With the development of next generation sequencing technologies in recent years, it has been demonstrated that many human infectious processes, including chronic wounds, cystic fibrosis, and otitis media, are associated with a polymicrobial burden. Research has also demonstrated that polymicrobial infections tend to be associated with treatment failure and worse patient prognoses. Despite the importance of the polymicrobial nature of many infection states, the current clinical standard for determining antimicrobial susceptibility in the clinical laboratory is exclusively performed on unimicrobial suspensions. There is a growing body of research demonstrating that microorganisms in a polymicrobial environment can synergize their activities associated with a variety of outcomes, including changes to their antimicrobial susceptibility through both resistance and tolerance mechanisms. This review highlights the current body of work describing polymicrobial synergism, both inter- and intra-kingdom, impacting antimicrobial susceptibility. Given the importance of polymicrobial synergism in the clinical environment, a new system of determining antimicrobial susceptibility from polymicrobial infections may significantly impact patient treatment and outcomes.

The checkerboard assay is a well-established tool used to determine the antimicrobial effects of two compounds in combination. Usually, data collected from the checkerboard assay use visible turbidity and optical density as a readout. While helpful in traditional checkerboard assays, these measurements become less useful in a polymicrobial context as they do not enable assessment of the drug effects on the individual members of the community. The methodology described herein allows for the determination of cell viability through selective and differential plating of each individual species in a community while retaining much of the high-throughput nature of a turbidity-based analysis and requiring no specialized equipment. This methodology further improves turbidity-based measurements by providing a distinction between bacteriostatic versus bactericidal concentrations of antibiotics. Herein, we use this method to demonstrate that the clinically used antibiotic combination of ceftazidime and gentamicin works synergistically against Pseudomonas aeruginosa in monoculture but antagonistically in a polymicrobial culture also containing Acinetobacter baumannii, Staphylococcus aureus, and Enterococcus faecalis, highlighting the fundamental importance of this methodology in improving clinical practices. We propose that this method could be implemented in clinical microbiology laboratories with minimal impact on the overall time for diagnosis.

Antimicrobial recalcitrance is a growing problem in today’s world. Not only are bacteria developing resistance at an alarming pace but the antibiotic discovery pipeline has gone dry, making antimicrobial stewardship essential for preserving the activity of the antibiotics still currently available for use. In addition to resistance, bacteria also display tolerance to certain treatments as they adapt to their body site-specific niche and cooperate with other organisms in polymicrobial communities. Thus, new and existing antibiotics must contend with altered bacterial metabolism, polymicrobial synergy, increased biofilm production, and nutrient-related adaptations present within specific infectious sites. Finally, these treatments must face the challenging process of moving to the infection site and doing their job without causing harm to the patient. This minireview explores the difficulties antimicrobials face when challenging organisms at different body sites, focusing on the niche-specific dynamics present at sites of infection.

Chronic wounds are persistent and difficult to treat. Often this is because they are colonized by polymicrobial communities which contribute to changes in antimicrobial susceptibilities, making these infections harder to effectively clear. We explored the role a community can play in individual members’ survival when challenged by antibiotics, specifically looking at a community consisting of Staphylococcus aureus , Pseudomonas aeruginosa , Enterococcus faecalis , and Acinetobacter baumannii . Our data shows that communities can contribute to both increases and decreases in susceptibilities depending on the species and the antibiotic. The changes in susceptibilities can be due to interspecies cooperation or competition, with identifiable mechanisms. We also demonstrated that current antimicrobial susceptibility testing (AST) methods used in hospitals, which focus on determining the minimum inhibitory concentration (MIC) via determination of visible turbidity breakpoints, are not able to truly indicate the clearance of bacteria, as species can persist in higher antibiotic concentrations after visible turbidity is gone. To combat decreases in antibiotic susceptibilities contributed to by the community, we used our data from individual antibiotics to determine a potentially effective antibiotic combination, similar to combinatorial therapy used in hospitals to treat recalcitrant infections. Our data proved useful, as the combination of gentamicin and cephalexin was able to overcome polymicrobial synergism and clear the desired bacteria. This demonstrates that it is possible to determine effective antibiotic treatments for polymicrobial infections, whether they be combinatorial in nature or not. One simply must account for the role of the community in order to prescribe the most effective treatment.

AIMS: To establish a methodology for identifying the effects of combinatorial antibiotic treatment on individual members of a polymicrobial community. METHODS AND RESULTS: Both gentamicin and ceftazidime were diluted to concentrations ranging from 0.06 μg ml ^-1 to 128 μg ml ^-1 . An equal ratio polymicrobial community of Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis , and Acinetobacter baumannii was inoculated into the combined antibiotics in the checkerboard and incubated for 18 hours in static conditions. After incubation, visible turbidity of the overall community was recorded, and bacteria from the wells were diluted to 10 ^-3 and then spot plated on selective and differential media. After 24 hours, colony-forming unit (CFU) counts were obtained for each species. CONCLUSIONS: Visible turbidity is not truly indicative of cell viability, and the polymicrobial community can decrease the antibiotic susceptibility of P. aeruginosa , rendering the clinically-established beneficial combination of gentamicin and ceftazidime ineffective. SIGNIFICANCE: Previous checkerboard methodology which focuses on using visible turbidity to determine monomicrobial antibiotic susceptibility fails to account for polymicrobial cooperation that has been shown to reduce antibiotic efficacy. Our new methodology could be implemented in clinical microbiology laboratories with minimal impact on the overall time for diagnosis.

Conjugated dienes are useful precursors to cascade reactions that undergo cycloadditions to access polycyclic compounds toward bioactive natural products. However, the synthesis of the diene precursor can be challenging due to instability of the diene due to isomerization or reaction conditions that do not yield the diene in high amounts. In this study, (‐)‐isopulegol was converted to a homoallylic bromide, which was eliminated with 1,8‐diazabicyclo(5.4.0)undec‐7‐ene to cleanly furnish the conjugated diene. The resulting diene was used as a precursor to various dienophiles to give tricyclic frameworks as well as other heterocycles. This new entry into the synthesis of conjugated dienes enabled the generation of a small group of 12 compounds that were tested for antimicrobial properties. Out of the 12 compounds, three showed consistent antimicrobial activities against JE2 and H99 cells (20–107 µg/mL) while the rest of the tested compounds did not possess any antimicrobial property (>128 µg/mL). The bioactive compounds possessed a Michael acceptor motif, suggesting an explanation for their antimicrobial activity.