Year: 2010
Type: dissertation
Abstract: The evolutionary success of bacteria depends on genetic variability. This variability may be the result of beneficial mutations in the genome or from the uptake of genetic elements that increase viability under stress conditions. Bacteria are repeatedly exposed to agents such as antibiotics and host immune responses, which may induce in bacteria a variety of survival strategies, including mutagenic mechanisms. The development of antibiotic resistance is one of the serious consequences of these mechanisms. Bacteria increase genetic variability and survive DNA damage by using horizontal gene transfer, competence and DNA repair mechanisms such as the SOS response, homologous recombination and mismatch repair. Besides genetic rearrangements, bacteria have other mechanisms to persist in hostile environments. Biofilms are sessile, often polysaccharide-encased bacterial growth forms that are particularly tolerant to mechanical shearing, antibiotics and host immune responses. Bovine udder infection, or mastitis, is an inflammation of the mammary gland caused by bacteria and it is usually recognized by clinical signs including abnormalities in the milk and the udder. Mastitis is the most common disease in the dairy industry and account for a significant proportion of the antibiotic treatments in dairy cows. Streptococcus uberis is a Gram-positive pathogenic bacterium and a member of the pyogenic group of the genus Streptococcus. S. uberis mainly infects the udder from environmental sources and is one of the most common causative agents of bovine mastitis. However, the molecular biology of S. uberis has been relatively unknown. The members of the Streptococcaceae family have been considered to lack the classical SOS response. In the first part of this research, the stress tolerance and mutagenic mechanisms of S. uberis were characterized. In study I, a novel self-regulated SOS -response gene cassette was identified and the expression of this cassette was induced by UV -light as well as the antibiotic ciprofloxacin. S. uberis was shown to perform mutagenic DNA -repair after UV -exposure and the repair was mediated by error-prone polymerase UmuC coded by the SOS -response gene cassette. This mutagenesis was shown to promote the development of antibiotic resistance and, according to the database searches, the homologs of this SOS gene cassette are found in several other streptococcal species. In study II, exposure to the fluoroquinolone antibiotic ciprofloxacin was also shown to induce antibiotic resistance-promoting mutations in S. uberis, but the mutagenesis was not UmuC-mediated. Unlike the model organism of the SOS response, Escherichia coli, S. uberis has distinct mechanisms for UV- and ciprofloxacin-induced mutagenesis. In study III, the effects of a sublethal concentration of ciprofloxacin on S. uberis were studied by proteomics. As a result of ciprofloxacin stress, S. uberis differentially expressed 20 proteins. The proteins were identified by mass spectrometry as enzymes involved in oxidative stress tolerance, NADH generation and nucleotide biosynthesis. The results suggest that ciprofloxacin exposure causes oxidative damage in S. uberis. The changes in enzymes involved in nucleotide balance suggest that nucleotide biosynthesis is a mechanism to stimulate mutagenesis leading to the development of antibiotic resistance. In the final part of the work, the ability of S. uberis to form biofilms was characterized and the biofilm formation of clinical and subclinical S. uberis isolates was investigated. The strains differed in their ability to form biofilms, ranging from low-producing strains to strains producing thick, multi-layered biofilms. To determine whether biofilm production by S. uberis is an inducible event, the effect of proteins from the host was tested. Milk, the natural growth medium of mastitis bacteria, induced biofilm formation in most strains, even at low concentrations. Further analyses indicated that the milk components casein and especially α- and β-casein are the primary inducing agents of biofilm production. The proteolytic activity of S. uberis is involved in this induction process, possibly by releasing peptides from caseins. Research on stress inducible systems provides us with information on how bacteria evolve, develop antibiotic resistance and escape the host immune system. The effects of antibiotic usage on bacteria are one of the most important questions in modern medicine, and knowledge of these effects helps in evaluating the necessity of antibiotic therapy. The biofilm formation of S. uberis provides a possible explanation for the ability of S. uberis to cause persistant mastitis, independent of antibiotic treatment.
Source: Työväentutkimus Vuosikirja
Author: Varhimo, Emilia
Language: English
Subfield: Genetics
Domain: Life Sciences
Open Access status: green
Funders: Helsingin Yliopisto, Åbo Akademi
OpenAlex ID: W1487722888