Biofilms facilitate cheating and social exploitation of β-lactam resistance in Escherichia coli

Gram-negative bacteria such as Escherichia coli commonly resist β-lactam antibiotics using plasmid-encoded β-lactamase enzymes. Bacterial strains that express β-lactamases have been found to detoxify liquid cultures and thus to protect genetically susceptible strains, constituting a clear laboratory example of social protection. These results are not necessarily general; on solid media, for instance, the rapid bactericidal action of β-lactams largely prevents social protection. Here, we tested the hypothesis that the greater tolerance of biofilm bacteria for β-lactams would facilitate social interactions. We used a recently isolated E. coli strain, capable of strong biofilm formation, to compare how cooperation and exploitation in colony biofilms and broth culture drives the dynamics of a non-conjugative plasmid encoding a clinically important β-lactamase. Susceptible cells in biofilms were tolerant of ampicillin—high doses and several days of exposure were required to kill them. In support of our hypothesis, we found robust social protection of susceptible E. coli in biofilms, despite fine-scale physical separation of resistant and susceptible cells and lower rates of production of extracellular β-lactamase. In contrast, social interactions in broth were restricted to a relatively narrow range of ampicillin doses. Our results show that β-lactam selection pressure on Gram-negative biofilms leads to cooperative resistance characterized by a low equilibrium frequency of resistance plasmids, sufficient to protect all cells.


Microscopy confirmation of void existence
Biofilms were stained with 10 µl of a 1000x dilution of stock of Rhodamine 6G chloride (Molecular Probes, Invitrogen), added underneath the polycarbonate membrane for a 2h exposure. This method was chosen in order to minimise the disturbance of the structure that a flow from the top could cause. Excitation for rhodamine was measured at 500 nm (set to 80% intensity) and emission 515-590 nm.
In contrast to images in the main MS these experiments used biofilms were grown on black polycarbonate membranes (Sterlitech, PCTB 0225100) for 24 h and then transferred to ampicillin-containing plates (0 or 1000 µg/ml) for another 24 h as before. Five locations in each of five biofilms were imaged for each type of biofilm.
Imaging was performed with a Leica SP5X upright microscope, equipped with a super continuum laser source set to 75% intensity. The system was controlled by the software LAS AF ver. 2.6.1. Images were recorded with a long working distance air lens 63x NA 0.7 in order to avoid any disturbance of the biofilm. Excitation at 611 nm (80% intensity) was used for the crimson protein and 475 nm (100% intensity) for the cyan protein. Emission for the crimson protein was measured between 625 -725 nm and for the cyan protein between 490 -550 nm. Imaris 8.1.2 (Imaris, Bitplane AG, available at http://bitplane.com) and JImageAnalyser 1.4, an in-house version of ImageJ at UFZ Magdeburg, were used for thresholding (Table S1), and Imaris was also used for image rendering. Images are shown in Supplementary Figure S1.  Table   S1. Mutants with a chromosomal integration of a fluorescence marker (cc11-1CyanΔLacZ) were constructed in order to facilitate imaging of the spatial structure of biofilms containing cells with and without plasmids. Primers bearing homology to the LacZ gene were used to amplify the region of the pCyanCTXM plasmid encoding bla  and the cyan fluorescence gene (  Table 7). Stability of the insertion was tested via overnight culture in LB and the relative fitness of chromosomal integrants and cells carrying pCRIMCTXM was calculated in overnight cultures in LB broth using the methods described for the liquid culture in the main MS.

Digestions and ligations. All PCR products (inserts and vectors) were
cleaned up with the Promega Wizard SV Gel and PCR Clean-Up System (Cat no A9281) before digesting with enzyme SacI-HF, chosen because this restriction site was not present in any of the vectors or inserts. Typical digestion volumes were 50 µl (to avoid star activity), containing 0.5 -1 µg DNA. The reactions were incubated at 37 • C for 30 min to 1.5 h. The vector was dephosphorylated with an alkaline phosphatase that was added into the vector digestion reaction after the first 30 min and the whole reaction was incubated at 37 • C for another 15 min. The digestions were cleaned up before proceeding to ligations with the same Promega cleanup system mentioned above. Typical vector: insert ratios were 1:2, 1:3, 1:5, or 1:6.
The ligations were incubated at room temperature for a time period of 2 h to 4 h and then inactivated as per the enzyme manufacturer's instructions. All enzymes used can be found in Table S3. PCR primers and cycle conditions can be found in Tables S4-S6.

Plasmid purifications and bacterial transformations.
Plasmid pCT was isolated using the Qiagen Highspeed midi plasmid kit (Qiagen, UK, Cat no 12643). Plasmid pTopo and the subsequently engineered plasmids (crimson or cyan with blaCTX-M-14 or CAT) were isolated with the Qiagen Highspeed mini plasmid kit (Qiagen UK, Cat no 27104). The engineered plasmids were transformed into DH10β-cells with electroporation using the high efficiency electro-transformation protocol for Escherichia coli, suggested by Bio-Rad (http://www.bio-rad.com/webroot/web/pdf/lsr/literature/4006174B.pdf). The transformed cc11-1 strains were recovered on IPTG-containing LB plates with the appropriate antibiotic selector. IPTG was used for inducing the transcription of the fluorescence gene. PCR was used for the confirmation of the transformants.

Sequencing constructed plasmids
The insertion in the plasmids was confirmed by sequencing. The constructed, cloned plasmids were purified from E.coli DH10β with the Qiagen Highspeed mini plasmid kit (Qiagen UK, Cat no 27104) and sent to Eurofins Genomics for sequencing. The sequencing primers used can be found in Table S7.

Supplementary Tables
Supplementary Table 1 Properties of the fluorescent proteins used in experiments Supplementary