Marjoram extract down-regulates the expression of Pasteurella multocida adhesion, colonization and toxin genes: A potential mechanism for its antimicrobial activity

doi:10.1016/j.cimid.2018.11.007

Comparative Immunology, Microbiology and Infectious Diseases

Volume 62, February 2019, Pages 101-108

https://doi.org/10.1016/j.cimid.2018.11.007 Get rights and content

Highlights

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This is novel study testing the antimicrobial and molecular anti-virulent mechanism of marjoram against P. multocida.
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Susceptibility of 16 P. multocida virulent isolates to herbal extracts was tested.
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The tested extracts showed efficient antimicrobial activity against P. multocida.
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The 5 screened extracts had synergistic antimicrobial activities with antibiotics.
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Marjoram extract down-regulated P. multocida putative virulence-associated genes.

Abstract

Due to the emergence of virulent and antibiotic-resistant microbes, natural antimicrobials from herbal origins have been given more attention as an alternative therapy. This study provides an in vitro research framework to investigate the antibacterial activities of 5 herbal (marjoram, garlic, onion, cinnamon and black seed) oil extracts against 16 multidrug-resistant (MDR) and virulent P. multocida serogroup A isolates recovered from dead and clinically diseased rabbits. Pathogenicity of the screened isolates was further proven experimentally and was verified by PCR analyses of 5 randomly selected virulence genes encoding attachment and colonization proteins (ptfA, pfhA, and omp87), sialidases (nanB) and dermonecrotoxin (toxA). A total of 12 P. multocida isolates were highly pathogenic with the possession of all examined virulence genes, while the other 4 isolates were of lower pathogenicity with expression of the target genes except toxA. In vitro anti-P. multocida activities of the 5 extracts and their synergism rates with 4 antibiotic drugs revealed that marjoram and cinnamon extracts had the highest antibacterial activities and the highest synergism rates against the screened isolates. Pasteurella multocida virulence gene expression profiles were assessed via real-time quantitative reverse transcription PCR (qRT-PCR) in response to marjoram extract. The quantitative analyses showed less than five-fold reduction in the targeted virulence genes expression in presence of marjoram extract compared with the control. The findings from this study document a novel molecular inhibitory activity of marjoram against P. multocida multiple virulence genes and provide a proof of concept for its implementation as an alternative candidate for the treatment of pasteurellosis in farm animals in future.

Introduction

Pasteurella multocida (P. multocida) is an important Gram-negative pathogen associated with a spectrum of animal diseases. Strains of P. multocida are designated into 5 capsular serogroups (A, B, D, E or F) and 16 lipopolysaccharides (LPS) somatic serotypes (1–16) [1]. Certain serotypes of P. multocida can cause pasteurellosis in rabbits (snuffles) resulting in considerable economic losses in rabbit production units [2]. The outcome of infections caused by P. multocida is influenced by the complex interactions of several hosts and pathogen-specific attributes [3]. The polysaccharide capsule and LPS are of major importance as virulence factors contributed in the pathogenesis of P. multocida in the host [4]. However, many other putative virulence determinants are related to pathogenicity including fimbriae, adherence and colonization factors, iron regulating and acquisition proteins, extracellular enzymes, exotoxins and a variety of outer membrane proteins [5].

Despite using the antimicrobial therapy as the most effective tool for controlling the infectious diseases caused by P. multocida, the used antibiotic agents are failing to bring an end to many infections due to the advent of MDR pathogens, which is recognized as an alarming threat to effective treatment and prevention of bacterial infections in humans and animals [6]. Increased resistance of P. multocida isolates to tetracyclines, erythromycin, trimethoprim/sulfamethoxazole, chloramphenicol, ciprofloxacin and cefotaxime antimicrobials was previously reported with the emergence of multidrug-resistant (MDR) strains [[7], [8], [9]]. In contrary, studies in France [10], Japan [11] and Hungry [12] demonstrated that cefotaxime, ciprofloxacin, tetracyclines and chloramphenicol were active drugs against P. multocida and recommended their use in pasteurellosis treatment. The undesirable effects and the high costs of antimicrobials administration still need to be considered in control of pasteurellosis. Thus, there is a dire need for developing alternative prophylactic strategies to combat the infections caused by these resistant strains. Currently, considerable researchers have evoked rekindled attentions towards the medicinal strength of some higher plants as a reasonable source for finding novel antimicrobial compounds [13], which have resulted in the development of alternative plant-based antimicrobial drugs with least side effects as compared to commercial antibiotics.

Indeed, few studies to date have focused on the antibacterial aspects of medicinal plants against P. multocida strains that target their cellular viability [14]. However, it is not yet known whether these anti-virulence potentials could be attributed to alterations of bacterial genes` expressions. This modulation of transcription can lead to subtle changes in the physiology of bacterial cell populations with consequences for their collective behaviors and thereby controlling pasteurellosis [15]. Considering the virulence and the MDR patterns within different strains of P. multocida [6], the informed selection of the virulence factors to be targeted for prevention of the struggle with infections caused by resistant P. multocida strains becomes vital. To the best of our knowledge, there is hardly any available report on the induced expression patterns of herbal oil extracts in P. multocida.

