| 1. |
- Achá Alarcón, L., et al.
(författare)
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Development and performance evaluation of an In-House ELISA for the detection of group A rotavirus in diarrheal stool samples from children and domestic South American camelids
- 2022
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934. ; 301
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Tidskriftsartikel (refereegranskat)abstract
- Group A rotavirus (RVA) is a prevalent pathogen causing acute gastroenteritis (AGE) in young children and animals. We developed an in-house ELISA (ROTA-GeFeK) for RVA detection, based on the expression of native recombinant VP6 protein in E. coli. To detect the RVA antigen, rabbit polyclonal IgG antibodies, produced against rVP6,were used as capture and detector antibodies in a sandwich ELISA. To validate the ROTA-GeFeK, 252 stool samples from children with AGE, were evaluated by conventional RT-PCR and commercial ELISA. Compared to RT-PCR, the ROTAGeFeK had a sensitivity of 88.2 % and a specificity of 94.4 %. Total detection rates with the ROTA-GeFeK, commercial ELISA and RT-PCR were 58 %, 58 % and 64 % respectively. The limit of detection was equal to 2.1 × 10 4 CCID 50 of the RVA strain RIX4414. No cross-reactivity with other enteric pathogens was observed. The RVApositive samples detected by the assay belonged to a diversity of G and [P] genotypes.This assay displayed reactivity and was proved to be useful for the detection of RVA in diarrheal samples of domestic South American Camelids. We suggest that the ROTAGeFeK can be used as an epidemiologic tool for rotavirus surveillance and for RVA detection in other animal species. © 2021
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| 2. |
- Birindwa, Archippe M., et al.
(författare)
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Bacteria and viruses in the upper respiratory tract of Congolese children with radiologically confirmed pneumonia
- 2021
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Ingår i: Bmc Infectious Diseases. - : Springer Science and Business Media LLC. - 1471-2334. ; 21:1
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Tidskriftsartikel (refereegranskat)abstract
- Background Acute pneumonia remains a leading cause of death among children below 5 years of age in the Democratic Republic of the Congo (DR Congo), despite introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) in 2013. Potential pathogens in the nasopharynx of hospitalised children with pneumonia have not been studied previously in DR Congo. Here we compare clinical characteristics, risk factors and nasopharyngeal occurrence of bacteria and viruses between children with severe and non-severe pneumonia. Methods Between June 2015 and June 2017, 116 children aged from 2 to 59 months hospitalised due to radiologically confirmed pneumonia at Panzi referral university hospital, Bukavu, Eastern DR Congo were included in the study and sampled from nasopharynx. A multiplex real-time PCR assay for detection of 15 different viruses and 5 bacterial species was performed and another multiplex PCR assay was used for pneumococcal serotype/serogroup determination. Results During the study period 85 (73%) of the children with radiologically confirmed pneumonia met the WHO classification criteria of severe pneumonia and 31 (27%) had non-severe pneumonia. The fatality rate was 9.5%. Almost all (87%) children were treated with antibiotics before they were hospitalised, in most cases with amoxicillin (58%) or trimethoprim-sulfamethoxazole (20%). The frequency of potential pathogens in the nasopharynx of the children was high, and any viral or bacterial nucleic acids present at high levels, irrespective of species or type, were significantly associated with severe pneumonia as compared with non-severe cases (52% versus 29%, p = 0.032). White blood cell count > 20,000/mu L and C-Reactive Protein > 75 mg/dL were associated with severe pneumonia at admission. Fatal outcome was in the multivariable analysis associated with having a congenital disease as an underlying condition. One or more pneumococcal serotypes/serogroups could be identified in 61 patients, and out of all identified serotypes 31/83 (37%) were non-PCV13 serotypes. Conclusions The occurrence of any bacteria or any viruses at high levels was associated with severe pneumonia at admission. Children with congenital disorders might need a higher attention when having symptoms of acute respiratory infection, as developed pneumonia could lead to fatal outcome.
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| 3. |
- Birindwa, Archippe M., et al.
(författare)
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High bacterial and viral load in the upper respiratory tract of children in the Democratic Republic of the Congo
- 2020
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 15:10
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Tidskriftsartikel (refereegranskat)abstract
- © 2020 Muhandule Birindwa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Background Respiratory pathogens including Streptococcus pneumoniae and Haemophilus influenzae, are implicated in the pathogenicity of acute lower respiratory infection (ALRI). These are also commonly found in both healthy and sick children. In this study, we describe the first data on the most frequent bacteria and viruses detected in the nasopharynx of children from the general population in the Eastern DR Congo. Methods From January 2014 to June 2015, nasopharyngeal samples from 375 children aged from 2 to 60 months attending health centres for immunisation or growth monitoring were included in the study. Multiplex real-time PCR assays were used for detection of 15 different viruses and 5 bacterial species and for determination of pneumococcal serotypes/serogroups in the nasopharyngeal secretions. Results High levels of S. pneumoniae were detected in 77% of cases, and H. influenzae in 51%. Rhinovirus and enterovirus were the most commonly found viruses, while respiratory syncytial virus (RSV) was rare (1%). Co-occurrence of both bacteria and viruses at high levels was detected in 33% of the children. The pneumococcal load was higher in those children who lived in a dwelling with an indoor kitchen area with an open fire, i.e. a kitchen with an open fire for cooking located inside the dwelling with the resultant smoke passing to the living room and/or bedrooms; this was also higher in children from rural areas as compared to children from urban areas or children not living in a dwelling with an indoor kitchen area with an open fire/not living in this type of dwelling. Immunization with 2–3 doses of PCV13 was associated with lower rates of pneumococcal detection. Half of the identified serotypes were non-PCV13 serotypes. The most common non-PCV13 serotypes/serogroups were 15BC, 10A, and 12F, while 5, 6, and 19F were the most prevalent PCV13 serotypes/serogroups. Conclusions The burden of respiratory pathogens including S. pneumoniae in Congolese children was high but relatively few children had RSV. Non-PCV13 serotypes/serogroups became predominant soon after PCV13 was introduced in DR Congo.
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| 4. |
- Birindwa, Archippe M., et al.
