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Antimicrobial Detection Illuminated: Developing Bioluminescent Antibiotic Biosensors Based on Bacterial Gene Regulatory Elements

Research output: Book/ReportDoctoral thesisCollection of Articles

Details

Original languageEnglish
PublisherTampere University of Technology
Number of pages98
ISBN (Electronic)978-952-15-3330-3
ISBN (Print)978-952-15-2895-8
Publication statusPublished - 7 Sep 2012
Publication typeG5 Doctoral dissertation (article)

Publication series

NameTampere University of Technology. Publication
PublisherTampere University of Technology
Volume1066
ISSN (Print)1459-2045

Abstract

Ever since World War II, antibiotics have been medicine’s number one asset in fighting microbial infection, one of the leading causes of death worldwide. Misuse of antibiotics has, however, led to rapid spread of antibiotic resistance among bacteria and ensuing development of multiple resistant pathogens. Therefore, antibiotics are rapidly losing their antimicrobial value, which can be seen a failure of society to protect one of its valuable resources.

The use of antibiotics in food production animals is strictly controlled by the European Union. Veterinary use is regulated to prevent spreading of resistance due to unwarranted use and to prevent antibiotic residues in food products. EU legislation establishes maximum residue limits (MRLs) of veterinary medicinal products in foodstuffs of animal origin, and enforces countries to establish and execute a national monitoring plan of animal products to implement food control measures. Among samples selected for monitoring, suspect noncompliant samples are screened for and then subjected to confirmatory analysis to establish the identity and concentration of the contaminant. Screening methods for antibiotic residues are typically based on microbiological growth inhibition, whereas physico-chemical methods are used for confirmatory analysis.

In this study, antibiotic whole-cell biosensor assays were examined as a novel screening method. Utilizing a tetracycline-specific bioluminescent whole-cell biosensor, a screening method for tetracycline residues in poultry meat was developed. Assay sensitization to meet the EU MRLs was achieved by improving tetracycline accumulation into the biosensor cells with a combination of membrane-permeabilizing agent polymyxin B and chelating agent EDTA. The result was a rapid, simple and cost-effective high-throughput screening method that could detect all four veterinary relevant tetracyclines and their 4-epimer metabolites in poultry meat with sensitivity below the MRLs. The study also provided proof of antimicrobial activity of tetracycline 4-epimer metabolites, a quality previously thought absent from 4-epidoxycycline.

Nisin is a lantibiotic, a peptide antibiotic produced by lactococci. The industrial use of nisin as a food preservative (E234) and maximum allowed levels set by the EU warrant developing methods for nisin quantification in foods. In this study, a bioluminescent whole-cell biosensor for nisin was constructed and utilized in determining nisin concentrations in milk. The developed assay was rapid and simple to perform, and required no sample pretreatment except dilution. Sensitivity of the assay was in the sub-picogram per ml level, exceeding the performance of all previously published methods. The assay was also used in determining nisin-production efficiency by quantifying nisin in growth medium of a nisin-producing Lactococcus strain. Simultaneously, nisin producers could be distinguished from non-producers. This idea was expanded in a follow-up study, which utilized the nisin biosensor in screening for nisin producers in raw milk. Screening was based on simple overlay of raw milk cultures and identification of nisin producers by a bioluminescent zone surrounding the nisinogenic colony. The seven identified nisinogenic colonies were divided in three groups by genetic fingerprinting,and characterized as nisin variant Z producing Lactococcus lactis subsp. lactis. In addition, four nisin A producers were identified in a panel of 91 dairy lactococcal strains. Specificity studies showed that only nisin and not other bacteriocin peptides induced bioluminescence in the sensor strain. Also, all nisin-gene harboring colonies induced bioluminescence, with the exception of one lactococcal strain shown to carry a nonfunctional nisin gene.

The development of novel inducible whole-cell biosensors for different groups of antimicrobials can be limited by the lack of regulatory elements specifically responsive for these substances. In this study, we characterized DNA and ligand binding of the macrolide antibiotic-responsive repressor protein, MphR(E). The protein was modified by rational design of mutations to improve DNA affinity and dimerization. DNA and ligand binding as well as macrolide-induced dissociation from DNA were studied by fluorescence anisotropy and mass spectrometry. Mutants with improved DNA affinity and retained ligand binding and dissociation characteristics were identified. One mutant surprisingly formed a covalent dimer through disulfide bridge formation. This was shown to improve DNA affinity, but ligand binding and induction was impaired. Ligand binding spectrum of MphR(E) was shown to cover macrolides with a 14-membered lactone ring structure, but macrolides with a 16-membered ring or lincosamides showed no binding. MphR(E) and its mutants showed interesting novel characteristics that could benefit biosensor design.

In conclusion, this study shows the applicability of whole-cell biosensors in developing simple, robust and cost-effective screening methods for antimicrobials in food products. These methods show high sensitivity and specificity towards the target analyte, and can be used in semi-quantitative to quantative analysis. In addition to residue monitoring, whole-cell biosensors can be used for producer identification. The identified nisin producers can find use as protective starter cultures in fermented food production. The modified repressor MphR(E) shows promise as an improved regulator of reporter gene production in whole-cell biosensor applications, and is an example of purposeful effort to develop regulatory elements for novel biosensor designs.

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