| 1. |
- Christerson, Linus, 1985-
(författare)
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High Resolution Genotyping of Chlamydia trachomatis
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Chlamydia trachomatis is an obligate intracellular bacterium of major human health concern, causing urogential chlamydia infections, lymphogranuloma venereum (LGV) and trachoma. Chlamydia is one of the most common sexually transmitted infections worldwide and can cause infertility. In the first four papers described herein we used a high resolution multilocus sequence typing (MLST) system to investigate the epidemiology of C. trachomatis, and showed that MLST is superior to conventional ompA genotyping with respect to resolution. In the fifth paper we simplified the methodology by developing and validating a multilocus typing (MLT) DNA microarray based on the MLST system. In more detail, MLST analysis of consecutive specimens from 2006 in Örebro County in Sweden, and comparison to specimens from 1999-2000, showed that the new variant C. trachomatis (nvCT) is monoclonal and likely has appeared in recent years. MLST analysis of LGV specimens from men who have sex with men (MSM) showed that the increase of LGV in Europe in the last decade indeed was a clonal outbreak, contrary to the USA where LGV might have been present all along. In the third paper, clinical symptoms could not be correlated with the MLST genotypes, suggesting, together with the combined results of all previous studies, that bacterial factors, if important, need to be understood in the context of host factors. MLST analysis of specimens from a high incidence C. trachomatis area in North Norway revealed interesting epidemiological details concerning unusual genetic variants, the nvCT and MSM, but found no significant difference in genetic diversity compared to two other geographic areas in Norway. Lastly, we developed a MLT array that provides high resolution while being rapid and cost-effective, which makes it an interesting alternative for C. trachomatis genotyping. In conclusion, the MLST system and the MLT array have proven to be useful tools and should now be applied in further investigations to improve our understanding of C. trachomatis epidemiology.
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| 2. |
- Bonnedahl, Jonas, 1970-
(författare)
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Antibiotic Resistance in Enterobacteriaceae Isolated from Wild Birds
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The presence and spread of clinically important antibiotic-resistant bacteria in reservoirs from natural environments are not well studied compared to the clinical environments. The overall aim of this project was to study the presence of clinically important antibiotic-resistant bacteria in a reservoir from natural environments. Wild birds were chosen not only as indicators of the level of antibiotic resistance in surrounding natural bacterial populations, but also since birds can act as vectors of several potential pathogens including enteropathogens and because they by migration have an ability to spread these pathogens across geographical regions. The studies in this thesis showed that wild birds carry antibiotic-resistant enterobacteriaceae. The levels and spectrum of antibiotic resistance varies between different bird populations and geographical regions. In bird populations without interaction with human activities throughout the year, antibiotic resistance is lacking. Antibiotic-resistant bacteria could however probably be dispersed to remote regions by bird migration. Extended-spectrum beta-lactamases (ESBLs) and especially CTX-M types are found in comparable high levels in gull populations considering the recent emergence of these resistance genes in clinical settings. The CTX-M types found in wild birds are the same types that are found in clinical settings and in food producing animals from the same regions. ESBL-producing E. coli isolated from Yellow-legged Gulls are genetically heterogenous, reflecting that these resistance genes are present across the full E. coli genetic diversity. In wild birds CTX-M are found both in E. coli strains with previously known “human signature” as well as “novel” strains. This indicates that these genes are indeed very mobile and rapidly dispersing both through horizontal gene transfer and through successful clones. The findings in this thesis indicate that bird colonies could act as melting pots and reservoirs for new resistance types and that wild birds could act as important indicators of the level of antibiotic resistance dispersal in natural environments.
