A microdillution assay is a particular experiment used to determine the minimum inhibitory concentration (MIC) of an antibiotic on a particular bacterial isolate. The MIC is the lowest concentration, typically reported in mg/L, of a drug required to inhibit visible bacterial growth.

1. Preparing an Antibiotic Gradient

• After having chosen a particular antibiotic, a specific concentration of the antibiotic is delivered into the A1 well of a 96 well plate. A small sample is aliquoted from the A1 well and placed into the A2 well and diluted with bacterial media to reach the next desired concentration. This process continues across the row until the final A12 well, culminating in a wide descending range of antibiotic concentrations across the plate. The process is then repeated with the next antibiotic starting in b1. It goes without saying that careful planning is required to ensure equal volume in each well in spite of the added bacterial media used in the dilutions.

2. Standardizing Bacterial cultures

   • After having cultivated the various samples of MRSA, each isolate was standardized to a Macfarland standard to create samples with approximately equal bacterial concentration based on solution turbidity.

     • MacFarland standards are used to eliminate false results of resistance or susceptibility of a microbial agent due to error from a sample either being too concentrated with bacteria or too dilute.

     • MacFarland standards are suspensions created either using latex particles or mixing barium chloride and sulfuric acid to create a barium sulfate precipitate, with standards reflecting the turbidity of cultures with 1.5, 3, 6, 9, and 12 1×1^8 colony forming unity (CFU) /ml of bacteria. The percent transmittance of each bacterial sample is measured via spectrometry against the associated MacFarland standard value and the sample is either concentrated or diluted accordingly. Sole visual inspection is also used to adjust the bacterial sample to the appropriate turbidity.

3. Loading Samples

   • Equal volumes of standardized cultures were placed into each well. The plate was then placed in an incubator with the appropriate cellular conditions, %CO2, and temperature to facilitate cell growth. The cells were allowed to grow for an allotted time frame for each species according to EUCAST recommendations.

4. Determining MIC

   • The bacteria were removed from the incubator and visually inspected. The well furthest to the right that does not contain bacterial growth corresponds to the MIC of that specific antibiotic and isolate.

5. Experimental Repeats

   • If the difference in antibiotic concentration between each well is large, the experiment can be repeated with a more focused range of antibiotic concentrations which provides a more refined MIC value. Additionally, replicates of each experiment are suggested to improve scientific accuracy.

Schaumberg et al. 2016 performed the above experiment in accordance with the International Organization for Standardization protocol for Susceptibility testing of infectious agents and evaluation of performance of antimicrobial susceptibility test devices (ISO 20776-1:2019).

Having now determined the antibiotic resistance of each strain, Schaumberg et al. 2016 began sequencing the isolates to try and determine potential novel mutations contributing to antibiotic resistance.

1. DNA Isolation and Purification

   • Bacterial DNA was isolated via a QiAamp DNA silica-membrane-based nucleic acid purification mini kit according to the prepackaged instructions.

     • Silica-membrane based nucleic acid mini kits bind nucleic acids to the silica membrane in the presence of included chaotropic salts. Polysaccharides and proteins do not bind well to the column and residual traces are removed during alcohol-based wash steps, along with the salts. After binding and washing, nucleic acids are selectively eluted under low-salt conditions, using water or TE buffer to purify the desired protein.

2. Polymerase Chain Reaction (PCR) of the PBP2a encoding mecA gene

   • PCR was utilized to amplify the PBP2a encoding gene mecA and then was sequenced.

   • The forward primer 5'-TAAGGGAGAAGTAACAGCAC-3' and the reverse primer 5′-ACCTTCTACACCTCCATATCAC-3′ were used to amplify the mecA. gene.

3. Comparison between MRSA isolates

   • The mecA sequence of each isolate was aligned with one another to another and against the industry standard wild type N315 (SA0038) MRSA strain using the MUSCLE program as implemented in Mega 6.

The figure above is the amalgamation of the two previously described experiments. Ceftaroline and ceftobiprole are two recently developed antibiotics used to treat MRSA infections that non-covalently bind to both the allosteric site and the active site of PBP2a. The MIC reported on both the x-axis and y-axis correspond to the same value obtained from the microdillution assay. MUSCLE analysis revealed the presence of three recurring mutations, those being G246E, S225R, and a N146K-N204K-G246E triple mutant. Each isolate was then plotted onto the figure, as one of the four shapes indicated by the legend, based on their relative MICs to both antibiotics. The shaded area indicates the MIC ranges for a MRSA isolate to be considered susceptible by EUCAST standard. A isolate located outside of this box is considered to not be susceptible and regarded as antibiotic resistant.

Analyzing the figure reveals that the wild type, G246E mutation, and the S225R mutation are all still considered susceptible to both ceftobiprole and ceftaroline. Its important to note that both mutated residues lie outside of both the allosteric and active sites, indicating that they don't interact directly with the bound antibiotic. Yet the figure doesn't reflect the theme of salt bridge cascade disruption that was observed in the H351N mutation. Instead, these two mutations made the strain more susceptible to both antibiotics as indicated by both MIC values being lower than those of the wildtype strains. A potential explanation relies on the idea that these mutations created a more efficient salt bridge cascade. The introduction of charged amino acids at these residues could potentially reorder or form new salt bridge interactions with surrounding residues. As a result, these new salt bridge interactions could potentially have widespread implications that reformat the salt bridge cascade or could bind previously obstructive residues that had the potential to divert the signal away from the active site. In either case, the efficiency of the salt bridge cascade would be improved, yielding the elevated response to both antibiotics seen in the figure.

The majority of the triple mutant N146K-N204K-G246E isolates are outside of the shaded region and on the upper range of MIC concentrations, indicating that they are antibiotic resistant. This is particularly noteworthy as the triple mutant isolate contains the G246E MIC lowering mutation. A potential explanation for this seemingly contradictory finding rests on the location of the various amino acid residues. Residue 146 is located directly on the allosteric site and residue 204 is in close proximity. The N146K and N204K mutations result in both residues having a positive charge, and in the context of the hypothesized allosterically displaced 146 residue, could undergo same charge repulsion which would shift residue 204 into a salt bridge interaction with residue 246. As a result, this triple mutant could potentially create a novel new salt bridge cascade pathway that diverts the signal away from the active site resulting in the observed antibiotic resistant phenotype.

This experiment revealed that the PBP2a salt bridge cascade is a unique construct that is not yet fully understood. However, it has highlighted the flexibility and versatility of charged amino acid residues in modifying and creating new salt bridge cascade pathways that have important implications for future antibiotic resistance studies. The same mutation of a single residue can result in completely different antibiotic susceptibilities, taken in the context of proximal mutations. The increased susceptibility of both the G246E and S225R mutations indicate the interconnectedness of the charged amino acids and salt bridge interactions of PBP2a, with a single charged amino acid mutation having widespread effects on the salt bridge cascade. Similarly, that same mutation can be repurposed with the appropriate leading mutations to form a novel salt bridge cascade that results in antibiotic resistance. One other key aspect to note is that the G246E mutation only results in antibiotic resistance when linked to the allosteric site, implicating the allosteric binding site as a critical determinant of antibiotic resistance. PBP2a's ability to form new salt bridge interactions with a single mutation due to the vast number of interactions among different charged residues ultimately implicates the versatility of the salt bridge cascade as a important tool in developing antibiotic resistance.