| 1. |
- 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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| 2. |
- 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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| 3. |
- Devi, Priya
(författare)
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Molecular characterization of the hepatitis C virus core protein
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Hepatitis C virus (HCV) is an RNA virus that causes chronic infection, which can lead to hepatocellular carcinomas in humans. Besides liver diseases, the chronic HCV infection causes a broad spectrum of extrahepatic complications such as lymphoproliferative, metabolic and autoimmune disorders. Notably, HCV encoded core (C) protein is the major virion component that is involved in the oncogenesis and immune subversion. Therefore, detailed molecular characterization of the C protein provides a rational starting point for identification of novel countermeasures against pathogenic HCV infections. In this thesis we have investigated the suppressive effect of the C protein on T cell functions in immortalized cell lines and clinical samples.In paper I, we found that the expression of the C protein enhanced overall tyrosine phosphorylation in immortalized T cells. Interestingly, stable expression of the C protein specifically reduced accumulation of the tyrosine phosphatase SHP-1 mRNA. Our detailed bisulfite sequencing (BS) studies revealed that the SHP-1 P2 promoter was particularly hypermethylated at CpG1 and proximal islands in these cells. In paper II, we presented a new high-throughput next generation bisulfite sequencing (NGS-BS) protocol for the analysis of locus specific CpG methylation in HCV-infected cells using SHP-1 P2 as a model promoter. In line with our data from the BS, the NGS-BS method showed similar methylation profile at CpG1 island in immortalized cells. Strikingly, peripheral blood mononuclear cells (PBMCs) isolated from healthy controls and HCV-positive (HCV+) patients, showed much lower levels of methylation at the CpG1 island with no significant difference in DNA methylation pattern. In paper III, we investigated the mechanism of the C protein-mediated release of Ca2+ from intracellular stores. We identified two distinct regions in the N- and C-terminal parts of the protein that were essential for activation of the Ca2+/NFAT pathway. Of these, the N-terminal region was required for self-association of the C protein into nucleocapsid-like structures whereas the C-terminal part is essential for anchoring the protein to the ER-membrane. In paper IV, we presented a PCR based diagnostic method for the specific detection of positive and negative strand HCV RNA using primers with a non-viral tag. The method was evaluated by analysing the plasma and PBMC samples from chronic HCV+ patients.Taken together, our studies provide more detailed molecular characterization of the HCV C protein functions in immortalized as well as in HCV+ T cells. Importantly, specific DNA methylation pattern of the SHP-1 gene promoter may function as a potential prognostic marker for the disease progression in HCV-induced tumors. In addition, our updated PCR-based HCV diagnostic method may provide a more specific tool to monitor HCV infections in minor reservoirs.
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| 4. |
- 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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| 5. |
- Eriksson, Per
(författare)
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Avian Influenza Virus : Deciphering receptor interactions and their role in interspecies transmission
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Influenza A virus (IAV) annually infects approximately 5–15 % of the human population, causing ~500,000 deaths globally. Novel IAVs have emerged and spread pandemically in the human population, but have over time established endemic circulation with reduced pathogenicity causing seasonal influenza. The natural reservoir of IAVs is wild waterfowl. The past pandemics have been associated with host switch and have partly or entirely originated from birds, or adapted via passage through pigs (postulated IAV mixing vessel). Understanding IAV interspecies transmission mechanisms is essential for pandemic preparedness. Enzootic circulation of avian IAV (AIV) is concentrated to a few waterfowl species, while other bird species seldom are infected. A species barrier preventing IAV interspecies transmission has been suggested. To investigate IAV host range and mixing vessels, histochemistry studies were conducted with tissues from avian species, pigs, and humans. Virus adaptation to new hosts was studied by challenging tufted ducks and chickens with mallard-derived AIVs, together with AIV receptor tropism and glycoproteomic analysis of receptor distribution. Finally, receptor and tissue tropism in ducks was studied systematically for AIV (H1–16). More abundant AIV attachment to human than pig tissues was observed, questioning the pig mixing vessel theory. Attachment patterns of AIVs to bird tissues was generally broad with abundant attachment to trachea. However, among ducks, pronounced attachment was observed to colon of Anas spp., suggesting that intestinal infection might be restricted to Anas spp., whereas other species may be susceptible to respiratory infection. Tufted ducks and chickens could not be infected by intraesophageal inoculation further supporting this hypothesis. Glycan array analysis revealed 3’SLN, 3’STF, and their fucosylated and sulfated analogues as main AIV receptors. Moreover, AIV Neu5Acα2,6 recognition was widespread. Avian respiratory and intestinal tracts glycoproteomic analysis revealed that avian and mammalian receptor structures are much more similar than earlier thought. Furthermore, observed AIV subtype titer variation in challenged tufted ducks and chickens did not correlate with virus receptor tropism. In summary, this thesis suggests that IAV receptor recognition, in particular α2,3 vs. α2,6 sialylated receptor structures, is less important for the IAV interspecies barrier than previously thought.
