The thesis describes the development of a number of novel mass spectrometric methods for the protein analysis of Gram-negative bacteria. These applications are developed with the aim of finding new... Show moreThe thesis describes the development of a number of novel mass spectrometric methods for the protein analysis of Gram-negative bacteria. These applications are developed with the aim of finding new and improved diagnostic routes for the typing of bacteria and their antibiotic resistance. The research is application driven and the focus is on utilizing high-end mass spectrometric instrumentation in diagnostic clinical microbiology, in a complimentary nature to already established techniques. Show less
H2: Hensgens MP, Goorhuis A, Notermans DW, van Benthem BH, Kuijper EJ. Decrease of hypervirulent Clostridium difficile PCR ribotype 027 in the Netherlands. Euro Surveill. 2009 H3: Hensgens MP,... Show moreH2: Hensgens MP, Goorhuis A, Notermans DW, van Benthem BH, Kuijper EJ. Decrease of hypervirulent Clostridium difficile PCR ribotype 027 in the Netherlands. Euro Surveill. 2009 H3: Hensgens MP, Keessen EC, Squire M, Riley TV, Koene MG, de Boer E, Lipman LJ, Kuijper EJ. Clostridium difficile infection in the community: a zoonotic disease? Clin Microbiol Infect. 2012 H4: Hensgens MP / Goorhuis A, van Kinschot CM, Crobach MJ, Harmanus C, Kuijper EJ. Clostridium difficile infection in an endemic setting in the Netherlands. Eur J Clin Microbiol Infect Dis. 2011 H5: Hensgens MP, Goorhuis A, Dekkers OM, Kuijper EJ. Time-interval of increased risk for Clostridium difficile infection after exposure to antibiotics. J Antimicrob Chemother. 2012 H7: Hensgens MP, Goorhuis A, Dekkers OM, van Benthem BH, Kuijper EJ. Outcome of nosocomial Clostridium difficile infections; results of a multicenter cohort study. Clin Infect Dis. 2013 H8: Hensgens MP / Bauer MP, Miller M, Gerding DN, Wilcox MH, Dale AP, Fawley WN, Kuijper EJ, Gorbach SL. Renal failure and leukocytosis are predictors of a complicated course of Clostridium difficile infection (CDI) if measured on day of diagnosis. Clin Infect Dis. 2012 H9: Hensgens MP, Kuijper EJ. Clostridium difficile infection due to binary toxin positive strains. Emerg Infect Dis. 2013 H10: Hensgens MP, Dekkers OM, Goorhuis A, Le Cessie S, Kuijper EJ. Predicting a severe course of Clostridium difficile infection at the bedside. Clin Microbiol Infect. 2012 Show less
Potato blackleg caused by pectinolytic Pectobacterium and Dickeya species is a bacterial disease creating serious economic losses in (seed)potato production worldwide. Effective management to... Show morePotato blackleg caused by pectinolytic Pectobacterium and Dickeya species is a bacterial disease creating serious economic losses in (seed)potato production worldwide. Effective management to control blackleg is absent and validated, cost-effective detection protocols for blackleg bacteria do not exist. This situation results partially from the lack of knowledge of the ecology of blackleg pathogens in general, and Dickeya spp. in particular. Since 2000 an increasing frequency of Dickeya spp. in seed potatoes in Europe has been observed which is connected to emergence of a new species belonging to biovar 3 but unclassified inside known and described six Dickeya species. This species was provisionally named __D. solani__ and isolated from potato in The Netherlands, Finland, Poland, Germany, Belgium, France, United Kingdom and Israel. The objectives of the study described in this thesis were to acquire knowledge on the ecology (i. e. distribution of Dickeya spp. in seed potato tubers, role of the soil-borne inoculum in disease incidence, role of the aerial haulm infection for the blackleg incidence in progeny tubers) of a new genetic clade of biovar 3 Dickeya spp.; and to find and characterize in detail the valuable biocontrol agent(s) (isolated from rotten potato tissue) to cure seed potato tubers from blackleg caused by biovar 3 Dickeya spp. (__D. solani__). Direct isolation of viable bacterial cells using (selective) plating techniques combined with molecular (16S rDNA and Rep-PCR), serological (DAS-ELISA) and biochemical (biovar determination) characterization of the isolates were used to evaluate the presence of Dickeya and Pectobacterium spp. in different plant parts and tissues. Bacterial population dynamics in planta was studied in greenhouse experiments. For the studies on colonization of potato tissues by biovar 3 Dickeya spp. (__D. solani__) and Serratia plymuthica A30, bacterial strains were transformed with plasmid-based genes coding for red or green fluorescent proteins (DsRed and GFP, respectively) that were constitutively expressed in bacterial cells. Epifluorescence stereo microscopy and confocal laser scanning microscopy were used to visualize