the submerged state fermentation results revealed that, except for isolate Ampb5, all crude extracts showed antibacterial activity against MDR S. aureus isolates. The crude extract from isolate Ampb29 showed the highest antibacterial activity against S. aureus with a mean zone of inhibition of 23.6 ± 0.1 mm. The maximum inhibition zone of 23.8 ± 0.1 mm was observed for Ampb32 against E. coli; 27.7 ± 0.3 and 20.3 ± 0.3 mm from Ampb30 against Salmonella spp. and Pseudomonas spp., respectively. The crude extract of isolate Ampb33 showed the highest activity against Klebsiella spp. with a mean zone of inhibition of 21 ± 0.5 mm. Moreover, isolate Ampb29 showed the highest activity against Citrobacter spp. and Proteus spp. with a mean zone of inhibition of 17.7 ± 0.3 and 22.7 ± 0.3 mm respectively.

On the other hand, crude extracts from isolates Ampb30 against E. coli and crude extracts from isolates Ampb5, Ampb31, Ampb32, and Ampb33 against Pseudomonas spp. did not show any antimicrobial activity. Isolates Ampb20, Ampb32, and Ampb33 also did not show any antimicrobial activity against Proteus spp. The results showed that there was a highly significant difference (P < .05) among the antagonistic activities of the isolates against each of the tested MDR bacterial isolates (Table 2).

Table 2.

Antibacterial Activity of Crude Extracts of Antimicrobial Producing Bacteria Against MDR Bacterial Isolates from Submerged Fermentation Using Agar Diffusion Method.

Isolates extracts	Mean zone of inhibition (mm)
Isolates extracts	S. aureus	E. coli	Salmonella spp.	Pseudomonas spp.	Klebsiella spp.	Citrobacter spp.	Proteus spp.	P value
Ampb5	0 ± 0.0	15.7 ± 0.2	23 ± 0.6	0 ± 0.0	18.3 ± 0.3	12.3 ± 0.8	14.7 ± 0.3	<.0001
Ampb8	18.8 ± 0.8	17.8 ± 0.1	10.3 ± 0.3	17.6 ± 0.1	19 ± 0.0	12.7 ± 0.6	20.3 ± 0.3	.003
Ampb17	21.4 ± 0.2	18.4 ± 0.1	15 ± 0.0	10 ± 0.1	13.3 ± 0.3	11 ± 0.5	12.3 ± 0.6	<.0001
Ampb18	20.8 ± 0.8	17.8 ± 0.4	14 ± 0.6	14.9 ± 0.1	18 ± 0.6	13.3 ± 0.3	12.7 ± 0.6	.001
Ampb20	17.8 ± 0.9	15.1 ± 0.1	15.3 ± 0.3	8.4 ± 0.2	15.3 ± 0.3	16.3 ± 0.8	0 ± 0.0	<.0001
Ampb28	20.4 ± 0.1	16.7 ± 0.1	10 ± 0.5	13.3 ± 0.4	20 ± 0.5	15.7 ± 0.3	15 ± 0.5	<.0001
Ampb29	23.6 ± 0.1	16.8 ± 0.2	18.3 ± 0.6	19.6 ± 0.2	17 ± 0.6	17.7 ± 0.3	22.7 ± 0.3	.001
Ampb30	20.3 ± 0.3	0 ± 0.0	27.7 ± 0.3	22.9 ± 0.3	20.3 ± 0.8	13.7 ± 0.3	15.3 ± 0.3	<.0001
Ampb31	16.8 ± 0.8	20.4 ± 0.2	22.3 ± 0.3	0 ± 0.0	17.7 ± 0.3	13 ± 0.6	13 ± 0.0	<.0001
Ampb32	22.9 ± 0.2	23.8 ± 0.1	19.3 ± 0.3	0 ± 0.0	14.3 ± 0.3	15 ± 0.0	0 ± 0.0	<.0001
Ampb33	22.5 ± 0.3	22.9 ± 0.1	20.3 ± 0.3	0 ± 0.0	21 ± 0.5	15.7 ± 0.3	0 ± 0.0	<.0001
Gen	23 ± 0.0	6.8 ± 0.0	9 ± 0.0	0 ± 0.0	0 ± 0.0	21 ± 0.0	8 ± 0.0	<.0001
P value	.001	<.0001	<.0001	<.0001	.001	.001	<.0001

Values are expressed as the mean of 3 replicates ± S.E.M (Kruskal-Wallis, P < .05).

