Methicillin-resistant Staphylococcus aureus in Finland: recent changes in the epidemiology, long-term facility aspects, and phenotypic and molecular detection of isolates

Staphylococcus aureus is one of the most important bacteria that cause disease in humans, and methicillin-resistant S. aureus (MRSA) has become the most commonly identified antibiotic-resistant pathogen in many parts of the world. MRSA rates have been stable for many years in the Nordic countries and the Netherlands with a low MRSA prevalence in Europe, but in the recent decades, MRSA rates have increased in those low-prevalence countries as well. MRSA has been established as a major hospital pathogen, but has also been found increasingly in long-term facilities (LTF) and in communities of persons with no connections to the health-care setting. In Finland, the annual number of MRSA isolates reported to the National Infectious Disease Register (NIDR) has constantly increased, especially outside the Helsinki metropolitan area. Molecular typing has revealed numerous outbreak strains of MRSA, some of which have previously been associated with community acquisition. In this work, data on MRSA cases notified to the NIDR and on MRSA strain types identified with pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and staphylococcal cassette chromosome mec (SCCmec) typing at the National Reference Laboratory (NRL) in Finland from 1997 to 2004 were analyzed. An increasing trend in MRSA incidence in Finland from 1997 to 2004 was shown. In addition, non-multi-drug resistant (NMDR) MRSA isolates, especially those resistant only to methicillin/oxacillin, showed an emerging trend. The predominant MRSA strains changed over time and place, but two internationally spread epidemic strains of MRSA, FIN-16 and FIN-21, were related to the increase detected most recently. Those strains were also one cause of the strikingly increasing invasive MRSA findings. The rise of MRSA strains with SCCmec types IV or V, possible community-acquired MRSA was also detected. With questionnaires, the diagnostic methods used for MRSA identification in Finnish microbiology laboratories and the number of MRSA screening specimens studied were reviewed. Surveys, which focused on the MRSA situation in long-term facilities in 2001 and on the background information of MRSA-positive persons in 2001-2003, were also carried out. The rates of MRSA and screening practices varied widely across geographic regions. Part of the NMDR MRSA strains could remain undetected in some laboratories because of insufficient diagnostic techniques used. The increasing proportion of elderly population carrying MRSA suggests that MRSA is an emerging problem in Finnish long-term facilities. Among the patients, 50% of the specimens were taken on a clinical basis, 43% on a screening basis after exposure to MRSA, 3% on a screening basis because of hospital contact abroad, and 4% for other reasons. In response to an outbreak of MRSA possessing a new genotype that occurred in a health care ward and in an associated nursing home of a small municipality in Northern Finland in autumn 2003, a point-prevalence survey was performed six months later. In the same study, the molecular epidemiology of MRSA and methicillin-sensitive S. aureus (MSSA) strains were also assessed, the results to the national strain collection compared, and the difficulties of MRSA screening with low-level oxacillin-resistant isolates encountered. The original MRSA outbreak in LTF, which consisted of isolates possessing a nationally new PFGE profile (FIN-22) and internationally rare MLST type (ST-27), was confined. Another previously unrecognized MRSA strain was found with additional screening, possibly indicating that current routine MRSA screening methods may be insufficiently sensitive for strains possessing low-level oxacillin resistance. Most of the MSSA strains found were genotypically related to the epidemic MRSA strains, but only a few of them had received the SCCmec element, and all those strains possessed the new SCCmec type V. In the second largest nursing home in Finland, the colonization of S. aureus and MRSA, and the role of screening sites along with broth enrichment culture on the sensitivity to detect S. aureus were studied. Combining the use of enrichment broth and perineal swabbing, in addition to nostrils and skin lesions swabbing, may be an alternative for throat swabs in the nursing home setting, especially when residents are uncooperative. Finally, in order to evaluate adequate phenotypic and genotypic methods needed for reliable laboratory diagnostics of MRSA, oxacillin disk diffusion and MIC tests to the cefoxitin disk diffusion method at both +35°C and +30°C, both with or without an addition of sodium chloride (NaCl) to the Müller Hinton test medium, and in-house PCR to two commercial molecular methods (the GenoType® MRSA test and the EVIGENETM MRSA Detection test) with different bacterial species in addition to S. aureus were compared. The cefoxitin disk diffusion method was superior to that of oxacillin disk diffusion and to the MIC tests in predicting mecA-mediated resistance in S. aureus when incubating at +35°C with or without the addition of NaCl to the test medium. Both the Geno Type® MRSA and EVIGENETM MRSA Detection tests are usable, accurate, cost-effective, and sufficiently fast methods for rapid MRSA confirmation from a pure culture.

