Antimicrobial Natural Products Produced by Soil-Derived Fungus Penicillium cremeogriseum W1-1

Abstract

Ten compounds (1–10) were obtained from soil-derived Penicillium cremeogriseum W1-1 with the antimicrobial guided isolation procedure. Among them, 4 presented broad-spectrum antimicrobial activities and its preliminary mechanisms were evaluated. Compound 4 showed growth inhibition on drug-resistant pathogenic strains Escherichia coli and Candida albicans with post-contact effect (PCE), changed the morphology and membrane structure, killed cells with leakage, inhibited the growth of C. albicans by eradicating biofilms. Interestingly, the fraction containing 4 presented in vivo anti-pathogenic activities in mice, indicating this indole diterpenoid alkaloid could been used as potential antimicrobial agent.

Introduction

Over 85 years, antibiotics have been playing important roles in combating diseases, such as microbial infections, cancers, hyperlipidemia, etc. Bacterial and fungal pathogens are a growing threat nowadays within the antimicrobial resistance crisis and new classes of antimicrobial pharmaceuticals are urgently needed. Therefore, many researchers tried to find novel chemicals from different bio-niches [1]. With great challenges in finding novel antimicrobial drugs, some advances have been achieved in past several years [1, 2]. The colleagues in our university have been working on the microbial strain library from different niches for more than 40 years. The strain collection supports us to develop researching on novel molecules. Herein, we report the antimicrobial compounds with broad-spectrum produced by a soil-derived fungus Penicillium cremeogriseum W1-1 and the preliminary mechanisms against target drug-resistant pathogens, Escherichia coli and Candida albicans.

The fungus W1-1 was isolated from a rhizospheric soil sample of Panax notoginseng collected in June, 2019, from a plantation in Wenshan, Yunnan Province, China. The isolation procedure was carried out according to the methods described by Miao et al. [3]. The strain was assigned as P. cremeogriseum on basis of the morphological characteristics and molecular phylogenic tree established by the ITS1/ITS4 gene sequence with accession number MW486650 in NCBI GeneBank. Conventional fermentation and extraction methods are used in this experiment (detail in SI). The dried crude extracts of mycelium and culture filtrate were then separated by silica gel column chromatography (CHCl₃-MeOH, gradient 80:1–10:1 (v/v)) into 23 fractions, respectively. The fractions were prepared at a concentration of 1.0 mg/ml using DMSO-H₂O, 1: 1 (v/v) and the active fractions were screened by filter paper antimicrobial method (detail in SI). The further isolation of active fractions were based on the TLC detection.

Ten compounds were obtained from the fractions with antimicrobial activities. Their structures (Fig. 1) were established on the detailed analysis of HR-ESI–MS, 1D and 2D NMR data and literatures. They were identified as 6-hydroxy-cyclopiamine B (1) [4], paspaline (2) [5], PC-M5' (3) [6], 4a-demethyl-paspaline-4a-carboxylic acid (4) [7], drechmerin A (5) [8], (3β,22E)-ergosta-5,7,22-trien-3-ol (6) [9], 5,8-epidioxy-(3β,5α,8α,22E)-ergosta-6,9,22-trien-3-ol (7) [10], (22E,24R)-ergosta-7,22-diene-3β,5α,6β-triol (8) [11], brefeldin A (9) [12] and 7'-O-acetylbrefeldin A (10) [13], including four indole diterpenoid alkaloids (2–5), three sterols (6–8) and two macrolides (9–10). Their antimicrobial activities were evaluated with minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) or minimum fungicidal concentration (MFC). The preliminary mechanisms of active compounds were investigated including post-contact effect (PCE), the morphological and membrane structure changes, killing cells with leakage by inhibiting efflux pump proteins activity, inhibiting the growth by eradicating biofilms.

Fig. 1.

Chemical structures of compounds

As presented in Table 1, the MIC values of compound 2, 4, 5 against E. coli and C. albicans were 12.5–25 μg/ml, respectively, better than the corresponding positive drugs, -- karamycin, ampicillin, fluconazole. Among them, only the antimicrobial activity of 5 was reported [8]. Compound 4 was firstly isolated from Penicillium camemberti OUCMDZ-1492 and its significant anti-viral activity against H1N1 activity was reported [7]. Little is known about its other bioactivity, such as antimicrobial activities. We carried out further antimicrobial assays of 4 against drug-resistant pathogens as it obtained from fractions as the fraction presented antimicrobial potential. And the action mechanisms were also observed in the investigation. Compound 2 was reported for its BK channel inhibition and tremorgenic effects in breast cancer cells [14].

Table 1.