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| 6. |
- Gillman, Anna, 1972-
(författare)
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Tamiflu in the Water : Resistance Dynamics of Influenza A Virus in Mallards Exposed to Oseltamivir
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The natural reservoir of influenza A virus (IAV) is wild waterfowl, and all human IAVs have their genetic origins from avian viruses. Neuraminidase inhibitors (NAIs) are currently the best drugs for treatment of human influenza; therefore, the orally available NAI oseltamivir (Tamiflu®) has been stockpiled worldwide as part of pandemic preparedness planning. Re-sistance to NAIs is related to worse clinical outcomes and if a new pandemic influenza virus would be oseltamivir-resistant its public health impact would be substantially worsened.The active metabolite oseltamivir carboxylate (OC) is not removed by sewage treatment and ends up in river water, where OC-concentrations up to 0.86µg/L have been detected.We hypothesize that occasional OC exposure of wild waterfowl carrying IAVs may result in circulation of resistant variants that may potentially evolve to become human-pathogenic.We tested the hypothesis in an in vivo Mallard (Anas platyrhynchos) model in which birds were infected with avian IAVs and exposed to OC. Excreted viruses were analyzed regarding genotypic and phenotypic resistance by neuraminidase (NA) sequencing and a functional NA inhibition assay.Two viruses with NAs of the phylogenetic N2-group, H6N2 and H7N9, acquired the NA substitutions R292K and I222T when host ducks were exposed to 12µg/L and 2.5µg/L of OC, respectively. Drug susceptibilities were at previously described levels for the substitutions. To test persistence of resistance, an OC resistant avian H1N1/H274Y virus (with a group N1 NA-protein) from a previous study, and three resistant H6N2/R292K variants were allowed to replicate in Mallards without drug pressure. Resistance was entirely maintained in the H1N1/H274Y virus, but the H6N2/R292K variants were outcompeted by wild type virus, indicating retained fitness of the resistant H1N1 but not the H6N2 variants.We conclude that OC in the environment may generate resistant IAVs in wild birds. Resistant avian IAVs may become a problem to humans, should the resistance trait become part of a new human pathogenic virus. It implies a need for prudent use of available NAIs, optimized sewage treatment and resistance surveillance of avian IAVs of wild birds.