MIC and MBC of Antimicrobial Producing Isolates

The present study showed that the lowest MIC value (0.13 mg/ml) was obtained from isolate Ampb29 extract against S. aureus, Ampb32 extract against E. coli, Ampb30 and Ampb32 against Salmonella spp., Ampb30 against Pseudomonas spp., and Ampb33 against Klebsiella spp. In addition, the lowest MIC value (0.25 mg/ml) was obtained from isolate Ampb29 against Citrobacter spp. and Proteus spp. The highest MIC value (>1 mg/ml) was obtained from the different isolates against the tested bacteria. The lowest MBC value (0.25 mg/ml) was obtained from Ampb29 extract against S. aureus, Ampb32 extract against E. coli, Ampb30 and Ampb5 against Salmonella spp., and Ampb30 against Pseudomonas spp. In addition, the lowest MBC value (0.5 mg/ml) was obtained from isolates Ampb33 against Klebsiella spp., Ampb29 against Citrobacter spp., and Proteus spp. (Table 3).

Table 3.

Minimum Inhibitory Concentration and Minimum Bactericidal Concentration of Crude Extracts.

Isolates	Tested bacteria (mg/ml)
	S. aureus		E. coli		Salmonella spp.		Pseudomonas spp.		Klebsiella spp.		Citrobacter spp.		Proteus spp.
	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC
Ampb5	>1	ND	1	>1	0.25	0.25	>1	ND	0.5	>1	>1	ND	>1	ND
Ampb8	0.5	1	0.5	0.5	>1	ND	0.25	1	0.5	1	>1	ND	0.5	1
Ampb17	0.25	0.5	0.25	0.5	1	>1	>1	ND	>1	ND	>1	ND	>1	ND
Ampb18	0.5	0.5	0.5	1	1	>1	1	ND	0.5	>1	>1	ND	>1	ND
Ampb20	0.25	0.5	1	>1	1	>1	>1	ND	>1	ND	0.5	>1	>1	ND
Ampb28	0.25	0.5	0.5	0.5	>1	ND	>1	ND	0.25	1	0.5	>1	1	>1
Ampb29	0.13	0.25	0.5	0.5	>1	ND	0.25	1	1	>1	0.25	0.5	0.25	0.5
Ampb30	0.25	0.5	>1	ND	0.13	0.25	0.13	0.25	0.25	1	>1	ND	1	>1
Ampb31	0.5	1	0.5	1	0.25	0.5	>1	ND	0.5	1	ND	ND	ND	ND
Ampb32	0.25	0.5	0.13	0.25	0.13	0.5	>1	ND	ND	ND	1	>1	ND	ND
Ampb33	0.25	0.5	0.25	1	0.25	0.5	>1	ND	0.13	0.5	1	>1	ND	ND

Discussion

Currently, the incidence of multidrug-resistant bacteria is increasing, affecting the treatment of several infectious diseases. Therefore, there is an urgent need to develop new drugs that are effective against antibiotic-resistant pathogens. Antimicrobial-producing microorganisms have been proven to be a potential source of bioactive compounds and are the richest source of secondary metabolites.¹⁹

The primary screening revealed that 11 (22.5%) of the bacterial isolates tested showed antimicrobial activity against one or more MDR bacteria. This result (22.5%) is inconsistent with the results reported by Al-Munawwarah et al.²⁰ and greater than the results reported by,²¹ who reported that 21.88% and 12.82% of the isolates showed antimicrobial activity against one or more tested bacteria, respectively. However, the result was less than that in previous reports, which were 26.7%,¹² 50%,²² and 59.09%, respectively.²³

Of these 11 isolates, 5 (45.5%) were isolated from poultry waste, 4 (36.4%) from soil, and 2 (18.2%) from dairy waste. Bizuye et al¹² reported that out of 8 isolates, 5 (62.5%) were isolated from industrial waste disposal areas, 2 that accounted for 25% of cattle breeding areas, and 1 that accounted for 12.5% from near the kitchen house.

Among the 11 isolates, all (100%) were shown to have antagonistic activity against MDR Klebsiella spp., 10 (62.5%) against MDR S. aureus, Salmonella spp., and Citrobacter spp., 9 (81.8%) against E. coli, and 8 (72.7%) against Proteus spp. Bizuye et al.¹² reported that of the 8 isolates, 5 (62.5%) were shown to have antagonistic activity against S. aureus ATCC29213, E. coli ATCC25922, S. typhi ATCC9289, and K. pneumonia ATCC7000603. However, Bizuye et al.¹² and Sapkota et al.²⁴ reported that none of the isolates showed activity against Pseudomonas aeruginosa (ATCC 27853). Our results indicated that the 5 isolates showed antagonistic activity against MDR Pseudomonas spp. Environmental isolates exhibit varying degrees of inhibition against multidrug resistant (MDR) bacteria due to differences in their ecological, genetic, and physiological adaptations. Isolates that are highly effective often come from competitive, nutrient-limited, or polluted environments, which drive the production of potent antimicrobials, specialized metabolites, and advanced competitive mechanisms.