MIC (μg/ml) against E. coli and C. albicans

Compounds	E.coli ML-35P (penicillin resistance)		C.albicans (08,030,401, resistance)
	MIC (μg/ml)	MBC (μg/ml)	MIC (μg/ml)	MFC (μg/ml)
1	200	400	50	100
2	12.5	50	50	200
3	50	100	50	200
4	25	50	50	100
5	12.5	50	50	100
9	100	200	100	400
Karamycin	25	50	100	400
Ampicillin	25	50
Fluconazole			50	100

Note: the MIC values of compounds > 200 μg/ml are not shown

Eight pathogenic strains including one fungus, two G⁻ bacteria and five G⁺ were employed in the antagonistic tests of 4. The MICs ranged from 25 μg/ml to 50 μg/ml against the targeted pathogens (Table 2), indicating 4 presented broad-spectrum antimicrobial ability.

Table 2.

MIC (μg/ml) of compound 4 against fungi and Gram-negative and Gram-positive bacteria

	Strains	Compound 4 MIC (μg/ml)	Ampicillin MIC (μg/ml)
	C. albicans (2,014,903)	50	100
G−	E. coli (ATCC 25,922)	25	25
	Pseudomona aeruginosa (ATCC 27,853)	50	12.5
G+	Staphylococcus aureus (SC005)	50	100
	S. epidermidis (CMCC 26,069)	25	25
	Bacillus cereus (CMCC 63,303)	25	25
	Enterococcus faecalis (SC009)	25	25
	Strepiococtus hemolyiicus (ATCC 19,615)	25	25

In the tests against E. coli and C. albicans, compound 4 showed concentration-dependent manner (Fig. 2a and b). The post-contact effect of 4 presented in the E. coli tests at high concentrations (> MIC). The growth of E. coli was inhibited after contact with 4 at 2MIC and 4MIC (Fig. 2c and d). The PCE results indicate that 4 could affect the growth of E. coli at high concentration with inhibition. The morphological changes of cells of E. coli and C. albicans were observed with scan electronical microscope (SEM) after treatment with 4 (Fig. 2e). The results showed that the outer surface of E. coli cells became markedly wrinkled, rough, even leakaged compared with the cells in control. The cells of C. albicans presented cyclic cleavage into two parts, indicating that 4 could kill pathogenic strains by destroying cells. The results of inhibition of the efflflux pump (Fig. 2f) indicated the possible usage of 4 as an adjuvant on new formulations in association with anti-biotics, mainly against bacterial infections. The changes of intracellular and extracellular structure will affect the structure of biofilm. Biofilm plays very important roles in antibiotic-resistance in treatment failures [15]. In our investigation, 4 could reduce both biofilm mass and viable cell counts after 24 h treatment (Fig. 2g and h). In the treatment, the biofilm architectures of E. coli and C. albicans showed morphological changes compared with the control under fluorescence microscopy. The biofilm of C. albicans could be destroyed and eradicated by 4 in 24 h at high concentration, while positive control fluconazole could not destroy biofilm. More efficient in disturbing the biofilm was observed in C. albicans than in E. coli. The results demonstrated that 4 also could disturb the fungal biofilm and kill persisting cells.

Fig. 2.

(a–b). Time-kill curves performed in TSB at 1 × , 2 × , 4 × MIC compound 4 against E. coli and C. albicans. Fig. 2c and d. Evaluation of PCE of 4 against E. coli and C. albicans. Fig. 2e. SEM images of E. coli and C. albicans. Fig. 2f. MIC of ethidium bromide or penicillin alone and in association with the standard inhibitors and compound 4 against the strain E. coli. Fig. 2g and h. Observation of biofilm by fiuorescence microscope and biofilm mass. Fig. 2i. Fraction B11 removed pathogenic bacteria efficiently in Kunming mice (Kunming mice peritoneal were infected with E. coli and treated with B11, Fluconazole and PBS at a dose of 4 mg/kg. Mice were harvested 3 days for bacteria colony counts. Data points represent each mouse, means SD from all mice. n = 6 mice per group.). All data were made using GraphPad Prism 5.0, then analyzed by t-test and P < 0.05 was considered statistically significant, after that results were expressed as mean ± SD

In order to evaluate bactericide efficiency of fraction B11 containing 4 against E. coli in vivo, mice were infected intraperitoneally with log-phase E. coli. Similar to in vitro findings, there was an significantly improvement in the reduction of microbacillary burden in the spleen, liver and kidney for B11 compared with Berberine (Fig. 2i, P < 0.05). The resulted suggested that B11 can be used to treat the deep infection of E. coli.

In summary, species in the genus of Penicillium are a reservoir of antimicrobial natural products. In this study, Penicillium cremeogriseum W1-1 produced some known compounds and a indole diterpenoid alkaloid (4) presented a broad-spectrum antimicrobial activity against tested pathogens. Compound 4 showed potential in destroying the pathogenic biofilm and inhibiting efflux pump proteins activity which causes many antibiotic treatment failures in clinical. More in-depth investigation should be carried out to evaluate this compound for drug development.

Supplementary Information

Below is the link to the electronic supplementary material.

Availability of data

The respiratory hospitalisation data were provided confidentially by Public Health Scotland (PHS), and others would need to apply to them to access the data.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12088-021-00957-z.