Thien-Fah Mah

Mechanisms of biofilm resistance to antimicrobial agents

Biofilms are communities of microorganisms attached to a surface. It has become clear that biofilm-grown cells express properties distinct from planktonic cells, one of which is an increased resistance to antimicrobial agents. Recent work has indicated that slow growth and/or induction of an rpoS-mediated stress response could contribute to biocide resistance. The physical and/or chemical structure of exopolysaccharides or other aspects of biofilm architecture could also confer resistance by exclusion of biocides from the bacterial community. Finally, biofilm-grown bacteria might develop a biofilm-specific biocide-resistant phenotype. Owing to the heterogeneous nature of the biofilm, it is likely that there are multiple resistance mechanisms at work within a single community. Recent research has begun to shed light on how and why surface-attached microbial communities develop resistance to antimicrobial agents.

A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance

Biofilms are surface-attached microbial communities with characteristic architecture and phenotypic and biochemical properties distinct from their free-swimming, planktonic counterparts. One of the best-known of these biofilm-specific properties is the development of antibiotic resistance that can be up to 1,000-fold greater than planktonic cells. We report a genetic determinant of this high-level resistance in the Gram-negative opportunistic pathogen, Pseudomonas aeruginosa. We have identified a mutant of P. aeruginosa that, while still capable of forming biofilms with the characteristic P. aeruginosa architecture, does not develop high-level biofilm-specific resistance to three different classes of antibiotics. The locus identified in our screen, ndvB, is required for the synthesis of periplasmic glucans. Our discovery that these periplasmic glucans interact physically with tobramycin suggests that these glucose polymers may prevent antibiotics from reaching their sites of action by sequestering these antimicrobial agents in the periplasm. Our results indicate that biofilms themselves are not simply a diffusion barrier to these antibiotics, but rather that bacteria within these microbial communities employ distinct mechanisms to resist the action of antimicrobial agents.

Biofilm-specific antibiotic resistance

Bacterial biofilms are the basis of many persistent diseases. The persistence of these infections is primarily attributed to the increased antibiotic resistance exhibited by the cells within the biofilms. This resistance is multifactorial; there are multiple mechanisms of resistance that act together in order to provide an increased overall level of resistance to the biofilm. These mechanisms are based on the function of wild-type genes and are not the result of mutations. This article reviews the known mechanisms of resistance, including the ability of the biofilm matrix to prevent antibiotics from reaching the cells and the function of individual genes that are preferentially expressed in biofilms. Evidence suggests that these mechanisms have been developed as a general stress response of biofilms that enables the cells in the biofilm to respond to all of the changes in the environment that they may encounter.

Pseudomonas aeruginosa tssC1 Links Type VI Secretion and Biofilm-Specific Antibiotic Resistance

Biofilm-specific antibiotic resistance is influenced by multiple factors. We demonstrated that Pseudomonas aeruginosa tssC1, a gene implicated in type VI secretion (T6S), is important for resistance of biofilms to a subset of antibiotics. We showed that tssC1 expression is induced in biofilms and confirmed that tssC1 is required for T6S.

A retrospective analysis of biofilm antibiotic susceptibility testing: a better predictor of clinical response in cystic fibrosis exacerbations.

BACKGROUND Bacteria grow as biofilms within CF airways. However, antibiotic susceptibility testing is routinely performed on planktonically-growing bacteria. This study assessed whether CF patients infected with multiresistant organisms had improved clinical outcomes if given antibiotics that inhibited their biofilm-grown bacteria. METHODS 110 patients with pulmonary exacerbations were treated with intravenous antibiotics based on susceptibility testing of planktonically-growing bacteria. A retrospective analysis was done using bacterial isolates grown from their sputum at exacerbation. Each isolate was grown as a biofilm and combination antibiotic susceptibility testing was performed. Clinical outcomes in patients treated with biofilm-susceptible antibiotics were compared to those that were not. RESULTS 66 of 110 patients (60%) were treated with antibiotic combinations that inhibited all of their planktonically-grown bacterial isolates, however, when the same isolates were grown as biofilms, only 24 patients (22%) had all of their biofilm-grown isolates remaining susceptible to the antibiotics (P=<0.001 ). When patients with at least one biofilm-grown susceptible isolate (n=61) were compared to those with none (n=49), there was a significant decrease in sputum bacterial density (P=0.02) and length of stay (P=0.04) and a non-significant decrease in treatment failure. Survival analyses of time to next exacerbation showed non-significant trends favoring patients treated with biofilm-effective antibiotics. CONCLUSIONS Most patients with CF exacerbations do not receive antibiotics that inhibit all biofilm-grown bacteria from their sputum at exacerbation. Patients treated with biofilm-effective therapy seemed to have improved clinical outcomes.