The community of medium-sized carnivores: the interactions between species, habitats and rabies

The composition of the carnivore community influences the different forms of inter-specific interactions. Furthermore, inter-specific interactions of carnivores have important implications for intra-guild competition, epidemiology and strategies of species-specific population management. Zoonooses, such as rabies, are diseases that can be transmitted from wildlife to people. Knowing the ecological characteristics of the species helps us to choose the right preventive actions and to time them accurately. In this thesis, I have studied how raccoon dogs Nyctereutes procyonoides, European badgers Meles meles, red foxes Vulpes vulpes and domestic cats Felis silvestris catus act as members of carnivore community, and how these interactions relate to the transmission risk of rabies. In the study area, these species form a community of medium-sized and rather generalist predators. They live in the same areas, in spatially and temporally overlapping home ranges and use the same habitats and dens and even have similar diets. However, there is no direct evidence of competition. Shared dens point to good tolerance of other species. Numerous observations of animals moving in each other’s proximity give similar clues. However, overlapping home ranges and similar habitat preferences lead to frequent inter-specific contacts, which increase the risk of possible rabies transmission. Also, the new insight of habitat use gained by this study illustrates the similar favouring of deciduous forests and fields by these sympatric medium-sized carnivores, creating a basis for contact zones, i.e. risky habitats for rabies transmission and spread. This study is so far the only simultaneous radio tracking study of raccoon dogs, badgers, foxes and cats. These results give new insight of the interactions in the carnivore community, as well as of the behaviour of each individual species. Also, these results have significant implications for the planning of rabies control. In order to reach viable management decisions, not only one or two species should be taken into consideration, but the whole community. In particular, this changes the perspective to inter-specific contacts, animal densities, densities of individuals susceptible to diseases and the magnitude of preventive actions. Rabies should be considered as a multi-vector disease, at least in Finland and the Baltic states. It is of interest for disease management to be able to model an epizootic with local parameters to reflect the real situation and also to suite best the local management needs.

Microbial diversity in the municipal composting process and development of detection methods