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| 3. |
- Elfving, Karin, 1982-
(författare)
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Epidemiological and Bacteriological Aspects of Spotted Fever Rickettsioses in Humans, Vectors and Mammals in Sweden
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Rickettsiae are obligate intracellular gram-negative bacteria transmitted by arthropod vectors. Rickettsiae sometimes cause disease in humans, typically with high fever, headache and occasionally an eschar.In Sweden, Rickettsia helvetica, belonging to the spotted fever group, is the only tick-transmitted rickettsia found free in nature. The pathogenic roll of R. helvetica has not been fully investigated, but it has been implicated in aneruptive fever and cardiac disease.This thesis describes parts of the transmission pathways of rickettsiae in Sweden. Rickettsia infection rates in ticks collected from birds were analysed, and the birds’ role as disseminators and reservoirs was studied. We found that more than one in ten ticks was infected with rickettsia bacteria, predominantly R. helvetica, and that migrating birds contribute not only to long-distance dispersion of bacteria, but also to an inflow of novel and potentially pathogenic rickettsia species, in this case R. monacensis and R. sp. strain Davousti-like species, into Sweden.Further, wild and domestic animals were found to have seroreactivity against R. helvetica, which shows that they are exposed and susceptible to rickettsia. Their role as reservoirs has not been determined, yet they may indirectly be involved in transmission of rickettsia to humans by infected ticks feeding on them.The seroreactivity in humans was also studied. Patients investigated for suspected Borrelioses and blood donors had detectable antibodies against Rickettsia spp., with the highest prevalence detected in the suspected Borreliosis group. This shows that humans in Sweden are exposed to and develop an immune response against rickettsia. The suspicion that R. helvetica may cause severe symptoms was verified by a patient with subacute meningitis where the bacterium was shown for the first time to cause an invasive infection with CNS involvement and where the bacterium was isolated from the patient’s cerebrospinal fluid.Growth characteristics and morphology of R. helvetica were studied to better understand invasiveness and virulence. The findings indicate that the invasiveness is comparable with other rickettsia, though R. helvetica seems to have a stable but slightly slower growth. Rickettsia helvetica is endemic in Sweden and therefore needs to be considered when investigating disease after a tick bite.
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| 4. |
- Hasan, Badrul, 1984-
(författare)
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Antimicrobial Resistance and Production of Extended Spectrum Beta-Lactamases in Enterobacteriaceae from Birds in Bangladesh
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The dissemination of members of the Enterobacteriaceae family with extended spectrum beta-lactamases (ESBLs) and metallo-beta-lactamases (MBLs) has become a global concern. ESBLs and MBLs have been reported in humans, domestic animals, wildlife and the environment, and their isolation frequencies are increasing rapidly worldwide. Most studies have been performed in developed countries and quite few in developing countries, where the antibiotic consumption is often poorly controlled. To explore the environmental contamination of antibiotic resistance in Bangladesh, and of ESBLs and MBLs in particular, fecal samples from poultry and wild birds were studied in this thesis.Samples were collected from both sick birds (poultry having Escherichia coli infections) and healthy birds (free-range poultry, seagulls and crows) residing in different environmental niches. Samples from patients and fresh/sea water were included, to follow the chain of antibiotic resistance in bacteria from humans to the environment. Information regarding the antibiotic usage in poultry production was also collected. The susceptibility of avian E. coli isolates cultured with and without selective pressure was tested against antibiotics commonly used in human and veterinary medicine in Bangladesh. Special attention was paid to ESBL-producing isolates, which were further characterized genetically.The results of the studies showed that E. coli isolates from commercial poultry, free-range poultry, gulls and crows were resistant to several classes of antibiotics, and that the level and spectrum of antibiotic resistance varied between different bird populations. There was no NDM-producer found among the birds, but ESBL-producing Enterobacteriaceae could be found in up to 59% of the crows, the birds with the highest carriage rate of multiresistant Enterobacteriaceae of all bird species studied. The most common ESBL-type was CTX-M-15, which also is the most common in the human population in Bangladesh. Birds also shared clinically important sequence types with humans, including E. coli clone O25b-ST131.In conclusion, ESBL-producing bacteria with multiresistance are easily spread to wild birds. Their opportunistic feeding behavior at poorly managed hospital waste dumps and nearby water bodies makes them into both reservoirs and active spreaders. The high level of antibiotic resistant and ESBL-producing bacteria in the bird population of Bangladesh is worrying, and there is no easy solution in sight. Nationwide programs are necessary to both improve the management of hospital waste and sewage and the control of the antibiotic usage to prevent further environmental contamination.