(författare)
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High rate of antibiotic resistance among pneumococci carried by healthy children in the eastern part of the Democratic Republic of the Congo
- 2018
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Ingår i: Bmc Pediatrics. - : Springer Science and Business Media LLC. - 1471-2431. ; 18
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundPneumococcal conjugate vaccines have been introduced in the infant immunisation programmes in many countries to reduce the rate of fatal pneumococcal infections. In the Democratic Republic of the Congo (DR Congo) a 13-valent vaccine (PCV13) was introduced in 2013. Data on the burden of circulating pneumococci among children after this introduction are lacking. In this study, we aimed to determine the risk factors related to pneumococcal carriage in healthy Congolese children after the vaccine introduction and to assess the antibiotic resistance rates and serotype distribution among the isolated pneumococci.MethodsIn 2014 and 2015, 794 healthy children aged one to 60months attending health centres in the eastern part of DR Congo for immunisation or growth monitoring were included in the study. Data on socio-demographic and medical factors were collected by interviews with the children's caregivers. Nasopharyngeal swabs were obtained from all the children for bacterial culture, and isolated pneumococci were further tested for antimicrobial resistance using disc diffusion tests and, when indicated, minimal inhibitory concentration (MIC) determination, and for serotype/serogroup by molecular testing.ResultsThe pneumococcal detection rate was 21%, being higher among children who had not received PCV13 vaccination, lived in rural areas, had an enclosed kitchen, were malnourished or presented with fever (p value <0.05). The predominant serotypes were 19F, 11, 6A/B/C/D and 10A. More than 50% of the pneumococcal isolates belonged to a serotype/serogroup not included in PCV13.Eighty per cent of the isolates were not susceptible to benzylpenicillin and non-susceptibility to ampicillin and ceftriaxone was also high (42 and 37% respectively). Almost all the isolates (94%) were resistant to trimethoprim-sulphamethoxazole, while 43% of the strains were resistant to 3 antibiotics.ConclusionsOur study shows alarmingly high levels of reduced susceptibility to commonly used antibiotics in pneumococci carried by healthy Congolese children. This highlights the importance of local antibiotic resistance surveillance and indicates the needs for the more appropriate use of antibiotics in the area. The results further indicate that improved living conditions are needed to reduce the pneumococcal burden, in addition to PCV13 vaccination.
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| 5. |
- Carvalheira, A., et al.
(författare)
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Acinetobacter portensis sp. Nov. and acinetobacter guerrae sp. nov., isolated from raw meat
- 2020
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Ingår i: International journal of systematic and evolutionary microbiology. - : Microbiology Society. - 1466-5026 .- 1466-5034. ; 70:8, s. 4544-4554
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Tidskriftsartikel (refereegranskat)abstract
- The taxonomic status of six strains of Acinetobacter obtained from meat samples, collected from supermarkets in Porto, Por-tugal, was investigated using polyphasic analysis. Partial rpoB sequence similarities lower than 95 % to other Acinetobacter species with validly published names led to the hypothesis that these strains represented novel species. This was confirmed based on comparative multilocus sequence analysis, which included the gyrB, recA and 16S rRNA genes, revealing that these strains represented two coherent lineages that were distinct from each other and from all known species. The names Acine-tobacter portensis sp. nov. (comprising four strains) and Acinetobacter guerrae sp. nov. (comprising two strains) are proposed for these novel species. The species status of these two groups was confirmed by low (below 95 %) whole-genome sequence average nucleotide identity values and low (below 70 %) digital DNA–DNA hybridization similarities between the whole-genome sequences of the proposed type strains of each novel species and the representatives of the known Acinetobacter species. Phylogenomic treeing from core genome analysis supported these results. The coherence of each new species lineage was supported by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry differentiation of the species at the protein level, by cellular fatty acid profiles, and by unique and differential combinations of metabolic and physiological properties shared by each novel species. The type strain of A. portensis sp. nov. is AC 877T (=CCUG 68672T=CCM 8789T) and the type strain of A. guerrae sp. nov. is AC 1271T (=CCUG 68674T=CCM 8791T). © 2020 The Authors.
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| 6. |
- Claverías, Fernanda, et al.
(författare)
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Corynebacterium alimapuense sp. nov., an obligate marine actinomycete isolated from sediment of Valparaíso bay, Chile.
- 2019
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Ingår i: International journal of systematic and evolutionary microbiology. - : Microbiology Society. - 1466-5034 .- 1466-5026. ; 69:3, s. 783-790
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Tidskriftsartikel (refereegranskat)abstract
- A novel Gram-positive, non-motile, non-spore-forming and aerobic bacterium, designated strain VA37-3T, was isolated from a marine sediment sample collected at 19.2m water depth from Valparaíso bay, Chile. Strain VA37-3T exhibits 97.6% 16S rRNA gene sequence similarity to Corynebacterium marinum D7015T, 96.4% to Corynebacterium humireducens MFC-5T and 96% to Corynebacterium testudinoris M935/96/4T; and a rpoB gene sequence similarity of 85.1% to Corynebacterium pollutisoli VMS11T, both analyses suggesting that strain VA37-3T represents a novel species of Corynebacterium. Physiological testing indicated that strain VA37-3T requires artificial sea water or sodium-supplemented media for growth, representing the first obligate marine actinomycete of the genus Corynebacterium. The genome of the proposed new species, along with the type strains of its most closely related species were sequenced and characterized. In silico genome-based similarity analyses revealed an ANIb of 72.8% (C. marinum D7015T), ANIm of 85.0% (Corynebacterium mustelae DSM 45274T), tetra of 0.90 (Corynebacterium callunae DSM 20147T) and ggdc of 24.7% (Corynebacterium kutscheri DSM 20755T) when compared with the closest related strains. The genomic DNA G+Ccontent of strain VA37-3T was 57.0%. Chemotaxonomic assessment of strain VN6-2T showed the major fatty acids were C18:1ω9c and C16:0. Menaquinones predominantly consisted of MK-8(II-H2). Polar lipids consisted of diphosphatidylglycerol, glycolipids, phosphatidylglycerol, phosphoglycolipid and phosphatidylinositol. Mycolic acids also were present. Overall, the results from phylogenetic, phenotypic and genomic analyses confirmed that strain VA37-3T represents a novel species of the genus Corynebacterium, for which the name Corynebacterium alimapuense sp. nov. is proposed, with VA37-3T as the type strain (=CCUG 69366T=NCIMB 15118T).
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| 7. |
- Emgård, Matilda, et al.
(författare)
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Carriage of penicillin-non-susceptible pneumococci among children in northern Tanzania in the 13-valent pneumococcal vaccine era.
- 2019
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Ingår i: International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases. - : Elsevier BV. - 1878-3511. ; 81, s. 156-166
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Tidskriftsartikel (refereegranskat)abstract
- To determine the antibiotic susceptibility and serotype distribution of colonizing Streptococcus pneumoniae in Tanzanian children. Serial cross-sectional surveys were performed following the national introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) in December 2012.A total of 775 children less than 2 years of age were recruited at primary health centres in Moshi, Tanzania between 2013 and 2015, and samples were obtained from the nasopharynx. S. pneumoniae were isolated by culture and tested for antibiotic susceptibility by disc diffusion and E-test methods; molecular testing was used to determine serotype/group.Penicillin non-susceptibility in the isolated pneumococci increased significantly from 31% (36/116) in 2013, to 47% (30/64) in 2014 and 53% (32/60) in 2015. Non-susceptibility to amoxicillin/ampicillin and ceftriaxone was low (n=8 and n=9, respectively), while 97% (236/244) of the isolates were non-susceptible to trimethoprim-sulfamethoxazole. The majority of the children (54%, n=418) had been treated with antibiotics in the past 3 months, and amoxicillin/ampicillin were overall the most commonly used antibiotics. Carriage of penicillin-non-susceptible pneumococci was more common in children with many siblings. The prevalence of PCV13 serotypes among the detected serotypes/groups decreased from 56% (40/71) in 2013 to 23% (13/56) in 2015.Penicillin non-susceptibility in S. pneumoniae colonizing Tanzanian children increased during an observation period shortly after the introduction of PCV13. Measures to ensure rational use of antibiotics and more effective systems for surveillance of antibiotic resistance and serotype distribution are needed to assure continued effective treatment of pneumococcal disease.