Composting refers to aerobic degradation of organic material and is one of the main waste treatment methods used in Finland for treating separated organic waste. The composting process allows converting organic waste to a humus-like end product which can be used to increase the organic matter in agricultural soils, in gardening, or in landscaping. Microbes play a key role as degraders during the composting-process, and the microbiology of composting has been studied for decades, but there are still open questions regarding the microbiota in industrial composting processes. It is known that with the traditional, culturing-based methods only a small fraction, below 1%, of the species in a sample is normally detected. In recent years an immense diversity of bacteria, fungi and archaea has been found to occupy many different environments. Therefore the methods of characterising microbes constantly need to be developed further. In this thesis the presence of fungi and bacteria in full-scale and pilot-scale composting processes was characterised with cloning and sequencing. Several clone libraries were constructed and altogether nearly 6000 clones were sequenced. The microbial communities detected in this study were found to differ from the compost microbes observed in previous research with cultivation based methods or with molecular methods from processes of smaller scale, although there were similarities as well. The bacterial diversity was high. Based on the non-parametric coverage estimations, the number of bacterial operational taxonomic units (OTU) in certain stages of composting was over 500. Sequences similar to Lactobacillus and Acetobacteria were frequently detected in the early stages of drum composting. In tunnel stages of composting the bacterial community comprised of Bacillus, Thermoactinomyces, Actinobacteria and Lactobacillus. The fungal diversity was found to be high and phylotypes similar to yeasts were abundantly found in the full-scale drum and tunnel processes. In addition to phylotypes similar to Candida, Pichia and Geotrichum moulds from genus Thermomyces and Penicillium were observed in tunnel stages of composting. Zygomycetes were detected in the pilot-scale composting processes and in the compost piles. In some of the samples there were a few abundant phylotypes present in the clone libraries that masked the rare ones. The rare phylotypes were of interest and a method for collecting them from clone libraries for sequencing was developed. With negative selection of the abundant phylotyps the rare ones were picked from the clone libraries. Thus 41% of the clones in the studied clone libraries were sequenced. Since microbes play a central role in composting and in many other biotechnological processes, rapid methods for characterization of microbial diversity would be of value, both scientifically and commercially. Current methods, however, lack sensitivity and specificity and are therefore under development. Microarrays have been used in microbial ecology for a decade to study the presence or absence of certain microbes of interest in a multiplex manner. The sequence database collected in this thesis was used as basis for probe design and microarray development. The enzyme assisted detection method, ligation-detection-reaction (LDR) based microarray, was adapted for species-level detection of microbes characteristic of each stage of the composting process. With the use of a specially designed control probe it was established that a species specific probe can detect target DNA representing as little as 0.04% of total DNA in a sample. The developed microarray can be used to monitor composting processes or the hygienisation of the compost end product. A large compost microbe sequence dataset was collected and analysed in this thesis. The results provide valuable information on microbial community composition during industrial scale composting processes. The microarray method was developed based on the sequence database collected in this study. The method can be utilised in following the fate of interesting microbes during composting process in an extremely sensitive and specific manner. The platform for the microarray is universal and the method can easily be adapted for studying microbes from environments other than compost.

Hard Custom, Hard Dance : Social Organisation, (Un)Differentiation and Notions of Power in a Tabiteuean Community, Southern Kiribati

Hard Custom, Hard Dance: Social Organisation, (Un)Differentiation and Notions of Power in a Tabiteuean Community, Southern Kiribati is an ethnographic study of a village community. This work analyses social organisation on the island of Tabiteuea in the Micronesian state of Kiribati, examining the intertwining of hierarchical and egalitarian traits, meanwhile bringing a new perspective to scholarly discussions of social differentiation by introducing the concept of undifferentiation to describe non-hierarchical social forms and practices. Particular attention is paid to local ideas concerning symbolic power, abstractly understood as the potency for social reproduction, but also examined in one of its forms; authority understood as the right to speak. The workings of social differentiation and undifferentiation in the village are specifically studied in two contexts connected by local notions of power: the meetinghouse institution (te maneaba) and traditional dancing (te mwaie). This dissertation is based on 11 months of anthropological fieldwork in 1999‒2000 in Kiribati and Fiji, with an emphasis on participant observation and the collection of oral tradition (narratives and songs). The questions are approached through three distinct but interrelated topics: (i) A key narrative of the community ‒ the story of an ancestor without descendants ‒ is presented and discussed, along with other narratives. (ii) The Kiribati meetinghouse institution, te maneaba, is considered in terms of oral tradition as well as present-day practices and customs. (iii) Kiribati dancing (te mwaie) is examined through a discussion of competing dance groups, followed by an extended case study of four dance events. In the course of this work the community of close to four hundred inhabitants is depicted as constructed primarily of clans and households, but also of churches, work co-operatives and dance groups, but also as a significant and valued social unit in itself, and a part of the wider island district. In these partly cross-cutting and overlapping social matrices, people are alternatingly organised by the distinct values and logic of differentiation and undifferentiation. At different levels of social integration and in different modes of social and discursive practice, there are heightened moments of differentiation, followed by active undifferentiation. The central notions concerning power and authority to emerge are, firstly, that in order to be valued and utilised, power needs to be controlled. Secondly, power is not allowed to centralize in the hands of one person or group for any long period of time. Thirdly, out of the permanent reach of people, power/authority is always, on the one hand, left outside the factual community and, on the other, vested in community, the social whole. Several forms of differentiation and undifferentiation emerge, but these appear to be systematically related. Social differentiation building on typically Austronesian complementary differences (such as male:female, elder:younger, autochtonous:allotochtonous) is valued, even if eventually restricted, whereas differentiation based on non-complementary differences (such as monetary wealth or level of education) is generally resisted, and/or is subsumed by the complementary distinctions. The concomitant forms of undifferentiation are likewise hierarchically organised. On the level of the society as a whole, undifferentiation means circumscribing and ultimately withholding social hierarchy. Potential hierarchy is both based on a combination of valued complementary differences between social groups and individuals, but also limited by virtue of the undoing of these differences; for example, in the dissolution of seniority (elder-younger) and gender (male-female) into sameness. Like the suspension of hierarchy, undifferentiation as transformation requires the recognition of pre-existing difference and does not mean devaluing the difference. This form of undifferentiation is ultimately encompassed by the first one, as the processes of the differentiation, whether transformed or not, are always halted. Finally, undifferentiation can mean the prevention of non-complementary differences between social groups or individuals. This form of undifferentiation, like the differentiation it works on, takes place on a lower level of societal ideology, as both the differences and their prevention are always encompassed by the complementary differences and their undoing. It is concluded that Southern Kiribati society be seen as a combination of a severely limited and decentralised hierarchy (differentiation) and of a tightly conditional and contextual (intra-category) equality (undifferentiation), and that it is distinctly characterised by an enduring tension between these contradicting social forms and cultural notions. With reference to the local notion of hardness used to characterise custom on this particular island as well as dance in general, it is argued in this work that in this Tabiteuean community some forms of differentiation are valued though strictly delimited or even undone, whereas other forms of differentiation are a perceived as a threat to community, necessitating pre-emptive imposition of undifferentiation. Power, though sought after and displayed - particularly in dancing - must always remain controlled.