bacterial presence in different tissues of potato tubers and plants. Studies on the distribution of Dickeya ( and __D. solani__) and Pectobacterium species in seed potatoes revealed that the pathogens were distributed unevenly inside tubers and that the stolon end tuber part harbored the highest bacterial inoculum, whereas deeper located tissues were usually not colonized. Studies on the importance of soil-borne biovar 3 Dickeya sp. (__D. solani__) inoculum for systemic colonization of potato plants and latent infection of progeny tubers unveiled that D. solani is able to systemically colonize progeny tubers from soil via the roots and to cause true blackleg symptoms in infected plants after soil infestation. Studies on the role of haulm infections for latent contamination of progeny tubers by D. solani presented that the injection of bacteria into stems can result in the downward vascular translocation and a colonization of underground plant parts and finally, latent contamination of progeny tubers. Potato leaves inoculation with a biovar 3 Dickeya sp., showed degradation of the inoculated plant material and spreading of the internal inoculum to the petiole and axil and finally to the main stem but bacteria were not detected in the underground plant parts. Studies on characterization of the bacteria antagonistic to biovar 3 Dickeya sp. (__D. solani__) allowed to obtain knowledge of the presence of such strains in rotten potato tissue and on possibility of their used in the biocontrol of Dickeya spp. in potato. Selection of a candidate strain (Serratia plymuthica A30) that was effective in protecting potato plant tissue from blackleg was performed on the basis of in vitro and greenhouse tests. In vitro tests proved that S. plymuthica A30 possess different mechanisms by which it can control Dickeya spp. (i. e. production of antibiotics and biosurfactants). The interaction of D. solani and S. plymuthica A30 under greenhouse conditions was investigated in detail using artificially co-inoculated tubers and by application of A30 to the soil. It was showed that both micro-organisms interact in situ and that S. plymuthica A30 is able to decrease or eradicate Dickeya sp. inoculum from infected potato plants by competition un the same niches. In conclusion, a new biovar 3 Dickeya spp. strain was present in high densities in stolon end of naturally infected progeny tubers. The bacterium efficiently colonized various plant tissues after artificial inoculation of soil or haulms and was able to systemically colonize entire potato plant in relatively short time. For biocontrol of biovar 3 Dickeya spp. in potato, a bacterial strain characterized as S. plymuthica A30 tested in in vitro and under greenhouse conditions proved to have great potential for controlling blackleg in conditions favorable for disease development and when Dickeya sp. was applied in the high inoculum. The A30 strain effectively protected potato plants against blackleg in greenhouse experiments, both in vacuum infiltrated tuber and when applied as superficial treatment during planting. Treatment with A30 reduced the disease incidence to 0%. S. plymuthica A30 was able to colonize the plants internally, and combat internal infections with D. solani. Show less
The study of bacterial-fungal interactions is essential to obtain a better understanding of terrestrial microbial ecology and may lie at the basis of novel applications in agriculture, food... Show moreThe study of bacterial-fungal interactions is essential to obtain a better understanding of terrestrial microbial ecology and may lie at the basis of novel applications in agriculture, food industry and human health. Nevertheless, the incentives, the genetic determinants and the mechanisms that underlie bacterial-fungal interactions are still poorly understood. Bacterial mycophagy is a trophic behaviour that takes place when bacteria obtain nutrients from living fungal hyphae, allowing the conversion of living fungal biomass into bacterial biomass (29). This trophic behavior was demonstrated for the first time for bacteria of the genus Collimonas, based on their ability to grow at the expenses of living fungal hyphae in a soil-like microcosm (28, 30). In this thesis I addressed the following research questions: (1) Which of the mechanisms putatively involved in Collimonas mycophagy are actually activated when Collimonas interact with a fungus (2) What is the fungal response to the presence of Collimonas bacteria? (3) What is the role played by plasmid pTer331, detected in the genome of the mycophagous bacterium C. fungivorans Ter331, in the ecology of this bacterium? Are the genes encoded on plasmid pTer331 involved in mycophagy? (4) Are the putative determinants of mycophagy uniformly distributed among Collimonas species? Show less