The current study showed that the most promising isolates against S. aureus were the Ampb28 and Ampb29 isolates (14.7 ± 0.5) mm when compared to other isolates. However, the isolate Ampb33 (19 ± 0.0) mm showed the highest antagonistic activity against E. coli when compared to others. Likewise, isolates Ampb17 and Ampb33 showed the highest antagonistic activity against Salmonella spp., while Ampb18 and Ampb30 also showed the highest antagonistic activity against Pseudomonas spp. Furthermore, isolate Ampb32 (19.3 ± 0.6 mm), Ampb29 (20.3 ± 0.3 mm), and Ampb29 (18.7 ± 0.3 mm) showed the highest antagonistic activity against Klebsiella spp., Citrobacter spp., and Proteus spp., respectively. There was a highly significant difference (P< .05) in the antagonistic activity of the isolates against each tested MDR bacterial isolate. Bizuye et al¹² reported that the most promising isolate against S. aureus was the Ab43 isolate (25 ± 1 mm), and isolate Ab43 (30 ±2 mm) showed the highest antagonistic activity against E. coli. The authors also reported that isolate Ab43 (32 ± 2 mm) showed the highest antagonistic activity against S. Typhi, and isolates Ab18 (20 ± 2 mm) and Ab43 (20 ± 1 mm) showed the highest antagonistic activity against K. pneumoniae.

The current study also indicated that isolate Ampb5 against S. aureus, isolates Ampb20 and Ampb30 against E. coli, and isolate Ampb8 against Salmonella spp. did not show any antimicrobial activity. Moreover, 5 isolates (Ampb5, Ampb17, Ampb20, Ampb31, Ampb32, and Ampb33) against Pseudomonas spp., Ampb30 to Citrobacter spp., and 3 isolates (Ampb20, Ampb32, and Ampb33) against Proteus spp. did not show any antimicrobial activity. Bizuye et al¹² reported that S. aureus showed resistance against Ab24, Ab41, and Ab44 isolates, and E. coli showed resistance against 3 isolates (Ab13, Ab24, and Ab41). They also reported that S. typhi showed high resistance to antibiotics in 3 isolates (Ab13, Ab24, and Ab44), and K. pneumonia showed resistance to Ab5, Ab41, and Ab44 isolates.

The crude extracts obtained from submerged state fermentation results revealed that, except for isolate Ampb5, all crude extracts of promising isolates showed antibacterial activity against MDR S. aureus isolates. Crude extracts from isolates Ampb29, Ampb32, and Ampb33 have shown the highest antimicrobial activity against S. aureus with a mean zone of inhibition of 23.6 ± 0.1, 22.9 ± 0.2, and 22.5 ± 0.3 mm respectively. The maximum inhibition zones of 23.8 ± 0.1 and 22.9 ± 0.1 mm were observed for Ampb32 and Ampb33 against E. coli. Crude extracts from isolates Ampb30, Ampb5, and Ampb31 have shown higher activity against Salmonella spp. with a mean zone of inhibition of 27.7 ± 0.3, 23 ± 0.6, and 22.3 ± 0.3 mm respectively. Pseudomonas spp. were more sensitive to the crude extract of isolates Ampb30 and Ampb29 with a mean zone of inhibition of 20.3 ± 0.3 and 19.3 ± 0.3 mm, respectively. The crude extracts of isolates Ampb33, Ampb30, and Ampb28 showed higher activity against Klebsiella spp. with a mean zone of inhibition of 21 ± 0.5, 20.3 ± 0.8, and 20 ± 0.5 mm, respectively. With a mean zone of inhibition of 17.7 ± 0.3 and 22.7 ± 0.3 mm, respectively, the crude extract of isolate Ampb29 demonstrated higher activity against Citrobacter spp. and Proteus spp.

The results obtained from this study indicated that crude extracts tested against individual MDR bacterial isolates showed a statistically significant difference (P < .05). This difference might be due to the ability of the antimicrobial producer to produce potential secondary metabolites associated with the breakdown of the crude extracts during the extraction process and the sensitivity of the tested bacterial isolates. Ilic et al.¹⁷ reported that the bioactivity of the isolates was dissimilar between gram-positive and gram-negative bacterial strains, and gram-positive bacteria were more susceptible to the tested crude extracts than gram-negative bacteria.

