Bacterial resistance to antibiotics has increased rapidly within recent years, and it has become a serious threat to public health. Infections caused by multi-drug resistant bacteria entail higher morbidity, mortality, and a burden to health care systems. The use of biocides, including silver compounds, may affect the resistance to both biocides and antibiotics and, thereby, can be a driving factor in this development. The aim of the following thesis was to investigate the frequency of silver resistance and the effects of silver exposure on bacterial populations being of clinical significance and from geographically different parts of the world. Furthermore, it explored the genetic background of silver resistance, and if silver could select directly or indirectly for antibiotic resistance. By a range of methods, from culture in broth to whole genome sequencing, bacterial populations from humans, birds and from the environment were characterized. The studies showed that sil genes, encoding silver resistance, occurred at a high frequency. Sil genes were found in 48 % of Enterobacter spp., in 41 % of Klebsiella spp. and in 21 % of all human Escherichia coli isolates with production of certain types of extended-spectrum beta-lactamases (CTX-M-14 and CTX-M-15). In contrast, silver resistance was not found in bird isolates or in bacterial species, such as Pseudomonas aeruginosa and Legionella spp., with wet environments as their natural habitat. One silver-resistant Enterobacter cloacae strain was isolated from a chronic leg ulcer after only three weeks of treatment with silver-based dressings. The in-vivo effects of these dressings were limited, and they failed to eradicate both Gram-positive and Gram-negative bacteria. The activity of silver nitrate in vitro was bacteriostatic on Gram-positive species such as S. aureus and bactericidal on Gram-negative species. In Enterobacteriaceae, sil genes were associated with silver resistance phenotypes in all but one case. Using whole genome sequencing, single nucleotide polymorphisms in the silS gene were discovered after silver exposure in isolates with expressed silver resistance. This resistance could co-select for resistance to beta-lactams, co-trimoxazole and gentamicin. The findings of this thesis indicate that silver exposure may cause phenotypic silver resistance, and it may reduce the susceptibility to mainly beta-lactams and select for bacteria with resistance to clinically important antibiotics.
Bacterial Responses to Silver Nanoparticle Treatment: Community Structure, Resistance, and Function.Gwin, Carley Ann January 2016 (has links)
<p>The antimicrobial properties of silver have been taken advantage of by societies for thousands of years. Its use has come back in favor in the form of silver nanoparticles, which are highly efficacious antimicrobial agents. Silver nanoparticles are incorporated into a myriad of products specifically designed for clinical use, but also for general use by consumers. Silver nanoparticles can be found in textiles such as clothing and stuffed toys, and in home appliances including washing machines and curling irons. A large number of products specifically marketed for use by children are also available to consumers, including pacifiers, sippy cups, and even breast milk storage bags. The hazards and toxicities associated with silver nanoparticles are not well understood, however modes of toxicity have been reported for ionic silver. It is assumed that the main mechanism of toxicity of silver nanoparticles relates to the release of ionic silver, however studies have indicated an additional nano-effect, likely due to nanoparticle size, differential coatings, and means of sustained dosing of ionic silver. However we are sure that these silver nanoparticles will accumulate in the waste stream, likely arriving during different stages of a product’s lifespan. A main sink of these nanoparticles travelling through both natural and engineered environments is wastewater treatment plants. As a society we rely on the biological removal of nutrients, which takes place primarily in the activated sludge of secondary treatment. Studies have already indicated possible, temporary decreases in removal efficiencies as well as changes in microbial communities, including losses of diversity, following exposure to silver nanoparticles. Therefore, it is of paramount importance to examine the effects of both silver nanoparticles and ionic silver on the community and function of wastewater bacteria. </p><p> Sequencing batch reactors were operated to mimic wastewater treatment. They were fed synthetic wastewater and after reaching acclimation, were dosed over time with varying concentrations of both ionic and nanosilver. Cell samples were collected periodically to assess the presence and identity of cultivable silver resistant bacteria and to map the microbial community changes taking place under different treatments using Next Generation Sequencing. Isolates were tested for the presence of known silver resistance (sil) genes as were activated sludge samples from a collection of domestic wastewater treatment plants, by designing TaqMan probe assays and performing quantitative PCR. The silver resistant isolates were also used to test the growth implications, as well as sil gene expression changes, following treatment with ionic silver and a variety of silver nanoparticles with various coatings, all at multiple concentrations. This was accomplished by performing multiple batch experiments and then using the TaqMan assays and reverse transcription-quantitative PCR.</p><p>Overall, microbial community changes were observed in the sequencing batch reactors, and there were differences noted based on treatment, including ionic silver versus nanosilver and between the two silver nanoparticle coatings. Most notably, the possibility of nitrification in wastewater treatment being particularly susceptible was strongly indicated. Individual wastewater bacteria isolates all contained sil genes, as did the majority of the wastewater treatment plant activated sludge, however the levels of actual sil gene expression were inconsistent. This particular finding supports a current body of work indicating that there are alternate modes of bacterial silver resistance in play that we are just becoming aware of.</p> / Dissertation
In the first study, three different transport systems for bacteria were evaluated. The CLSI M40-A guideline was used to monitor the maintenance of both mono- and polymicrobial samples during a simulated transportation at room temperature that lasted 0-48 h. All systems were able to maintain the viability of all organisms for 24 h, but none of them could support all tested species after 48 h. The most difficult species to recover was Neisseria gonorrhoeae, and in polymicrobial samples overgrowth was an observed problem. The aim of the second study was to study the presence of TSST-1 and three other important toxin genes in invasive isolates of Staphylococcus aureus collected during the years 2000-2012 at two tertiary hospitals. The genes encoding the staphylococcal toxins were detected by PCR, and whole-genome sequencing was used for analyzing the genetic relatedness between isolates. The results showed that the most common toxin was TSST-1, and isolates positive for this toxin exhibited a clear clonality independent of year and hospital. The typical patient was a male aged 55-74 years and with a bone or a joint infection. The third study was a clinical study of the effect of silver-based wound dressings on the bacterial flora in chronic leg ulcers. Phenotypic and genetic silver-resistance were investigated before and after topical silver treatment, by determining the silver nitrate MICs and by detecting sil genes with PCR. The silver-based dressings had a limited effect on primary wound pathogens, and the activity of silver nitrate on S. aureus was mainly bacteriostatic. A silver-resistant Enterobacter cloacae strain was identified after only three weeks of treatment, and cephalosporin-resistant members of the Enterobacteriaceae family were relatively prone to developed silver-resistance after silver exposure in vitro. The last study was undertaken in order to develop an easy-to-use method for simulating the laundering process of hospital textiles, and apply the method when evaluating the decontaminating efficacy of two different washing temperatures. The laundering process took place at professional laundries, and Enterococcus faecium was used as a bioindicator. The results showed that a lowering of the washing temperature from 70°C to 60°C did not affect the decontamination efficacy; the washing cycle alone reduced the number of bacteria with 3-5 log10 CFU, whereas the following tumble drying reduced the bacterial numbers with another 3-4 log10 CFU, yielding the same final result independent of the washing temperature. To ensure that sufficient textile hygiene is maintained, the whole laundering process needs to be monitored. The general conclusion is that all developmental work in the bacterial field requires time and a large strain collection.
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