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| 8. |
- Emgård, Matilda, 1984, et al.
(författare)
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Co-occurrence of bacteria and viruses and serotype distribution of Streptococcus pneumoniae in the nasopharynx of Tanzanian children below 2 years of age following introduction of the PCV13
- 2024
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Ingår i: FRONTIERS IN PUBLIC HEALTH. - 2296-2565. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: Pneumococcal conjugate vaccines have reduced severe disease attributed to vaccine-type pneumococci in children. However, the effect is dependent on serotype distribution in the population and disease development may be influenced by co-occurrence of viral and bacterial pathogens in the nasopharynx. Methods: Following introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) in Tanzania we performed repeated cross-sectional surveys, including 775 children below 2 years of age attending primary healthcare centers. All children were sampled from nasopharynx and pneumococci were detected by single-target PCR. Pneumococcal serotypes/groups and presence of viruses and other bacteria were determined by two multiplex PCR assays. Results: The prevalence of PCV13 vaccine-type pneumococci decreased by 50%, but residual vaccine-types were still detected in 21% of the children 2 years after PCV13 introduction. An increase in the non-vaccine-type 15 BC was observed. Pneumococci were often co-occurring with Haemophilus influenzae, and detection of rhino/enterovirus was associated with higher pneumococcal load. Discussion: We conclude that presence of residual vaccine-type and emerging non-vaccine-type pneumococci in Tanzanian children demand continued pneumococcal surveillance. High co-occurrence of viral and bacterial pathogens may contribute to the disease burden and indicate the need of multiple public health interventions to improve child health in Tanzania.
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| 9. |
- Gonzales-Siles, Lucia, et al.
(författare)
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A Pangenome Approach for Discerning Species-Unique Gene Markers for Identifications of Streptococcus pneumoniae and Streptococcus pseudopneumoniae
- 2020
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Ingår i: Frontiers in Cellular and Infection Microbiology. - : Frontiers Media SA. - 2235-2988. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Correct identifications of isolates and strains of the Mitis-Group of the genus Streptococcus are particularly difficult, due to high genetic similarity, resulting from horizontal gene transfer and homologous recombination, and unreliable phenotypic and genotypic biomarkers for differentiating the species. Streptococcus pneumoniae and Streptococcus pseudopneumoniae are the most closely related species of the clade. In this study, publicly-available genome sequences for Streptococcus pneumoniae and S. pseudopneumoniae were analyzed, using a pangenomic approach, to find candidates for species-unique gene markers; ten species-unique genes for S. pneumoniae and nine for S. pseudopneumoniae were identified. These species-unique gene marker candidates were verified by PCR assays for identifying S. pneumoniae and S. pseudopneumoniae strains isolated from clinical samples. All determined species-level unique gene markers for S. pneumoniae were detected in all S. pneumoniae clinical isolates, whereas fewer of the unique S. pseudopneumoniae gene markers were present in more than 95% of the clinical isolates. In parallel, taxonomic identifications of the clinical isolates were confirmed, using conventional optochin sensitivity testing, targeted PCR-detection for the "Xisco" gene, as well as genomic ANIb similarity analyses for the genome sequences of selected strains. Using mass spectrometry-proteomics, species-specific peptide matches were observed for four of the S. pneumoniae gene markers and for three of the S. pseudopneumoniae gene markers. Application of multiple species-level unique biomarkers of S. pneumoniae and S. pseudopneumoniae, is proposed as a protocol for the routine clinical laboratory for improved, reliable differentiation, and identification of these pathogenic and commensal species.
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| 10. |
- Gonzales-Siles, Lucia, et al.
(författare)
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Identification and capsular serotype sequetyping of Streptococcus pneumoniae strains
- 2019
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Ingår i: Journal of Medical Microbiology. - : Microbiology Society. - 0022-2615 .- 1473-5644. ; 68:8, s. 1173-1188
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Tidskriftsartikel (refereegranskat)abstract
- Purpose. Correct serotype identification of Streptococcus pneumoniae (pneumococcus) is important for monitoring disease epidemiology and assessing the impacts of pneumococcal vaccines. Furthermore, correct identification and differentiation of the pathogenic S. pneumoniae from closely related commensal species of the mitis group of the genus Streptococcus are essential for correct serotype identification. Methodology. A new protocol for determining the existing 98 serotypes of pneumococcus was developed, applying two PCR amplifications and amplicon sequencing, using newly designed internal primers. The new protocol was validated using S. pneumoniae genome sequences, reference strains with confirmed serotypes and clinical isolates, and comparing the results with those from the traditional Quellung reaction or antiserum panel gel precipitation, in addition to real-time PCR analysis. The taxonomic identifications of 422 publicly available (GenBank) genome sequences of S. pneumoniae, Streptococcus pseudopneumoniae and Streptococcus mitis were assessed by whole-genome sequence average nucleotide identity based on blast (ANIb) analysis. Results. The proposed sequetyping protocol generates a 1017 bp whole cpsB region sequence, increasing resolution for serotype identification in pneumococcus isolates. The identifications of all GenBank genome sequences of S. pneumoniae were confirmed, whereas most of the S. pseudopneumoniae and almost all of the S. mitis genome sequences did not fulfil the ANIb thresholds for species-level identification. The housekeeping biomarker gene, groEL, correctly identified S. pneumoniae but often misclassified S. pseudopneumoniae and S. mitis as S. pneumoniae. Conclusions. These studies affirm the importance of applying reliable identification protocols for S. pneumoniae before serotyping; our protocols provide reliable diagnostic tools, as well as an improved workflow, for serotype identification of pneumococcus and differentiation of serogroup 6 types.
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| 11. |
- Gonzales-Siles, Lucia, et al.