The results of a previous study revealed that the highest zone of inhibition of 25 mm was recorded from LT005 against S. aureus, 14 mm from LT008 against S. Typhi,18 mm from LT003 against P. aeruginosa, and 17 and 13 mm from LT005 against E. coli and K. pneumonia, respectively.¹³ Another study also indicated that the highest zone of inhibition (34 ± 1 mm) was recorded in the crude extract of isolate Ab43 against S. aureus. In addition, 35 ± 1, 25 ± 2, and 14 ± 1 mm were recorded from the crude extracts of Ab18 against E. coli, S. Typhi, and K. pneumonia, respectively.¹²

On the other hand, crude extracts of Ampb30 against E. coli isolates (Ampb5, Ampb31, Ampb32, and Ampb33) against Pseudomonas spp., and isolates (Ampb20, Ampb32, and Ampb33) against Proteus spp. did not show any antimicrobial activity. Crude extracts from isolate Ab28 did not show any antimicrobial activity against S. aureus, E. coli, S. Typhi, and K. pneumoniae.¹²

The present study showed that the lowest MIC value (0.13 mg/ml) was obtained from the crude extract of isolate Ampb29 against S. aureus, Ampb32 against E. coli, Ampb30, and Ampb32 against Salmonella spp., Ampb30 against Pseudomonas spp., and Ampb33 against Klebsiella spp. In addition, the lowest MIC value (0.25 mg/ml) was obtained from extracts of isolate Ampb29 against Citrobacter spp. and Proteus spp. The highest MIC value was >1 mg/ml and was obtained from different isolate extracts against all tested bacteria. The MIC of the crude extracts ranged from 1.46 to 2.52 mg/ml against Gram-positive bacteria (S. aureus) and from 1.84 to 2.82 mg/ml against Gram-negative bacteria (E. coli).¹³

The lowest MBC value (0.25 mg/ml) was obtained from Ampb29 crude extract against S. aureus, Ampb32 extract against E. coli, Ampb30 and Ampb5 extracts against Salmonella spp., and Ampb30 extract against Pseudomonas spp. In addition, the lowest MBC value (0.5 mg/ml) was obtained from crude extract of isolate Ampb33 against Klebsiella spp., Ampb29 against Citrobacter spp., and Proteus spp. A study conducted by Gebreyohannes et al¹³ reported that among the 11 crude extracts tested, the MBC ranged from 2.92 to 7.56 mg/ml against S. aureus and E. coli. The authors also reported that MBC ranged from 3.80 to 8.46 mg/ml and the MIC and MBC varied among the tested isolates against S. aureus and E. coli. There was a difference among the zones of inhibition, MIC, and MBC results obtained from this study, which differed from previous reports.^12,13 This might be due to the difference in the tested bacteria, type, and concentration of antimicrobial-producing bacteria and the methods used to test the antibacterial activity.

Conclusion

Antibiotic resistance, which causes substantial morbidity and mortality, is a significant challenge in modern medicine. To overcome this problem, there is an urgent need for the development of novel, safe, and effective antibiotics. The study findings revealed that wastes of dairy and poultry have the potential to isolate bacteria exhibiting antibacterial activity against tested multidrug-resistant bacterial isolates. These results suggested that microbial isolates from these hotspot environments can be used commercially to produce antibiotics after purification and proper standardization. Future work will focus on the molecular identification of isolates, the isolation and characterization of bioactive compounds, and understanding the mechanism of action of bioactive compounds isolated from these bacteria.

Footnotes

Acknowledgements

We would like to thank Habtamu M., Lamenew F., Dr. Baye S., and Misganaw L. for their support during sample collection and laboratory work and Mr. Teshager Zerihun for his support in data analysis. The authors also thank the Department of Biology, BDU, for providing the facilities for conducting this research

Abbreviations

ATCC American type culture collection

CFU colony-forming unit

MBC minimum bactericidal concentration

MDR multidrug-resistant

MHA Mueller-Hinton agar

MIC minimum inhibitory concentration

NA nutrient agar

rpm revolutions per minute

SCA starch casein agar

Ampb antimicrobial producing bacteria

ORCID iD

Kindu Geta

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Bahir Dar and Debre Tabor Universities support financial and material to conduct this research.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data Availability Statement

Please contact the corresponding author for data requests.*

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