Microbial identification by detection of ligation probes on DNA microarray

Microbes in natural and artificial environments as well as in the human body are a key part of the functional properties of these complex systems. The presence or absence of certain microbial taxa is a correlate of functional status like risk of disease or course of metabolic processes of a microbial community. As microbes are highly diverse and mostly notcultivable, molecular markers like gene sequences are a potential basis for detection and identification of key types. The goal of this thesis was to study molecular methods for identification of microbial DNA in order to develop a tool for analysis of environmental and clinical DNA samples. Particular emphasis was placed on specificity of detection which is a major challenge when analyzing complex microbial communities. The approach taken in this study was the application and optimization of enzymatic ligation of DNA probes coupled with microarray read-out for high-throughput microbial profiling. The results show that fungal phylotypes and human papillomavirus genotypes could be accurately identified from pools of PCR amplicons generated from purified sample DNA. Approximately 1 ng/μl of sample DNA was needed for representative PCR amplification as measured by comparisons between clone sequencing and microarray. A minimum of 0,25 amol/μl of PCR amplicons was detectable from amongst 5 ng/μl of background DNA, suggesting that the detection limit of the test comprising of ligation reaction followed by microarray read-out was approximately 0,04%. Detection from sample DNA directly was shown to be feasible with probes forming a circular molecule upon ligation followed by PCR amplification of the probe. In this approach, the minimum detectable relative amount of target genome was found to be 1% of all genomes in the sample as estimated from 454 deep sequencing results. Signal-to-noise of contact printed microarrays could be improved by using an internal microarray hybridization control oligonucleotide probe together with a computational algorithm. The algorithm was based on identification of a bias in the microarray data and correction of the bias as shown by simulated and real data. The results further suggest semiquantitative detection to be possible by ligation detection, allowing estimation of target abundance in a sample. However, in practise, comprehensive sequence information of full length rRNA genes is needed to support probe design with complex samples. This study shows that DNA microarray has the potential for an accurate microbial diagnostic platform to take advantage of increasing sequence data and to replace traditional, less efficient methods that still dominate routine testing in laboratories. The data suggests that ligation reaction based microarray assay can be optimized to a degree that allows good signal-tonoise and semiquantitative detection.