(författare)
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Mass Spectrometry Proteotyping for detection, identification characterization and diagnostics of infectious bacteria in clinical respiratory-tract samples
- 2016
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Ingår i: 11th International Meeting on Microbial Epidemiological Markers (IMMEM XI) 9 - 12 March 2016, Estoril, Portugal.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Background. Lower respiratory tract infection (LRTI) is the leading cause of childhood deaths in most developing countries and the world (?) and are the most common causes of hospital and out-patient visits within the EU, comprising 1 of 3 admissions annually. In general, the over-prescription and use of broad-spectrum antibiotics are common practices that lead to the evolution and development of resistance in infectious bacteria and will lead to loss of time and resources in patient handling and adverse patient outcomes. Conventional approaches have depended upon cultivation of bacteria with subsequent testing for antibiotic sensitivity. Therefore, reliable and time-effective microbiological diagnostics are essential for more effective treatment of respiratory infections. In this project, we apply state-of-the-art proteomics techniques for identifications of pathogens and antibiotic resistance from clinical samples, without prior cultivation. Material and methods. Nasopharyngeal swab samples were collected, in commercial Amies medium supplemented with 5x STGG, as a part of the EU-TAILORED-Treatment project (www.tailored-treatment.eu/). Samples were stored at -20°C until analyses. Different protocols for removal of human cells and mucus were tested, including non-ionic detergents, i.e., Igepal, Saponin, Urea-Chaps, as well as cytolysis. Samples were concentrated and analyzed by ‘proteotyping’ (1), using a Lipid-based Protein Immobilization (LPITM) technology (WO2006068619), in which intact bacterial cells or cell fractions are bound to a surface. Peptides were generated, using enzymatic digestion, and then separated and analyzed, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The mass spectra profiles were compared to a database of reference peptide sequences, consisting of all complete genomes of the NCBI Reference Sequence (RefSeq) Database. Results were confirmed by standard microbiology, including cultivation of bacteria in selective media, MALDI-TOF MS analyses and qPCR. Results. Proteotyping applied to clinical samples demonstrated that the number of viable bacteria and detected proteins determined were ten-times higher when nasal swabs were stored in Amies media supplemented with STGG 5X media compared to Amies media without STGG, after 1 and 2 months of storage at -70C. Among the different protocols tested to remove human biomaterial, all treatments proved effective to varying degrees, although the Igepal treatment was able to retain the highest number of discriminatory peptides. Using proteotyping, we were able to identify the pathogenic bacteria directly within clinical samples (nasopharyngeal and nasal swabs) that had been identified to be positive for respiratory infectious bacteria by standard methodologies at clinical bacteriology laboratories at Sahlgrenska University Hospital (Sweden) or Universitair Medisch Centrum Utrecht (Netherlands). Conclusions. Proteotyping of infectious bacteria, using tandem LC-MS/MS enabled the differentiation and identification of infectious bacteria in clinical samples from LRTIs. It has high levels of resolution and highly reproducible detection of protein biomarkers. Proteotyping identified biomarkers for species- and sub-species-level strain discrimination and antibiotic resistance, all from single MS analyses. 1) Karlsson et al., 2015. Syst. Appl. Microbiol. 38 :246-257.
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| 12. |
- Gonzales-Siles, Lucia, et al.
(författare)
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Mass Spectrometry Proteotyping of Streptococcus pneumoniae and commensal Streptococcus: identification of biomarkers for infectious strain characterization
- 2016
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Ingår i: 26th ECCMID 2016 Amsterdam, The Netherlands. 9 - 12 April 2016.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Background: Streptococcus pneumoniae (pneumococcus) is the leading cause of community-acquired pneumonia, with morbidity and mortality worldwide. S. pneumoniae belongs to the S. mitis-Group (viridans streptococci), phenotypically and genotypically similar to commensal species of the upper respiratory tract, S. mitis, S. oralis, and S. pseudopneumoniae, causing problems for identifications in clinical laboratories. In this project, we apply state-of-the-art proteomics for Streptococcus spp. 'proteotyping'; identifying and characterizing protein biomarkers for species-level identification, antibiotic resistance, virulence and strain typing for epidemiological analyses (1). Material/methods: Bacterial proteins, from intact bacteria or cell fractions, are bound to a membrane surface, using patented (WO2006068619) FlowCell (LPITM) technology. Peptides are generated from the bound proteins, by enzymatic digestion, separated and analyzed, using LC-MS/MS. The mass spectra profiles are compared to reference peptide sequences and whole genome sequence (wgs) data of the NCBI RefSeq Database. The S. mitis-Group specie, S. pneumoniae, S. mitis, S. oralis, S. psedopneumoniae, as well as the more distantly-related, Group A Streptococcus (GAS) species, S. pyogenes , were analyzed individually and in mixtures, to demonstrate the resolution of proteotyping for differentiating bacteria. Results: Using proteotyping protocols, S. pneumoniae were detected and differentiated from other streptococci, S. mitis, S. oralis, S. psedopneumoniae and the more distant relative, S. pyogenes, by identification of unique discriminatory peptides. Metabolic protein biomarkers were identified, including for antibiotic resistance and virulence. It was possible to find discriminatory biomarkers for a target species when analyzing 1:1 mixes of S. pneumoniae and other species from the S. mitis-Group. The different strains of S. pneumoniae, analyzed in different ratio combinations, were successfully differentiated and identified. For successful proteotyping, a comprehensive and accurate genomic database was observed to be key for obtaining reliable peptide matching and proteotyping data. Importantly, because of observed high rates of misclassified wgs data in the public databases, the taxonomic classifications of genomes in GenBank were analyzed against reference type strain genomes of target species by calculating wgs similarities, using Average Nucleotide Identity with BLAST (ANIb). While wgs data for S. pneumoniae were confirmed to be classified correctly, approximately one-third of wgs data for other species of the S. mitis-Group were determined to be misclassified. Streptococci strains that could not be identified, using standard genotypic and phenotypic approaches, were characterized by proteotyping and genome sequencing to establish their taxonomy and biomarker features to enhance species database matching. Conclusions: Proteotyping enables differentiation, identification and characterization of pneumococcus from the most closely related species attaining, as well, strain-level discrimination from single LC-MS/MS analyses. The protocol enhances identification and characterization of pathogenic bacterial isolates through identifications of expressed biomarkers, ultimately for cultivation-independent analyses of clinical samples. 1) Karlsson et al., 2015. Syst Appl Microbiol. 38:246-257.
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| 13. |
- Gonzales-Siles, Lucia, et al.
(författare)
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Proteomic analysis of enterotoxigenic Escherichia coli (ETEC) in neutral and alkaline conditions
- 2017
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Ingår i: Bmc Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 17:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in children and travelers to endemic areas. Secretion of the heat labile AB5 toxin (LT) is induced by alkaline conditions. In this study, we determined the surface proteome of ETEC exposed to alkaline conditions (pH 9) as compared to neutral conditions (pH 7) using a LPI Hexalane FlowCell combined with quantitative proteomics. Relative quantitation with isobaric labeling (TMT) was used to compare peptide abundance and their corresponding proteins in multiple samples at MS/MS level. For protein identification and quantification samples were analyzed using either a 1D-LCMS or a 2D-LCMS approach. Results: Strong up-regulation of the ATP synthase operon encoding F1Fo ATP synthase and down-regulation of proton pumping proteins NuoF, NuoG, Ndh and WrbA were detected among proteins involved in regulating the proton and electron transport under alkaline conditions. Reduced expression of proteins involved in osmotic stress was found at alkaline conditions while the Sec-dependent transport over the inner membrane and outer membrane protein proteins such as OmpA and the beta-Barrel Assembly Machinery (BAM) complex were upregulated. Conclusions: ETEC exposed to alkaline environments express a specific proteome profile characterized by upregulation of membrane proteins and secretion of LT toxin. Alkaline microenvironments have been reported close to the intestinal epithelium and the alkaline proteome may hence represent a better view of ETEC during infection.
