Short Description

Worldwide, multidrug-resistant germs are on the rise and provoke the intensive search for novel effective compounds. To simplify the search for new antibiotics and to track the antibiotic pollution in water samples, whole-cell biosensors constitute a helpful investigative tool. In this subproject, we developed a functional and complete heterologous Beta-lactam biosensor in Bacillus subtilis. By the time these specified cells sense a compound of the beta-lactam family, they will respond by producing a measurable luminescence signal. Thereby, we analyzed the detection range and sensitivity of the biosensor in response to six different Beta-lactam antibiotics from various subclasses. The evaluated Biosensor was then encapsulated into Peptidosomes to proof the concept of our project EncaBcillus. The trapping of engineered bacteria thus will allow for increased control and simplified handling, potentially raising the chances for their application in e.g. sewage treatment plants.

Background

Antibiotics represent the most effective treatment against bacterial infections. Since the discovery of penicillin by Alexander Fleming in 1928, many new antibiotics have been constantly developed and were successfully applied to treat life-threatening diseases. This significant advancement in medicine saved millions of lives and still does today. However, fighting microorganisms has never been a completed task, but rather an ongoing race between drug discovery and pathogens developing resistances. Thus, multi-drug resistant bacteria still constitute a major threat for humanity, as infectious diseases represent the second leading cause of death worldwide. [1][2]

One major reason, for the steady increase of antimicrobial resistances is the “inappropriate use of antimicrobials”. Due to excessive prescription and application in livestock farming, little amounts of antibiotics are nearly found everywhere, even in drinking water. These low-dose and non-lethal concentrations containing habitats, allow bacteria to adjust and develop resistances.[2][3]

As Beta-lactams make up a large percentage of all antibiotics used, the project preferentially focused on this class of broad-spectrum antibiotics. Carbapenems, penicillin derivatives, cephalosporins and monobactams represent the four main classes of the beta-lactams that sum up to over 100 different active substances. All compounds of this particular group can be easily identified by their common chemical structure: the beta-lactam ring (see Figure1).[4]

To address the increasing development of multi-drug resistant bacteria our iGEM Team aims at developing a novel beta-lactam biosensor in Bacillus subtilis based on the genetics of the bla-operon found in Staphylococcus aureus (for a detailed description consider our Design section below). The genetically engineered Biosensor will help to (I) reliably detect even minimal antibiotic concentrations of compounds from the beta-lactam family in waste and drinking water and (II) unravel producer strains of yet unknown Beta-lactam related antibiotics. After extensive characterization of the detection range and sensitivity, the greater goal is to combine the functional Beta-lactam biosensor with our Peptidosomes. Thereby, we would proof the applicability of EncaBcillus as a completely new cultivation platform. Encapsulation of this whole-cell biosensor, will allow an easier and safer handling of the bacteria and thus making them more appealing for field applications, like for example in sewage treatment plants. As a proof of principle we used six Beta-lactams and two controls (water and bacitracin) to evaluate our Biosensor (table 1).

Design

Genetic engineering

To achieve our goal of encapsulating bacteria into Peptidosomes that can sense antibiotics of the beta-lactam family, we first needed to develop a reliable biosensor strain. In Staphylococcus aureus the bla-operon encodes a one-component system, which is responsible for sensing and mediating resistance against beta-lactam antibiotics. The idea was to transfer the regulatory elements of this operon to Bacillus subtilis and replace the native output – being the beta-lactamase BlaZ – by an easy detectable signal. Thus, making Bacillus subtilis a beta-lactam sensing biosensor. (see Figure 2).

For the creation of our biosensor in B. subtilis, the bla-operon from S. aureus was split into three genetic constructs: (A) The Receptor gene blaR1 under control of a strong constitutive promotor (Pveg), (B) the Repressor gene blaI under control moderate strong constitutive promoter (P_lepA) and (C) the target promoter region of the bla-operon (P_blaZ and P_blaR1I) in front of the lux-operon (luxABCDE) (see Figure 3). In addition, an inducible version of the blaR1 construct was made by placing the P_xylA promoter upstream of the blaR1 gene (A).[5]

All genetic constructs and plasmids have been created using the RFC10 RFC10 and/or RFC25 cloning standard. Enzymes used were obtained from New England BioLabs©. Cloning procedures were carried out according to the manufacturer`s protocols.