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| 7. |
- Gullsby, Karolina
(författare)
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Molecular detection and epidemiological studies of atypical bacteria causing respiratory tract infections
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Respiratory infections are common causes of morbidity and mortality. Chlamydia pneumoniae, Mycoplasma pneumoniae and Bordetella pertussis cause respiratory infection, often with similar symptoms. Molecular diagnostic methods are preferred since these bacteria are difficult to culture. The aim of this thesis was to evaluate and improve the diagnostics and knowledge of the epidemiology of these bacteria.A real-time polymerase chain reaction (PCR) method targeting the IS481 element present in the genome of B. pertussis was compared to culture and serology results, and a duplex real-time PCR method was constructed for detecting C. pneumoniae and M. pneumoniae, which was compared to two endpoint PCR methods. Both real-time PCR methods showed high sensitivity and specificity.Typing of 624 M. pneumoniae samples, collected from 1996 to 2017 from four counties, was performed by P1 typing and multiple-locus variable number tandem repeat analysis (MLVA). A polyclonal distribution of strains was seen over all epidemic periods, but strains of P1 type 2/variant 2 and MLVA types 3-5-6-2 and 4-5-7-2 predominated in 2010−2013. A shift from type 2 strains to different variant 2 strains was seen and a new variant, 2e, was detected in 2016−2017. An A2063G mutation associated with macrolide resistance was detected by a fluorescence resonance energy transfer (FRET) PCR method in one (0.16%) of 608 M. pneumoniae strains.Molecular characterisation using whole-genome sequencing of 93 B. pertussis isolates, collected between 1986 and 2016 from three counties showed that there were polyclonal strains in the county of Dalarna, Gävleborg and Uppsala in the years 2014−2016. Changes in virulence-related genes were detected: a shift from isolates harbouring the ptxP3 allele in favour of ptxP1 was seen, and almost all isolates had a disrupted prn gene. No detection of macrolide resistance in B. pertussis was detected.In conclusion, the validated real-time PCR methods for detection of B. pertussis, C. pneumoniae and M. pneumoniae have led to improved diagnostic methods for use in clinical laboratories. The molecular characterisation of M. pneumoniae and B. pertussis strains has contributed to the wider understanding of the genetic changes that has occurred over the epidemic periods, but further studies is needed.
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| 8. |
- 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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| 9. |
- 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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| 10. |
- Hoffman, Tove
(författare)
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Dispersal of ticks and their microorganisms by African-Western Palaearctic migratory birds
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In Europe, tick-borne diseases are the most widespread and common vector-borne diseases and their geographical distribution is increasing. The dispersal of ticks depends on the movements of their vertebrate hosts. Avian hosts are more likely to be involved in long-distance range expansion of ticks due to their migration pattern. Billions of birds in the African-Palaearctic migration system migrate biannually between breeding grounds in the Palaearctic and wintering grounds in Africa and thereby create natural links between Africa, Europe, and Asia. In this thesis the dispersal of ticks and their microorganisms by northbound migratory birds utilizing flyways in the African-Western Palaearctic region has been investigated and the association between bird ecology and tick taxon addressed. The results suggest that long-distance migratory birds with wintering regions in Africa are involved in northward dispersal of the tick species Hyalomma rufipes, a known vector or Crimean-Congo hemorrhagic fever virus, and that birds with an open or wetland habitat have more H. rufipes in comparison to birds with a winter habitat comprising forest and shrubs. The results also suggest a role for birds in the ecology of Alkhurma hemorrhagic fever virus, a hemorrhagic flavivirus, and a potential mechanism for dispersal of the virus to new regions, including Europe and Asia Minor. The results did not provide evidence for immature ticks of the Hyalomma marginatum complex and birds having a major role in the ecology and northward dispersal of tick-borne Anaplasma phagocytophilum, a zoonotic bacterium causing febrile illness in humans and domestic animals. However, the results give support to the idea of a divergent enzootic cycle of A. phagocytophilum involving birds as hosts. Finally, the results of this thesis suggest that H. rufipes do not serve as vectors or contribute to the transmission of the tularemia-causing bacterium Francisella tularensis and that migratory birds do not contribute to northward dispersal of F. tularensis-infected ticks. However, the results suggest that migratory birds contribute to northward dispersal of H. rufipes carrying both Francisella and spotted fever group Rickettsia species, including Francisella-like endosymbionts and Rickettsia aeschlimannii. In conclusion, this thesis helps to clarify the knowledge about the dispersal of ticks and the microorganisms they carry by northbound migrating birds in the African-Western Palaearctic region. Furthermore, it highlights the need of establishing surveillance programs for monitoring the risk of introduction and establishment of important exotic tick species, such as H. rufipes, and tick-borne pathogens in the Western Palaearctic.
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