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Selection of Resistance at very low Antibiotic ConcentrationsGullberg, Erik January 2014 (has links)
The extensive medical and agricultural use and misuse of antibiotics during the last 70 years has caused an enrichment of resistant pathogenic bacteria that now severely threatens our capacity to efficiently treat bacterial infections. While is has been known for a long time that high concentrations of antibiotics can select for resistant mutants, less is known about the lower limit at which antibiotics can be selective and enrich for resistant bacteria. In this thesis we investigated the role of low concentrations of antibiotics and heavy metals in the enrichment and evolution of antibiotic resistance. Selection was studied using Escherichia coli and Salmonella enterica serovar Typhimurium LT2 with different resistance mutations, different chromosomal resistance genes as well as large conjugative multidrug resistance plasmids. Using very sensitive competition experiments, we showed that antibiotic and heavy metal levels more than several hundred-fold below the minimal inhibitory concentration of susceptible bacteria can enrich for resistant bacteria. Additionally, we demonstrated that subinhibitory levels of antibiotics can select for de novo resistant mutants, and that these conditions can select for a new spectrum of low-cost resistance mutations. The combinatorial effects of antibiotics and heavy metals can cause an enrichment of a multidrug resistance plasmid, even if the concentration of each compound individually is not high enough to cause selection. These results indicate that environments contaminated with low levels of antibiotics and heavy metals such as, for example, sewage water or soil fertilized with sludge or manure, could provide a setting for selection, enrichment and transfer of antibiotic resistance genes. This selection could be a critical step in the transfer of resistance genes from environmental bacteria to human pathogens.
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Trade-offs in CRISPR Immunity against Mobile Genetic ElementsCederblad, Johanna January 2022 (has links)
The prokaryotic adaptive immune system CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a defense mechanism that helps to protect the prokaryotic cell from invading mobile genetic elements. This project was performed at Uppsala University and served to answer whether the expression of Cascade, which is part of the CRISPR defense system, will have a negative effect on the cell that expresses it and to also determine whether the CRISPR defense system is effective enough to stop the spread of a conjugative plasmid. A microfluidic system was used in order to perform the experiments and images were taken with the help of fluorescent microscopy. Three different donor strains from E.coli were used. These strains had their own version of the RP4 conjugative plasmid which had the ability to infect recipient E.coli cells with said plasmid. The recipient cells had the ability to express the CRISPR system in order to defend themselves from the plasmid and CRISPR was also inducible with the help of IPTG. The different versions of the RP4 conjugative plasmid had different amounts of spacer targets that Cascade, the recognition complex in the CRISPR system, could recognize. When the recipient cells were induced and had a known target sequence of the plasmid they were able to defend themselves and keep the number of transconjugant cells low. When the recipient cells did not know the target the amount of transconjugant cells were higher. It was also noted that when the cells were induced inside the microfluidic PDMS chip they had a slower generation time. It was also noted that recipient cells had begun to die towards the end of the microfluidic experiments when the cells were induced. This raised the question as to whether the CRISPR defense system was targeting itself as well as the RP4 conjugative plasmid.
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The Processing of Replication Initiation Protein PrgW in Enterococcus faecalis is Necessary for Activity and Stable Maintenance of pCF10Massie-Schuh, Ella January 2013 (has links)
Enterococcus faecalis are Gram-positive bacteria that colonize the gastrointestinal tracts of mammals, birds and invertebrates and are also found in sewage, soil, food and water. In addition to being commensal organisms, Enterococci can also cause nosocomial infections in humans including urinary tract infections, septicemia and endocarditis. Hospital-acquired infections often present a challenge in treatment due to the emergence of multi-drug resistant strains. Enterococcal plasmids may act as extremely stable reservoirs for resistance genes and other virulence factors. Pheromone responsive plasmids such as pCF10 mediate efficient transfer of genetic material within the species E. faecalis but may also be capable of transferring resistance genes across species and genus boundaries. Polymicrobial environments often found in nosocomial infections may expose plasmid-harboring enterococci to pathogenic species, poising cells for this type of promiscuous horizontal gene transfer of resistance determinants. Previous studies showed that prgW, which encodes the pCF10 replication initiation protein PrgW, is the minimal origin of replication for this plasmid. The replicon, which is usually limited to Enterococcal spp., can replicate in Lactococcus lactis if it is engineered to produce pre-cCF10. Three conserved cysteines (C78/C275/C307) are important for plasmid stability and allow for replication of the pCF10 replicon in L. lactis in the absence of pre-cCF10. PrgW has a predicted molecular weight of 38,635. Four polyclonal antibodies targeting PrgW at the N-terminus (aa 1-20), C-terminus (aa 314-333) and two internal regions (aa 64-80 and aa 250-271) were used in current experiments and retrospective studies. When PrgW was overexpressed in E. faecalis, four different apparent approximate molecular weights were detected by Western blotting (p40*, p36*, p24* and p18*), suggestive of processing. In Enterococci where the replicon is active, p36* was consistently detected by all four antisera; when PrgW was overexpressed in Streptococcus mutans where the replicon is non-functional, p49* and p40* were detected but p36* was not observed. PrgW p24* was detected by a mixture of the internally targeting antibodies as well as the C-terminal targeting antibody, but not the N-terminal targeting antibody, suggesting that the N-terminal domain of PrgW has been cleaved off in p24*. The p24* form may play a role in pCF10 stability. Mutations to three cysteines in PrgW (C78/C275/C307), which reduce the stability of pCF10, result in the loss of p24*. Enterococcal conjugative plasmids have been previously implicated in the transfer of antibiotic resistance genes. The pCF10 plasmid contains the conjugative transposon Tn925, which possesses the tetM tetracycline resistance gene. Proximity of donor and recipient cells is a key part of pheromone-responsive conjugation. Aggregation substance allows for formation of clumps of E. faecalis in liquid mating experiments. E. faecalis forms biofilms; in contrast to filter mating experiments, polymicrobial biofilms provide an in vitro model of a natural scenario during which horizontal gene transfer may occur. Rates of cross-genus genetic transfer of tetM between E. faecalis OG1RF(pCF10) donor cells and Staphylococcus aureus recipient cells growing on glass coverslips as mixed-species biofilm populations were determined to be 10-8 after pheromone induction of pCF10 conjugation. This biofilm transfer model also holds potential to test the efficacy of synthetic peptides in the reduction or even prevention of pCF10 transfer, and the consequential dissemination of antibiotic resistance determinants throughout the genus Enterococcus and beyond. / Microbiology and Immunology
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