A Facile High-Throughput Model of Surface-Independent Staphylococcus aureus Biofilms by Spontaneous Aggregation.

Many microbes in their natural habitats are found in biofilm ecosystems attached to surfaces and not as free-floating (planktonic) organisms. Furthermore, it is estimated that nearly 80% of human infections are associated with biofilms. Biofilms are traditionally defined as three-dimensional, structured microbial communities that are attached to a surface and encased in a matrix of exopolymeric material. While this view of biofilm largely arises from in vitro studies under static or flow conditions, in vivo observations have indicated that this view of biofilms is essentially true only for foreign-body infections on catheters or implants where biofilms are attached to the biomaterial. In mucosal infections such as chronic wounds or cystic fibrosis or joint infections, biofilms can be found unattached to a surface and as three-dimensional aggregates. In this work, we describe a high-throughput model of aggregate biofilms of methicillin-resistant Staphylococcus aureus (MRSA) using 96-well plate hanging-drop technology. We show that MRSA forms surface-independent biofilms, distinct from surface-attached biofilms, that are rich in exopolymeric proteins, polysaccharides, and extracellular DNA (eDNA), express biofilm-related genes, and exhibit heightened antibiotic resistance. We also show that the surface-independent biofilms of clinical isolates of MRSA from cystic fibrosis and central catheter-related infections demonstrate morphological differences. Overall, our results show that biofilms can form by spontaneous aggregation without attachment to a surface, and this new in vitro system can model surface-independent biofilms that may more closely mimic the corresponding physiological niche during infection.IMPORTANCE The canonical model of biofilm formation begins with the attachment and growth of microbial cells on a surface. While these in vitro models reasonably mimic biofilms formed on foreign bodies such as catheters and implants, this is not the case for biofilms formed in cystic fibrosis and chronic wound infections, which appear to present as aggregates not attached to a surface. The hanging-drop model of biofilms of methicillin-resistant Staphylococcus aureus (MRSA), the major causative organism of skin and soft tissue infections, shows that these biofilms display morphological and antibiotic response patterns that are distinct from those of their surface-attached counterparts, and biofilm growth is consistent with their in vivo location. The simplicity and throughput of this model enable adoption to investigate other single or polymicrobial biofilms in a physiologically relevant setting.

Antimicrobial and Antibiofilm Activity of Synergistic Combinations of a Commercially Available Small Compound Library With Colistin Against Pseudomonas aeruginosa.

Biofilm-associated Pseudomonas aeruginosa infections remain a significant clinical challenge since the conventional antibiotic treatment or combination therapies are largely ineffective; and new approaches are needed. To circumvent the major challenges associated with discovery of new antimicrobials, we have screened a library of compounds that are commercially available and approved by the FDA (Prestwick Chemical Library) against P. aeruginosa for effective antimicrobial and anti-biofilm activity. A preliminary screen of the Prestwick Chemical Library alone did not yield any repositionable candidates, but in a screen of combinations with a fixed sub-inhibitory concentration of the antibiotic colistin we observed 10 drugs whose bacterial inhibiting activity was reproducibly enhanced, seven of which were enhanced by more than 50%. We performed checkerboard assays of these seven drugs in combination with colistin against planktonic cells, and analysis of their interactions over the complete combination matrix using the Zero Interaction Potency (ZIP) model revealed interactions that varied from highly synergistic to completely antagonistic. Of these, five combinations that showed synergism were down-selected and tested against preformed biofilms of P. aeruginosa. Two of the five combinations were active against preformed biofilms of both laboratory and clinical strain of P. aeruginosa, resulting in a 2-log reduction in culturable cells. In summary, we have identified synergistic combinations of five commercially available, FDA-approved drugs and colistin that show antimicrobial activity against planktonic P. aeruginosa (Clomiphene Citrate, Mitoxantrone Dihydrochloride, Methyl Benzethonium Chloride, Benzethonium Chloride, and Auranofin) as well as two combinations (Auranofin and Clomiphene Citrate) with colistin that show antibiofilm activity.