Molecular mechanisms of biofilm-based antibiotic resistance and tolerance in pathogenic bacteria

Biofilms are surface-attached groups of microbial cells encased in an extracellular matrix that are significantly less susceptible to antimicrobial agents than non-adherent, planktonic cells. Biofilm-based infections are, as a result, extremely difficult to cure. A wide range of molecular mechanisms contribute to the high degree of recalcitrance that is characteristic of biofilm communities. These mechanisms include, among others, interaction of antimicrobials with biofilm matrix components, reduced growth rates and the various actions of specific genetic determinants of antibiotic resistance and tolerance. Alone, each of these mechanisms only partially accounts for the increased antimicrobial recalcitrance observed in biofilms. Acting in concert, however, these defences help to ensure the survival of biofilm cells in the face of even the most aggressive antimicrobial treatment regimens. This review summarises both historical and recent scientific data in support of the known biofilm resistance and tolerance mechanisms. Additionally, suggestions for future work in the field are provided.

Identification of Genes Involved in Pseudomonas aeruginosa Biofilm-Specific Resistance to Antibiotics

Pseudomonas aeruginosa is a key opportunistic pathogen characterized by its biofilm formation ability and high-level multiple antibiotic resistance. By screening a library of random transposon insertion mutants with an increased biofilm-specifc antibiotic susceptibility, we previously identified 3 genes or operons of P. aeruginosa UCBPP-PA14 (ndvB, PA1875–1877 and tssC1) that do not affect biofilm formation but are involved in biofilm-specific antibiotic resistance. In this study, we demonstrate that PA0756–0757 (encoding a putative two-component regulatory system), PA2070 and PA5033 (encoding hypothetical proteins of unknown function) display increased expression in biofilm cells and also have a role in biofilm-specific antibiotic resistance. Furthermore, deletion of each of PA0756, PA2070 and PA5033 resulted in a significant reduction of lethality in Caenorhabditis elegans, indicating a role for these genes in both biofilm-specific antibiotic resistance and persistence in vivo. Together, these data suggest that these genes are potential targets for antimicrobial agents.

Safety and efficacy of composite collagen–silver nanoparticle hydrogels as tissue engineering scaffolds

The increasing number of multidrug resistant bacteria has revitalized interest in seeking alternative sources for controlling bacterial infection. Silver nanoparticles (AgNPs), are amongst the most promising candidates due to their wide microbial spectrum of action. In this work, we report on the safety and efficacy of the incorporation of collagen coated AgNPs into collagen hydrogels for tissue engineering. The resulting hybrid materials at [AgNPs] < 0.4 μM retained the mechanical properties and biocompatibility for primary human skin fibroblasts and keratinocytes of collagen hydrogels; they also displayed remarkable anti-infective properties against S. aureus, S. epidermidis, E. coli and P. aeruginosa at considerably lower concentrations than silver nitrate. Further, subcutaneous implants of materials containing 0.2 μM AgNPs in mice showed a reduction in the levels of IL-6 and other inflammation markers (CCL24, sTNFR-2, and TIMP1). Finally, an analysis of silver contents in implanted mice showed that silver accumulation primarily occurred within the tissue surrounding the implant.

Inhibition of Bacterial Quorum Sensing and Biofilm Formation by Extracts of Neotropical Rainforest Plants

Bacterial biofilms are responsible for many persistent infections by many clinically relevant pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa. Biofilms are much more resistant to conventional antibiotics than their planktonic counterparts. Quorum sensing, an intercellular communication system, controls pathogenesis and biofilm formation in most bacterial species. Quorum sensing provides an important pharmacological target since its inhibition does not provide a selective pressure for resistance. In this study, we investigated the quorum sensing and biofilm inhibitory activities of 126 plant extracts from 71 species collected from neotropical rainforests in Costa Rica. Quorum sensing and biofilm interference were assessed using a modified disc diffusion bioassay with Chromobacterium violaceum ATCC 12,472 and a spectrophotometric bioassay with Pseudomonas aeruginosa PA14, respectively. Species with significant anti-quorum sensing and/or anti-biofilm activities belonged to the Meliaceae, Melastomataceae, Lepidobotryaceae, Sapindaceae, and Simaroubaceae families. IC50 values ranged from 45 to 266 µg/mL. Extracts of these active species could lead to future development of botanical treatments for biofilm-associated infections.

Anti-microbiological and Anti-infective Activities of Silver

Silver nanoparticles are the latest version of silver preparations that have been revived and tested as anti-microbials, particularly as an alternative to antibiotics since the emergence of drug resistant bacteria. Silver nanoparticles share commonalities with other silver preparations and other metallic nanoparticles, but also several significant differences in their interactions with microbes. Their mechanism of action is not completely understood but their potential utility has led to the high level of research activity to determine the safety and efficacy of these nanoparticles for clinical applications.