For submission of our parts to the registry, all Biobricks were cloned into the pSB1C3 backbone. The created genetic constructs were verified by sequencing (Eurofins or GATC sequencing services). All designed plasmids were stored in Escherichia coli DH10β (see Experiments and Protocols for details). In this project, we used integrative single-copy B. subtilis specific vectors that stably integrate into the genome at designated loci.[6]

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Biosensor Characterization

First, we investigated the detection range towards different beta-lactam families as well as the sensitivity of the created biosensor. Therefore, we conducted plate reader experiments and disk diffusion assays to test our biosensor in liquid as well as on solid conditions. We recorded the luminescence signal and growth behavior (see Experiments and Protocols for details) of our biosensor strains in the presence of six different beta-lactam antibiotics. We also included physiological controls that lack one or two of the genetic constructs of the complete biosensor machinery. Furthermore, we analyzed the impact of deleting the Bacillus subtilis gene penP - encoding a beta-lactamase (which has not been studied intensively yet) - on the luminescence output. The strain W168 penP::kan^R was created via Long-Flanking Homology PCR (see Experiments and Protocols for details). We also investigated, if the different beta-lactam antibiotics induce the promoter driving PenP.[7]

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Encapsulation into Peptidosomes

Finally, we could demonstrate a fully functional biosensor encapsulated in Peptidosomes. For this we used the strain showing the strongest response, broadest detection range and the highest viability when tested with different beta-lactams. Peptidosomes containing the biosensor, were exposed to different Beta-lactams and development of the luminescence signal was obtain every hour over a time period of 5 hours.

Results

1. Determination of Inhibitory Antibiotic Concentrations

Before starting the actual tests regarding the functionality of the beta-lactam biosensor, several pretests have been conducted to determine the optimal antibiotic concentration for the subsequent experiments. Therefore, we analyzed the concentration dependent effect of six different beta-lactam antibiotics on the growth of Bacillus subtilis W168 and a strain missing the functional beta-lactamase PenP (W168 penP::kan^R). The beta-lactams tested in this and the following assays were: Ampicillin, Carbenicillin, Cefoperazone, Cefoxitin, Cefalexin and Penicillin G. As controls we chose water(dH₂O) and the antibiotic Bacitracin, which does not belong to the group of beta-lactams.

Several preliminary plate reader experiments have been implemented using a 96 well plate format, in which we tested at least six different concentrations of each antibiotic. These assays were not performed in triplicates. Finally, the concentrations were narrowed down to two concentrations per antibiotic (see Table 3) that would cause a slight growth inhibition, to use in the following assay performed in triplicates (see Figure 4). All tests regarding antibiotic effects were implemented with Mueller Hinton Medium. The strains were induced after 1 hour of growth at 37°C in the plate reader. The growth was followed by measuring the optical density at OD=600nm for 18 hours every 5 minutes.

We expected a higher growth inhibition at higher antibiotic concentrations and a higher sensitivity to the antibiotics of the penP mutant. Adding distilled water to the culture should not show any effect on the growth of both tested strains.

As expected, the data from the pretest performed in triplicates in Figure 4 show, that the presence of the beta-lactamase PenP plays a key role in facilitating survival at higher antibiotic concentrations. The wild type strain B. subtilis W168 is therefore able to grow at higher antibiotic concentrations as the mutant W168 penP::kan^R when treated with ampicillin, carbenicillin, cefoperazone, cefoxitin and penicillin G (see Figure 4). However, this was not the case for cefalexin and bacitracin. Here, we could not observe any difference in growth inhibition between the wild type and the penP mutant in regard to the tested concentrations (see Figure 4). Cefalexin showed a very strong inhibitory effect on the growth of both wildtype and the mutant. For this reason, we chose a relatively weak final concentration (see Table 4 below). Furthermore, we noticed a growth inhibition of the mutant W168 penP::kan^R during the stationary phase, especially when treated with Carbenicillin, Cefalexin and Penicillin G (data not shown). For this reason, we selected weaker antibiotic concentrations for the upcoming experiments with this mutant.

From these first experiments, we selected the following antibiotic concentrations for the upcoming plate reader experiments with the biosensor strains: