Experimental evolution of silver nanoparticle resistance in Escherichia coli

The recent exponential increase in the use of engineered nanoparticles (eNPs) means both greater intentional and unintentional exposure of eNPs to microbes. Intentional use includes the use of eNPs as biocides; unintentional exposure results from the fact that eNPs are included in a variety of commercial products (paints, sunscreens, cosmetics.) Many of these eNPs include heavy metals or metal oxides such as titanium dioxide, silver, gold, zinc and zinc oxide. The fact that early studies of the impact of metallic nanoparticles achieved approximately 90% lethality to Ag, Cu eNPs, suggests that genetic variants are already circulating in bacteria that can be co-opted to provide heavy metal eNP resistance. This project has utilized laboratory experimental evolution to evolve eNP resistance in the bacterium Escherichia coli (K12 MG1655 strain.). This is currently being validated by demonstrating the greater fitness of evolved strains versus ancestral strains in the presence of different sized and coated silver nanoparticles (10nm, 40nm, citrate-coated, PVP-coated) as well as phenotypic changes in the bacterial cell wall (as measured by Atomic Force Microscopy, AFM.). Finally, the bacterial genomes of the evolved and ancestral strains were resequenced. The genomic basis of this complex phenotype was determined. The practical application of such knowledge cannot be underestimated since nature is already evolving nanoparticle resistant bacteria. Thus knowledge of the nature of the physiological, morphological, and genomic mechanisms of resistance will be essential to deploy sustainable use of NPs as biocides, and to prevent unintentional environmental damage

Mechanobiology of Antimicrobial Resistant Escherichia coli and Listeria innocua

A majority of antibiotic-resistant bacterial infections in the United States are associated with biofilms. Nanoscale biophysical measures are increasingly revealing that adhesive and viscoelastic properties of bacteria play essential roles across multiple stages of biofilm development. Atomic Force Microscopy (AFM) applied to strains with variation in antimicrobial resistance enables new opportunities for investigating the function of adhesive forces (stickiness) in biofilm formation. AFM force spectroscopy analysis of a field strain of Listeria innocua and the strain Escherichia coli K-12 MG1655 revealed differing adhesive forces between antimicrobial resistant and nonresistant strains. Significant increases in stickiness were found at the nanonewton level for strains of Listeria innocua and Escherichia coli in association with benzalkonium chloride and silver nanoparticle resistance respectively. This advancement in the usage of AFM provides for a fast and reliable avenue for analyzing antimicrobial resistant cells and the molecular dynamics of biofilm formation as a protective mechanism

Rapid Evolution of Silver Nanoparticle Resistance in Escherichia Coli

The recent exponential increase in the use of engineered nanoparticles (eNPs) means both greater intentional and unintentional exposure of eNPs to microbes. Intentional use includes the use of eNPs as biocides. Unintentional exposure results from the fact that eNPs are included in a variety of commercial products (paints, sunscreens, cosmetics). Many of these eNPs are composed of heavy metals or metal oxides such as silver, gold, zinc, titanium dioxide, and zinc oxide. It is thought that since metallic/metallic oxide NPs impact so many aspects of bacterial physiology that it will difficult for bacteria to evolve resistance to them. This study utilized laboratory experimental evolution to evolve silver nanoparticle (AgNP) resistance in the bacterium Escherichia coli (K-12 MG1655), a bacterium that does not harbor any known silver resistance elements. After 225 generations of exposure to the AgNP environment, the treatment populations demonstrated greater fitness vs. control strains as measured by optical density (OD) and colony forming units (CFU) in the presence of varying concentrations of 10 nm citrate-coated silver nanoparticles (AgNP) or silver nitrate (AgNO3). Genomic analysis shows that changes associated with AgNP resistance were already accumulating within the treatment populations by generation 100, and by generation 200 three mutations had swept to high frequency in the AgNP resistance stocks. This study indicates that despite previous claims to the contrary bacteria can easily evolve resistance to AgNPs, and this occurs by relatively simple genomic changes. These results indicate that care should be taken with regards to the use of eNPs as biocides as well as with regards to unintentional exposure of microbial communities to eNPs in waste products

CRISPR-Cas Systems in Bacteroides fragilis, an Important Pathobiont in the Human Gut Microbiome

Background: While CRISPR-Cas systems have been identified in bacteria from a wide variety of ecological niches, there are no studies to describe CRISPR-Cas elements in Bacteroides species, the most prevalent anaerobic bacteria in the lower intestinal tract. Microbes of the genus Bacteroides make up ~25% of the total gut microbiome. Bacteroides fragilis comprises only 2% of the total Bacteroides in the gut, yet causes of >70% of Bacteroides infections. The factors causing it to transition from benign resident of the gut microbiome to virulent pathogen are not well understood, but a combination of horizontal gene transfer (HGT) of virulence genes and differential transcription of endogenous genes are clearly involved. The CRISPR-Cas system is a multi-functional system described in prokaryotes that may be involved in control both of HGT and of gene regulation.Results: Clustered regularly interspaced short palindromic repeats (CRISPR) elements in all strains of B. fragilis (n = 109) with publically available genomes were identified. Three different CRISPR-Cas types, corresponding most closely to Type IB, Type IIIB, and Type IIC, were identified. Thirty-five strains had two CRISPR-Cas types, and three strains included all three CRISPR-Cas types in their respective genomes. The cas1 gene in the Type IIIB system encoded a reverse-transcriptase/Cas1 fusion protein rarely found in prokaryotes. We identified a short CRISPR (3 DR) with no associated cas genes present in most of the isolates; these CRISPRs were found immediately upstream of a hipA/hipB operon and we speculate that this element may be involved in regulation of this operon related to formation of persister cells during antimicrobial exposure. Also, blood isolates of B. fragilis did not have Type IIC CRISPR-Cas systems and had atypical Type IIIB CRISPR-Cas systems that were lacking adjacent cas genes.Conclusions: This is the first systematic report of CRISPR-Cas systems in a wide range of B. fragilis strains from a variety of sources. There are four apparent CRISPR-Cas systems in B. fragilis—three systems have adjacent cas genes. Understanding CRISPR/Cas function in B. fragilis will elucidate their role in gene expression, DNA repair and ability to survive exposure to antibiotics. Also, based on their unique CRISPR-Cas arrays, their phylogenetic clustering and their virulence potential, we are proposing that blood isolates of B. fragilis be viewed a separate subgroup