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| 14. |
- Gonzales-Siles, Lucia, et al.
(författare)
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The different ecological niches of enterotoxigenic Escherichia coli (ETEC).
- 2016
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Ingår i: Environmental microbiology. - : Wiley. - 1462-2920 .- 1462-2912. ; 18:3, s. 741-751
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Tidskriftsartikel (refereegranskat)abstract
- Enterotoxigenic Escherichia coli (ETEC) is a water and food borne pathogen that infects the small intestine of the human gut and causes diarrhea. ETEC adheres to the epithelium by means of colonization factors and secretes two enterotoxins, the heat labile toxin (LT) and/or the heat stable toxin (ST) that both de-regulate ion channels and cause secretory diarrhea. ETEC, as all E. coli, is a versatile organism able to survive and grow in different environments. During transmission and infection ETEC is exposed to various environmental cues that have an impact on survivability and virulence. The ability to cope with exposure to different stressful habitats is probably shaping the pool of virulent ETEC strains that cause both endemic and epidemic infections. This review will focus on the ecology of ETEC in its different habitats and interactions with other organisms as well as abiotic factors.
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| 15. |
- Guzman-Otazo, J., et al.
(författare)
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Diarrheal bacterial pathogens and multi-resistant enterobacteria in the Choqueyapu River in La Paz, Bolivia
- 2019
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- Water borne diarrheal pathogens might accumulate in river water and cause contamination of drinking and irrigation water. The La Paz River basin, including the Choqueyapu River, flows through La Paz city in Bolivia where it is receiving sewage, and residues from inhabitants, hospitals, and industry. Using quantitative real-time PCR (qPCR), we determined the quantity and occurrence of diarrheagenic Escherichia coli (DEC), Salmonella enterica, Klebsiella pneumoniae, Shigella spp. and total enterobacteria in river water, downstream agricultural soil, and irrigated crops, during one year of sampling. The most abundant and frequently detected genes were gapA and eltB, indicating presence of enterobacteria and enterotoxigenic E. coli (ETEC) carrying the heat labile toxin, respectively. Pathogen levels in the samples were significantly positively associated with high water conductivity and low water temperature. In addition, a set of bacterial isolates from water, soil and crops were analyzed by PCR for presence of the genes bla(CTX-M), bla(KPC), bla(NDM), bla(VIM) and bla(OXA-48). Four isolates were found to be positive for bla(CTX-M) genes and whole genome sequencing identified them as E. coli and one Enterobacter cloacae. The E. coli isolates belonged to the emerging, globally disseminated, multi-resistant E. coli lineages ST648, ST410 and ST162. The results indicate not only a high potential risk of transmission of diarrheal diseases by the consumption of contaminated water and vegetables but also the possibility of antibiotic resistance transfer from the environment to the community.
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| 16. |
- Jaen-Luchoro, Daniel, et al.
(författare)
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Corynebacterium genitalium sp. nov., nom. rev. and Corynebacterium pseudogenitalium sp. nov., nom. rev., two old species of the genus Corynebacterium described from clinical and environmental samples
- 2023
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Ingår i: Research in Microbiology. - : Elsevier BV. - 0923-2508. ; 174:1-2
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Tidskriftsartikel (refereegranskat)abstract
- Two Corynebacterium species were proposed decades ago, isolated from clinical samples and divided into biovars: "Corynebacterium genitalium" biovars I-V and "Corynebacterium pseudogenitalium" biovars C1 -C6. Several biovars have been re-classified as new species. Nevertheless, biovar I and C5, together with their respective specific epithets "Corynebacterium genitalium" and "Corynebacterium pseudogenitalium", remained not validly published after more than 40 years. Several more strains, temptatively classified as "C. genitalium" biovar I and "Corynebacterium pseudogenitalium" C5, have been isolated from clinical and environmental samples. Both species presented Gram-positive, non-spore forming rod-shaped cells, able to grow aerobically with CO2. Core-genome analysis identified "C. genitalium" to be most closely related to Corynebacterium tuscaniense, Corynebacterium urinipleomorphum, Corynebacterium aquatimens and C appendicis, and Corynebacterium gottingense as the most closely related species to "C. pseudogenitalium". Comprehensive genomic, genotypic, phenotypic analyses, as well as chemotaxonomic, support the proposal for "C. genitalium" and "C. pseudogenitalium" as distinct species within the genus Corynebacterium. The designated type strains of the two species are Furness 392-1T = ATCC 33030T = CCUG 38989T = CCM 9178T = DSM 113155T for C. genitalium sp. nov., nom. rev., and Furness 162-C2T = ATCC 33039T = CCUG 27540T = CCM 9177T = DSM 113154T for C. pseudogenitalium sp. nov., nom. rev. (c) 2022 The Author(s). Published by Elsevier Masson SAS on behalf of Institut Pasteur. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
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| 17. |
- Jaén-Luchoro, Daniel, et al.
(författare)
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Corynebacterium sanguinis sp. nov., a clinical and environmental associated corynebacterium.
- 2020
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Ingår i: Systematic and applied microbiology. - : Elsevier BV. - 1618-0984 .- 0723-2020. ; 43:1
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Tidskriftsartikel (refereegranskat)abstract
- Clinical and environmental-associated strains (n=17), genotypically related to Corynebacterium spp., yet distinct from any species of the genus Corynebacterium with validly published names, have been isolated during the last 20 years and tentatively identified as Corynebacterium sanguinis, although the combination, "Corynebacterium sanguinis" was never validly published. The comprehensive genotypic and phenotypic characterisations and genomic analyses in this study support the proposal for recognizing the species within the genus Corynebacterium, for which the name, Corynebacterium sanguinis sp. nov., is reaffirmed and proposed. Strains of Corynebacterium sanguinis are Gram-positive, non-motile, non-spore-forming, short, pleomorphic and coryneform bacilli, growing aerobically, with CO2. They contain mycolic acids, major respiratory menaquinones, MK-8 (II-H2) and MK-9 (II-H2), and polar lipids, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, phosphoglycolipid, glycolipids and a novel lipid that remains to be characterized and identified. Strains of Corynebacterium sanguinis are genotypically most similar to Corynebacterium lipophiliflavum, with 16S rRNA gene sequence similarities of 98.3% and rpoB sequence similarities of 94.9-95.2%. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis were able to clearly differentiate Corynebacterium sanguinis from the most closely related species. The genome size of Corynebacterium sanguinis is 2.28-2.37Mbp with 65.1-65.5mol% G+C content. A total of 2202-2318 ORFs were predicted, comprising 2141-2251 protein-encoding genes. The type strain is CCUG 58655T (=CCM 8873T=NCTC 14287T).