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| 5. |
- Järhult, Josef D., 1975-
(författare)
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Tamiflu® - Use It and Lose It?
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Influenza A viruses cause seasonal and pandemic outbreaks that range from mild infections to the disastrous Spanish Flu. Resistance to neuraminidase inhibitors (NAIs) is a growing problem as these drugs constitute a vital part of treatment strategies and pandemic preparedness plans worldwide. Oseltamivir (Tamiflu®) is the mostly used NAI. Its active metabolite, oseltamivir carboxylate (OC), is excreted from treated patients and degrades poorly in sewage treatment plants and surface water. Thus, OC can enter aquatic environments where the natural influenza reservoir, dabbling ducks, can be exposed to the substance and resistance could develop. If NAI resistance is established in influenza viruses circulating among wild birds, the resistance can form part of a virus re-entering the human population either by reassortment or by direct transmission. In this thesis, evidence is presented that OC is present in the waterways during a seasonal influenza outbreak in Japan, a country in which oseltamivir is liberally used. Furthermore, when mallards were infected with an influenza A/H1N1 virus and subjected to low, environmental-like concentrations of OC, resistance developed through acquisition of the well-known resistance mutation H274Y. The influenza infection in the mallards was mainly intestinal, had a rapid onset and was progressing in a longitudinal fashion in the intestine. Finally, influenza A viruses isolated from wild mallards in Sweden and containing resistance-related mutations were examined by a neuraminidase inhibition assay. The viruses did not have a decreased sensitivity to NAIs, but had mutations with a resistance-enhancing potential. Thus, OC is present in the environment and environmental-like concentrations of OC induce resistance in influenza viruses of dabbling ducks. The present resistance situation among wild birds is not well understood but the existence of H274Y among wild birds, though rare, and the spread of the former seasonal A/H1N1 virus containing H274Y among humans indicate that resistance mutations could establish themselves also among wild birds. An oseltamivir-resistant pandemic or a human-adapted highly-pathogenic avian influenza virus are frightening scenarios as oseltamivir is a cornerstone in the defense in those situations. There is a need for further studies, surveillance in wild birds and for a prudent use of antivirals.
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| 6. |
- Orozovic, Goran
(författare)
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Resistance to neuraminidase inhibitors in influenza A virus isolated from mallards
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Influenza A virus belongs to the Orothomyxoviridae family of viruses and is one of the most common pathogens that cause infections of the respiratory tract. The aim of this thesis was to investigate if neuraminidase inhibitor (NAI) Tamiflu® (oseltamivir, OC) and Relenza® (zanamivir, ZA) - related resistance mutations exist in the neuraminidase (NA) gene of viruses collected from wild birds.A new set of degenerate primers was designed for the sequencing procedure, which resulted in a protocol that reduced time and costs of NA sequencing. This protocol was employed for subtyping of 120 NA genes (i.e. influenza viruses). Altogether, 230 NA sequences from avian influenza viruses originating from wild mallards (Ottenby, Sweden) were scanned for NAI-related mutations together with 5,490 avian, 379 swine and 122 environmental NA sequences from the NCBI dataset. The screening showed a distinction between the numbers of mutants found in avian virus sequences derived from NCBI (2.4%) as compared to virus sequences form mallards (6.5%). This is the first report of NAI resistance mutations in viruses isolated from wild birds.The mutants carrying NAI resistance-related and resistance-unrelated mutations were screened using NA inhibition assay (NAIA) with ZA and OC inhibitors. The majority of mutations assayed showed IC50 values indicating an inhibitor sensitive phenotype. One H12N3 mutant showed a cross-resistant phenotype, i.e. insensitive to both ZA and OC treatment. Protein structure homology-modeling indicated that this cross-resistance might be associated to a D151K mutation, possibly supported by changes in NA residue 149, 150, 152 and 153. In addition, an OC resistance-related emergence of H274Y mutants was revealed in an experimental set up where mallard ducks, subjected to different concentrations of OC ( 0.28, 3.5 and 280 nM) in their water pool, were infected with avian H1N1 virus.In conclusion, this thesis provides new insights into the field of NAI resistance in avian influenza virus as well as indicating the evolutionary forces modern drug design has to confront. This thesis also emphasizes the importance of a continuous search for new means of protecting the human population from this potentially devastating pathogen.
