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).

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.

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 final antibiotic concentrations for the upcoming plate reader experiments with the biosensor strains (see Table 4)

In our first experiment, we performed plate reader assays in a 96 well plate format and measured growth (OD600) and luminescence output for 18 hours every 5 minutes. Induction with the Beta-lactam antibiotics occurred after 1 hour. All strains have been tested in triplicates under the same conditions. Strains with the genotype penP::kan^R have been induced with lower concentrations compared to the wild type strain W168 (see Table 4 above).

After induction, we anticipate a strong increase in luminescence signal for strains containing the full set of constructs (P_blaZ_lux or P_blaR1I_lux, P_veg_blaR1 or P_xyl_blaR1, P_lepA_blaI), thus representing functional biosensors. Besides the biosensor constructs, we also tested all physiological controls missing one essential composite of the biosensor`s heterologous one-component system (data not shown). The control strain W168 (wild type) and control 1, will not show any luminescence output, while the positive control 2 is expected to show a steady luminescence signal regardless of the presence of any antibiotic compound.

As the beta-lactamase PenP confers resistance to beta-lactam antibiotics in B. subtilis, a rising luminescence signal is estimated for the PpenP_lux constructs post induction. Generally, the Pxyl promoter is activated by adding Xylose as an inducer to the medium. In the plate reader experiments, 0.2% xylose was added to the cells starting from the day culture step.

Further we propose biosensor strains carrying the genotype remark penP::kan^R to give a stronger signal in presence of beta-lactam compounds, as they cannot be degraded by the PenP enzyme.

The bar charts in Figure 5 illustrate the best biosensor constructs identified in the plate reader experiments and compares the relative luminescence units of the strains reached 2 hours after induction.

As shown in figure 5, the wildtype W168 (black with white dots) shows no increase in RLU values when induced with the different beta-lactam antibiotics and controls. Control 1 (black tight stripes) behaves similarly to the wild type strain. The slight decrease of Control 2 (light grey) in the bar chart where induction with ampicillin and carbenicillin happened, is mostly explained by the higher growth inhibition caused by the chosen concentrations for W168 (with penP). Most of the times, the constitutive expression of the lux operon resulted in an RLU of over 1.3 million for control 2 (see Figure 5).

Biosensor 1 gives an overall good signal for all beta-lactam antibiotics tested, but also shows a higher basal activity in absence of the beta-lactam compounds of 40.000- 90.000 RLU (see Figure 5, bar chart with bacitracin and dH2O). Further, we could observe a difference in signal intensity dependent on the beta-lactam antibiotic tested. Therefore, biosensor 1 gives the highest signal in presence of penicillin G, cefoxitin and cefoperazone with up to 2.7 million RLU. Ampicillin and penicillin G again show a weaker increase in signal produced by biosensor 1, which could be due to the same reason as for control 2 (see Figure 5).

For Biosensor 2, the detection range and sensitivity is comparable to Biosensor 1, This strain strongly senses cefoxitin, ampicillin and cefoperazone reaching up to 2.4 million RLU. Even the basal activity of the PblaZ promoter in biosensor 2, as shown in the bar charts with bacitracin and dH2O, conforms with the one from biosensor 1.

The activity of biosensor 3 can be induced by adding Xylose (0.2% final concentration) to the sample. In absence of Xylose, the signal is very weak as can be seen in the bar with the dark blue stripes (see Figure 5). This is due to the fact, that without xylose, nearly no receptor molecules are localized at the bacteria`s cell surface and the signal cannot be detected and amplified. For induction with cefoperazone though, the weak receptor density on the surface because of the leaky expression of blaR1, seems to be enough to cause a quite powerful signal already. From this finding can be concluded, that the receptor seems to have a high affinity to cefoperazone. In the bar charts of bacitracin and dH2O (see Figure 5), Biosensor 3 shows the lowest basal promoter activity compared to the other two Biosensors tested. Here, we could also observe a high sensitivity for penicillin G, cefoxitin, cefoperazone and ampicillin. In general, all Biosensors show good performance in liquid MH-Medium. They able to detect the six beta-lactams reliably by giving a signal way stronger than the basal luminescence.