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| 18. |
- Jakobsson, Hedvig E, et al.
(författare)
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Draft Genome Sequence of Moraxella catarrhalis Type Strain CCUG 353T.
- 2016
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Ingår i: Genome Announcements. - 2169-8287. ; 4:3
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Tidskriftsartikel (refereegranskat)abstract
- Moraxella catarrhalis is a Gram-negative commensal and pathogenic bacterium found in the human respiratory tract. It is associated with otitis media and respiratory tract infections. Here, we report the draft genome sequence of M. catarrhalis type strain CCUG 353(T), composed of 18 contigs and a total size of 1.89 Mb.
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| 19. |
- Jakobsson, Hedvig E, et al.
(författare)
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Draft Genome Sequences of Six Strains of Streptococcus pneumoniae from Serotypes 5, 6A, 6B, 18C, 19A, and 23F
- 2017
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Ingår i: Genome Announcements. - 2169-8287. ; 5:14
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Tidskriftsartikel (refereegranskat)abstract
- Streptococcus pneumoniae is a pathogenic bacterium found most commonly in the respiratory tract of humans and is a common cause of pneumonia and bacterial meningitis. Here, we report the draft genome sequences of six S. pneumoniae strains: CCUG 1350, CCUG 7206, CCUG 11780, CCUG 33774, CCUG 35180, and CCUG 35272.
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| 20. |
- Jakobsson, Hedvig E, et al.
(författare)
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Proteotyping of Streptococcus pneumoniae, using tandem mass spectrometry for identification of biomarkers for species and strain differentiation
- 2016
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Ingår i: 11th International Meeting on Microbial Epidemiological Markers (IMMEM XI) 9 - 12 March 2016, Estoril, Portugal. - : European Society for Clinical Microbiology and Infectious Diseases.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Background. Streptococcus pneumoniae (pneumococcus) is the leading cause of community-acquired pneumonia and a major cause of morbidity and mortality worldwide. S. pneumoniae is phenotypically and genotypically similar to commensal species of the upper respiratory tract of the Streptococcus mitis-Group (viridans streptococci), S. mitis, S. oralis, and S. pseudopneumoniae, causing problems of identification in clinical microbiology laboratories. We have applied state-of-the-art proteomics techniques for Streptococcus spp. proteotyping; to detecting and characterizing expressed protein biomarkers for species-level identification, determination of antibiotic resistance and virulence biomarkers and strain typing for epidemiological analyses. Material and methods. The proteins of intact bacteria or cell fractions are bound to a membrane surface, using patented (WO2006068619) Lipid-based Protein Immobilization (LPITM) technology. Peptides are generated from the bound proteins, using enzymatic digestion, separated and analyzed, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The mass spectra profiles are compared to a database of reference peptide sequences. Subsequently, the identified peptides are compared to a database of reference genome sequences, all complete genomes of the NCBI Reference Sequence (RefSeq) Database. In this study, the type strains of the close-related mitis complex species S. pneumoniae (CCUG 28588T), S. mitis (CCUG 31611T), S. oralis (CCUG 13229T), S. psedopneumoniae (CCUG 49455T) and the more distantly-related S. pyogenes (CCUG 4207T) were analysed individually and in mixtures, to demonstrate proteotyping capability and differentiate closely related species,. Additionally, mixes containing different S. pneumoniae strains were analyzed. Results. Using proteotyping protocols, it was possible to detect and correctly identify S. pneumoniae from the closely related bacterial species, S. mitis, S. oralis S. psedopneumoniae and S. pyogenes, as well as different strains of S. pneumoniae by identification of unique discriminatory peptides. For successful proteotyping,a comprehensive and accurate genomic database is the key to obtaining reliable proteotyping data. Importantly, because of questionable classifications of sequenced genomes in the public databases, before incorporation of reference genomic sequence data for proteotyping, the genome sequences should be verified and confirmed for accurate classifications. Furthermore, it is also essential to include all relevant species with as many as 25 genomes in order to obtain a comprehensive coverage of coding sequences for accurate peptide matching and to be able to discriminate between the most closely related species. In this study, all genomes of the S. mitis-Group in the database were analyzed, using Average Nucleotide Identity Blast (ANIb) and S. mitis-Group strains that cannot be identified to the species level, using standard genotypic and phenotypic approaches, where characterized by proteotyping and whole genome sequencing to describe their taxonomy and to improve the database matching. Conclusions: Proteotyping, using LC-MS/MS, enabled the differentiation and identification of pneumococcus from its closely related species and sub-species-level strain discrimination, all from single MS analyses. The whole method will enhance the identification and characterization of microorganisms, allowing high-resolution discrimination of closely related species through the confident identification of new biomarkers, ultimately for cultivation-independent application to the analyses of clinical samples.
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| 21. |
- Karlsson, Roger, 1975, et al.
(författare)
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Discovery of Species-unique Peptide Biomarkers of Bacterial Pathogens by Tandem Mass Spectrometry-based Proteotyping
- 2020
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 19:3, s. 518-528
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Tidskriftsartikel (refereegranskat)abstract
- Mass spectrometry (MS) and proteomics offer comprehensive characterization and identification of microorganisms and discovery of protein biomarkers that are applicable for diagnostics of infectious diseases. The use of biomarkers for diagnostics is widely applied in the clinic and the use of peptide biomarkers is increasingly being investigated for applications in the clinical laboratory. Respiratory-tract infections are a predominant cause for medical treatment, although, clinical assessments and standard clinical laboratory protocols are time-consuming and often inadequate for reliable diagnoses. Novel methods, preferably applied directly to clinical samples, excluding cultivation steps, are needed to improve diagnostics of infectious diseases, provide adequate treatment and reduce the use of antibiotics and associated development of antibiotic resistance. This study applied nano-liquid chromatography (LC) coupled with tandem MS, with a bioinformatics pipeline and an in-house database of curated high-quality reference genome sequences to identify species-unique peptides as potential biomarkers for four bacterial pathogens commonly found in respiratory tract infections (RTIs): Staphylococcus aureus; Moraxella catarrhalis; Haemophilus influenzae and Streptococcus pneumoniae. The species-unique peptides were initially identified in pure cultures of bacterial reference strains, reflecting the genomic variation in the four species and, furthermore, in clinical respiratory tract samples, without prior cultivation, elucidating proteins expressed in clinical conditions of infection. For each of the four bacterial pathogens, the peptide biomarker candidates most predominantly found in clinical samples, are presented. Data are available via ProteomeXchange with identifier PXD014522. As proof-of-principle, the most promising species-unique peptides were applied in targeted tandem MS-analyses of clinical samples and their relevance for identifications of the pathogens, i.e. proteotyping, was validated, thus demonstrating their potential as peptide biomarker candidates for diagnostics of infectious diseases.