Over the last few decades, the use of natural compounds such as medicinal plants has gained attention due to increasing concerns about the safety of synthetic chemicals and emerging antibiotic resistance in bacteria. The current in vitro study is, therefore, the first to evaluate the antimicrobial activities of five common herbal oil extracts against MDR and virulent P. multocida isolates recovered from naturally infected rabbits suffering from respiratory diseases in Egypt giving a new safe approach for its treatment as a bio-control agent. In addition, the focus of this study was targeted to estimate the impact of the effective medicinal extract at the molecular level through the application of a wide scale genomic analysis using specific real-time quantitative reverse transcription PCR (real-time qRT-PCR) assay to investigate the expression levels of five critical virulence-associated genes putatively involved in modulating the pathogenesis of P. multocida.

Section snippets

Bacterial isolates

Sixteen field bacterial isolates recovered from 200 rabbits were used in this study. They were previously isolated from clinical samples consisting of nasal swabs (3) from live rabbits with a clinical evidence of snuffles and lung tissue samples (13) those were collected from dead rabbits at different localities in Sharkia Governorate, Egypt. These isolates were definitively identified on the basis of standard laboratory findings [16]. For reliable and specific detection, the identity of P.

Characteristics of P. multocida isolates

Standard conventional laboratory tests and PCR amplification of kmt1 gene were used for confirmation of the isolates as P. multocida. In capsular PCR assays, all the isolates amplified a product of approximately 1044 bp, which is a specific product for serogroup A.

Pasteurella multocida experimental infection

Pathogenicity tests were performed for the 16 P. multocida field isolates obtained from naturally infected rabbits. After being intranasally inoculated with P. multocida isolates, 12 rabbits died within 24 h postinfection with the

Discussion

The existence of antimicrobial resistance among P. multocida isolates considers a big problem in the veterinary medical field, specifically in rabbit industry. The implication of this problem can result in increasing the treatment cost, prolongation of illness due to treatment failure and it sometimes can lead to death. In this study, efforts were exerted to find an effective and safe antimicrobial(s) from natural sources such as herbal extracts with focusing on exploring their effects on the

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed a potential conflict of interest.

Acknowledgments

The authors would like to thank Dr. Hala I Mahmoud, Animal Wealth Development Department, Faculty of Veterinary Medicine, Zagazig University, Egypt for her help in the statistical analysis of the data.

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¹: Both authors contributed equally to this manuscript.

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Marjoram extract down-regulates the expression of Pasteurella multocida adhesion, colonization and toxin genes: A potential mechanism for its antimicrobial activity

Highlights

Abstract

Introduction

Section snippets

Bacterial isolates

Characteristics of P. multocida isolates

Pasteurella multocida experimental infection

Discussion

Conflict of interest statement

Acknowledgments

Vet. Microbiol.

Curr. Opin. Microbiol.

J. Comp. Pathol.

Methods.

Comp. Immunol. Microbiol. Infect. Dis.

Braz. J. Microbiol.

Microb. Pathog.

Microb. Pathog.

Microb. Pathog.

Vet. Clin. North Am. Food Anim. Pract.

Zentralbl. Bakteriol.

J. Food Prot.

Int. J. Food Microbiol.

Food Control

Metabolism

Vet. Microbiol.

Capsular serotyping of Pasteurella multocida from various animal hosts - a comparison of phenotypic and genotypic methods

Trop. Biomed.

Pasteurella: pathogenesis of bacterial infections in animals

Pathog. Bact. Infect. Anim.

Profiling of virulence associated genes of Pasteurella multocida isolated from cattle

Vet. Res. Commun.

In vitro and in vivo pathogenicity studies of Pasteurella multocida strains harbouring different ompA

Vet. Res. Commun.

Occurrence of virulence factors and antimicrobial resistance in Pasteurella multocida strains isolated from slaughter cattle in Iran

Front. Microbiol.

Virulence gene profiles of Pasteurella multocida strains isolated from cattle and buffalo

Vet. Arh.

Phenotypic characteristics of Pasteurella multocida isolated from commercial chickens affected by fowl cholera in Jos, Nigeria

Niger. J. World’s Poult. Res.

Isolation, Identification and Antibiogram of Pasteurella multocida Isolates of Rabbits sunffing from Pasterllosis

Int. J. Agro Vet. Med. Sci.

Antimicrobial susceptibility of Pasteurella multocida isolated from swine and poultry

Acta Vet. Hung.

Antimicrobial resistance in Pasteurella and Mannheimia: epidemiology and genetic basis

Vet. Res.