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| 7. |
- Hernández, Jorge, 1955-
(författare)
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Human Pathogens and Antibiotic Resistant Bacteria in Polar Regions
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Coincident with human activity in recent decades, human-associated microorganisms have arrived to the Antarctic region, possibly linked to increasing presence of scientific bases and ship-borne tourists. In the Arctic, humans have been present for a very long time, and the few parts of the Arctic without human activities is decreasing with time. The studies in this thesis investigate the occurrence of different pathogens in Antarctic and Arctic wildlife, especially in birds. The first study shows the existence of Enteropatogenic Escherichia coli (EPEC) in Antarctic fur seals. The EPEC isolates were so called atypical EPECs, carrying the eae gene but lacking the bfp gene. This is the first record of a diarrheogenic E. coli in wild animals in the Antarctic. The second study displays that spreading of antibiotic resistance mechanisms appears to be much more efficient than previously was known. Enterococcus faecium isolated from Alaskan birds showed high resistance to vancomycin and teicoplanin, but also to ampicillin and ciprofloxacin. These isolates also carried vanA genes and the virulent esp gene, which places the isolates in the clinical clone CC17 and indicates the isolates had a human origin. Bacteria from birds that reside in the Bering Strait region in the third study, demonstrates that only six of 145 E. coli from 532 birds had reduced antibiotic susceptibility. Despite this, selective screen on E. coli showed only four ESBL-producing isolates. The four E. coli isolates carried CTX-M genes. One isolate belonged to the E. coli O25b - ST131 genotype, which is a successful clone with a global spread. In the fourth study, 123 seawater samples and 400 fresh penguin feces were analyzed. From these, 71 E. coli strains were isolated and only one E. coli from penguins was resistant to one antibiotic (cloramfenicol), whereas in E. coli from seawater, resistance against ampicillin, tetracycline, streptomycin and trim-sulfa were detected. E. coli carrying ESBL type CTX -M genes were also detected and Multilocus Sequencing Typing (MLST) showed six different sequence types (ST) previously reported in humans: ST131, ST227, ST401, ST410, ST685 and ST937. In the short time interval between the second study (2005) and the third study (2010) in relation to the fifth study (2012) we found a dramatic increase in antibiotic-resistant genes in the Arctic region. Enterococci, E. coli, and Kl. pneumoniae carried antibiotic resistance genes to an extent and variety not previously reported. E. coli from Arctic birds showed resistant to 1, 2, 3, 4, and 5 different antibiotics. Resistant gene type vanA was confirmed in enterococci and ESBL genes type TEM, SHV and CTX-M in E. coli and Kl. pneumoniae was detected. Multilocus Sequencing typing (MLST), indicating that both E. coli and Kl. pneumoniae carrying ESBL markers that connects them to the humans. In summary, the combined studies strengthen that bacteria that cause infections in humans could spread to relatively pristine environments. We concluded that human and associated antibiotic-resistant bacteria has reached a global level, then we showed that ESBL- carrying bacteria circulating nowadays also in the last ESBL-free continent, Antarctica.
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