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| 22. |
- Karlsson, Roger, 1975, et al.
(författare)
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Proteotyping bacteria: Characterization, differentiation and identification of pneumococcus and other species within the Mitis Group of the genus Streptococcus by tandem mass spectrometry proteomics
- 2018
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Ingår i: Plos One. - : Public Library of Science (PLoS). - 1932-6203. ; 13:12
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Tidskriftsartikel (refereegranskat)abstract
- A range of methodologies may be used for analyzing bacteria, depending on the purpose and the level of resolution needed. The capability for recognition of species distinctions within the complex spectrum of bacterial diversity is necessary for progress in microbiological research. In clinical settings, accurate, rapid and cost-effective methods are essential for early and efficient treatment of infections. Characterization and identification of microorganisms, using, bottom-up proteomics, or "proteotyping", relies on recognition of species-unique or associated peptides, by tandem mass spectrometry analyses, dependent upon an accurate and comprehensive foundation of genome sequence data, allowing for differentiation of species, at amino acid-level resolution. In this study, the high resolution and accuracy of MS/MS-based proteotyping was demonstrated, through analyses of the three phylogenetically and taxonomically most closely-related species of the Mitis Group of the genus Streptococcus: i.e., the pathogenic species, Streptococcus pneumoniae (pneumococcus), and the commensal species, Streptococcus pseudopneumoniae and Streptococcus mitis. To achieve high accuracy, a genome sequence database used for matching peptides was created and carefully curated. Here, MS-based, bottom-up proteotyping was observed and confirmed to attain the level of resolution necessary for differentiating and identifying the most-closely related bacterial species, as demonstrated by analyses of species of the Streptococcus Mitis Group, even when S. pneumoniae were mixed with S. pseudopneumoniae and S. mitis, by matching and identifying more than 200 unique peptides for each species.
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| 23. |
- Karlsson, R., et al.
(författare)
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Proteotyping: Proteomic characterization, classification and identification of microorganisms - A prospectus
- 2015
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Ingår i: Systematic and Applied Microbiology. - : Elsevier BV. - 0723-2020. ; 38:4, s. 246-257
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Forskningsöversikt (refereegranskat)abstract
- Modern microbial systematics requires a range of methodologies for the comprehensive characterization, classification and identification of microorganisms. While whole-genome sequences provide the ultimate reference for defining microbial phylogeny and taxonomy, selected biomarker-based strategies continue to provide the means for the bulk of microbial systematic studies. Proteomics, the study of the expression of genes, as well as the structure and function of the resulting proteins, offers indirect measures of genome sequence data. Recent developments in applications of proteomics for analyzing microorganisms have paralleled the growing microbial genome sequence database, as well as the evolution of mass spectrometry (MS) instrumentation and bioinformatics. MALDI-TOF MS, which generates proteomic mass patterns for 'fingerprint'-based characterizations, has provided a marked breakthrough for microbial identification. However, MALDI-TOF MS is limited in the number of targets that can be detected for strain characterization. Advanced methods of tandem mass spectrometry, in which proteins and peptides generated from proteins, are characterized and identified, using LC-MS/MS, provide the ability to detect hundreds or thousands of expressed microbial strain markers for high-resolution characterizations and identifications. Model studies demonstrate the application of proteomics-based analyses for bacterial species- and strain-level detection and identification and for characterization of environmentally relevant, metabolically diverse bacteria. Proteomics-based approaches represent an emerging complement to traditional methods of characterizing microorganisms, enabling the elucidation of the expressed biomarkers of genome sequence information, which can be applied to 'proteotyping' applications of microorganisms at all taxonomic levels. (C) 2015 Elsevier GmbH. All rights reserved.
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| 24. |
- Karlsson, Roger, et al.
(författare)
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Proteotyping: Tandem Mass Spectrometry Shotgun Proteomic Characterization and Typing of Pathogenic Microorganisms
- 2016
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Ingår i: MALDI‐TOF and Tandem MS for Clinical Microbiology. - Chichester, UK : John Wiley & Sons, Ltd. - 9781118960226 ; , s. 419-450
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Proteotyping provides the means to identify and quantify the actual expression patterns of proteins and their associated pathways, which provides a more accurate picture of infectious agents and their pathogenic potential. Proteotyping, as an analytical method, is intimately correlated with genotypic or genomic data and offers an approach for a holistic characterization of microorganisms. Bioinformatics is vital for the analysis of the data generated by shotgun proteomics. This chapter describes the complete bioinformatics workflow necessary for proteotyping. Mass spectrometry (MS)-based shotgun proteomics analyses offer more detailed and comprehensive analyses of microorganisms. Two approaches may be applied: the so-called top-down and bottom-up proteomics. A major driver for the development and use of tandem MS and proteotyping in clinical settings will be the rapidly growing databases of whole genome reference sequences, which will refine microbial phylogeny and provide a foundation for proteomics-based identification.
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| 25. |
- Moore, Edward R.B. 1954, et al.
(författare)
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PROTEOTYPING: Tandem Mass Spectrometry Proteomics and Whole Genome Sequence-Based Diagnostics of Infectious Bacteria is Depedent upon a Reliable and Comprehensive Systematic Framework
- 2016
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Ingår i: Third Meeting on “Microbial Systematics and Metagenomics”. Bergey’s International Society for Microbial Systematics (BISMiS). September 12 - 15, 2016, Pune, India.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The global expansion of anti-microbial resistance (AMR) in bacteria, including human pathogens, presents major challenges for treatment and preventing the spread of infection. The World Health Organisation (WHO) has predicted the advent of infectious diseases for which no antibiotic treatment will be available [1]. With this outlook in the escalation of AMR, combined with continuing decline in new antibiotic discovery, development of innovative, reliable, rapid and costefficient analytical techniques for effective diagnostics and characterisations of infectious microorganisms is increasingly essential to prevent rising mortality and to reduce the costs associated with antibiotic-resistant infections. However, the routine methodologies used today for diagnosing infectious bacteria depend upon protocols that require prior cultivation from samples. Faced with patients exhibiting symptoms of infection, physicians typically resort to prescribing broad-spectrum antibiotics while they wait days or weeks for results from the laboratory. With increasing whole-genome DNA sequence data becoming available, MS-based proteomics also have increasingly been applied to biological studies. Proteomic analyses of bacterial cells may be considered indirect analyses of the genomes of bacteria. The ‘proteome’ comprises the entire set of proteins expressed by a cell, an organism or a biological system. ‘Proteotyping’ [2], using state-ofthe-art LC-MS/MS analyses of generated cellular peptides, enables identification of the most closely related bacterial species and sub-species-level strain discrimination, AMR- and virulence-factors, from single MS analyses. Comprehensive and accurate genome sequence data is the key to obtaining accurate peptide matching and to be able to discriminate between the most closely related species. In this study, genome sequences were analysed, using Average Nucleotide Identity Blast (ANIb) and taxon-specific MLSA to assess their reliabilities. Critically, significant numbers of sequenced genomes in the public databases exhibited questionable identifications. Characterisations and identifications of responsible agents of infectious disease have relied heavily upon established systematic frameworks and the documented features of well-described microbial species. As methodologies, such as whole-genome sequencing and MS proteomics are developed to enable more comprehensive, detailed and complex analyses, comprehensive databases derived from a reliable systematic framework are essential. References [1] World Health Organization (WHO). (2014) Antimicrobial Resistance: Global Report on Surveillance. ISBN: 978-92-4-15674-8. [2] Karlsson et al. [2015) Proteotyping: Proteomic characterization, classification and identification of microorganisms – A prospectus. Syst. Appl. Microbiol. 38: 246-257.