High-throughput microarray for antimicrobial susceptibility testing.

We describe the development of a novel, high-throughput, nano-scale microarray platform for antimicrobial susceptibility testing (AST). The platform allows to process 480 samples at 50 nL volume on a single chip, analyze by fluorescence read-out with an easy dunk-and-rinse step, and the ability to process multiple samples and chips simultaneously. We demonstrate the applicability of this chip for culturing community acquired methicillin resistant Staphylococcus aureus (CA-MRSA), and perform AST against clinical isolates of CA-MRSA. The chip platform holds promise for an impact in microbial biotechnology as an attractive high-throughput, lower sample volume and quicker alternative to conventional AST such as the traditional broth microdilution or the newer automated systems.

nBioChip, a Lab-on-a-Chip Platform of Mono- and Polymicrobial Biofilms for High-Throughput Downstream Applications.

Current in vitro techniques for the culture of microorganisms, and particularly of delicate microbial biofilms, are still mostly limited to low-density plates and manual labor and are not amenable to automation and true high-throughput (HT) applications. We have developed a novel fully automated platform for the formation of mono- and polymicrobial biofilms of Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans at the nanoscale level. The nBioChip is robotically printed, robustly handled, and scanned using a standard microarray reader. Using this technique, hundreds to thousands of identical nanobiofilms encapsulated in hydrogel spots were cultured on microscope slides. The spots can withstand the washing steps involved in screening assays. The miniaturized biofilms demonstrated characteristics similar to those displayed by conventionally formed macroscopic biofilms, including (i) three-dimensional architectural features, (ii) synthesis of exopolymeric matrix material, and (iii) elevated resistance to antibiotic treatment. On the basis of our results, the nBioChip can generate reliable high-throughput antimicrobial susceptibility testing (HT-AST) in 12 to 18 h. The chip serves as a proof-of-concept universal platform for high-throughput drug screening and other downstream applications and furthers understanding of microbial interactions in mixed-species communities at the nanoscale level. IMPORTANCE With an estimated 80% of infections being associated with a biofilm mode of growth and the ensuing recalcitrance of these biofilms with respect to conventional antibiotic treatment leading to high mortality rates, there is a dire and unmet need for the development of novel approaches to prevent, treat, and control these infections. Both bacteria and fungi are capable of forming biofilms that are inherently fragile and often polymicrobial in nature, which further complicates treatment. In this work, we showcase a nanobiofilm chip as a convenient platform for culturing several hundreds of mono- or polymicrobial biofilms and for susceptibility testing. This platform enables true ultra-high-throughput screening for antimicrobial drug discovery or diagnostics or for addressing fundamental issues in microbiology.

Screening a Commercial Library of Pharmacologically Active Small Molecules against Staphylococcus aureus Biofilms.

It is now well established that bacterial infections are often associated with biofilm phenotypes that demonstrate increased resistance to common antimicrobials. Further, due to the collective attrition of new antibiotic development programs by the pharmaceutical industries, drug repurposing is an attractive alternative. In this work, we screened 1,280 existing commercially available drugs in the Prestwick Chemical Library, some with previously unknown antimicrobial activity, against Staphylococcus aureus, one of the commonly encountered causative pathogens of burn and wound infections. From the primary screen of the entire Prestwick Chemical Library at a fixed concentration of 10 µM, 104 drugs were found to be effective against planktonic S. aureus strains, and not surprisingly, these were mostly antimicrobials and antiseptics. The activity of 18 selected repurposing candidates, that is, drugs that show antimicrobial activity that are not already considered antimicrobials, observed in the primary screen was confirmed in dose-response experiments. Finally, a subset of nine of these drug candidates was tested against preformed biofilms of S. aureus We found that three of these drugs, niclosamide, carmofur, and auranofin, possessed antimicrobial activity against preformed biofilms, making them attractive candidates for repurposing as novel antibiofilm therapies.