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| 26. |
- Salvà-Serra, Francisco, 1989, et al.
(författare)
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Complete genome sequences of Streptococcus pyogenes type strain reveal 100%-match between PacBio-solo and Illumina-Oxford Nanopore hybrid assemblies
- 2020
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- We present the first complete, closed genome sequences of Streptococcus pyogenes strains NCTC 8198(T) and CCUG 4207(T), the type strain of the type species of the genus Streptococcus and an important human pathogen that causes a wide range of infectious diseases. S. pyogenes NCTC 8198(T) and CCUG 4207(T) are derived from deposit of the same strain at two different culture collections. NCTC 8198(T) was sequenced, using a PacBio platform; the genome sequence was assembled de novo, using HGAP. CCUG 4207(T) was sequenced and a de novo hybrid assembly was generated, using SPAdes, combining Illumina and Oxford Nanopore sequence reads. Both strategies yielded closed genome sequences of 1,914,862 bp, identical in length and sequence identity. Combining short-read Illumina and long-read Oxford Nanopore sequence data circumvented the expected error rate of the nanopore sequencing technology, producing a genome sequence indistinguishable to the one determined with PacBio. Sequence analyses revealed five prophage regions, a CRISPR-Cas system, numerous virulence factors and no relevant antibiotic resistance genes. These two complete genome sequences of the type strain of S. pyogenes will effectively serve as valuable taxonomic and genomic references for infectious disease diagnostics, as well as references for future studies and applications within the genus Streptococcus.
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| 27. |
- Salvà-Serra, Francisco, 1989, et al.
(författare)
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Detection of "Xisco" gene for identification of Streptococcus pneumoniae isolates
- 2018
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Ingår i: Diagnostic Microbiology and Infectious Disease. - : Elsevier BV. - 0732-8893 .- 1879-0070. ; 90:4, s. 248-250
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Tidskriftsartikel (refereegranskat)abstract
- We describe a PCR-assay differentiating Streptococcus pneumoniae from closely-related species of the Mitis group of the genus Streptococcus and identification of pneumococcus clinical isolates, based on the "Xisco" gene discriminatory marker. The complete "Xisco" gene sequence was observed in all S. pneumoniae genomes analyzed and absent in all non-pneumococcus genomes.
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| 28. |
- Salvà-Serra, Francisco, 1989, et al.
(författare)
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Draft Genome Sequence of Streptococcus gordonii Type Strain CCUG 33482T.
- 2016
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Ingår i: Genome Announcements. - 2169-8287. ; 4:2
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Tidskriftsartikel (refereegranskat)abstract
- Streptococcus gordoniitype strain CCUG 33482(T)is a member of theStreptococcus mitisgroup, isolated from a case of subacute bacterial endocarditis. Here, we report the draft genome sequence ofS. gordoniiCCUG 33482(T), composed of 41 contigs of a total size of 2.15 Mb with 2,061 annotated coding sequences.
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| 29. |
- Salvà-Serra, Francisco, 1989, et al.
(författare)
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Proteotyping for Rapid Identifications of Clinically-Relevant Infectious Bacteria
- 2016
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Ingår i: Programme of the XXXV ECCO Meeting 2016.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Aims The development and application of novel identifications and diagnostics of pathogenic bacteria, virulence and antibiotic resistance factors, to enhance treatment of infectious diseases and to address the pandemic of antimicrobial resistance (1). To apply mass spectrometry (MS)-based ‘proteotyping’, a rapid proteomic-genomic method to identify and use cell biomarkers for pathogen identification and detection of targeted metabolic functions (www.tailored-treatment.eu/). Methods and results The proteins of intact bacterial cells or cell-fractions are bound to a membrane surface, using the patented (WO2006068619) Lipid-based Protein Immobilization (LPI) technology. Peptides are generated from bound proteins, using enzymatic digestion, separated and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The MS profiles are compared to those of reference peptide sequences and the peptide sequences are compared against a curated in-house database of genome sequences. Conclusions Analyses of bacterial cell peptides identified protein biomarkers of infectious bacteria, at the species-level, virulence and antibiotic resistance factors. Model samples have been ‘proteotyped’, using well-characterized reference strains, and an enhanced whole-genome sequence database, to demonstrate ‘proof-of-concept’, and the method been applied directly to the analyses of clinical samples, without prior cultivation. Significance of study Proteotyping demonstrates the potential for proteomics-based analyses for detecting expressed genomic markers of bacterial species, virulence and antibiotic resistance, for the identifications and diagnostics of infectious microorganisms. References Cohen A, Bont L, Engelhard D, Moore E, Fernández D, Kreisberg-Greenblatt R, Oved K, Eden E, Hayes J (2015). A multifaceted 'omics' approach for addressing the challenge of antimicrobial resistance. Future Microbiol. 10:365-376
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| 30. |
- Skovbjerg, Susann, 1973, et al.
(författare)
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Sequetyping Validation of Streptococcus pneumoniae Clinical Isolates
- 2016
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Ingår i: 10th International Symposium on Pneumococci and Pneumococcal Diseases. Glasgow, UK; 20160626-30.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Introduction: Serotyping of Streptococcus pneumoniae (pneumococcus) is required to monitor epidemiological trends following introductions of pneumococcal conjugate vaccines. However, new techniques are needed to lower the costs and work-load for obtaining pneumococcal serotype information, especially in low-income settings. Here, we validated a modified protocol of sequetyping for S. pneumoniae serotype identification. Methods: Forty-six strains of S. pneumoniae, isolated from blood cultures at the Sahlgrenska University Hospital, Gothenburg, Sweden, were analyzed. Serotypes of the strains were identified by Sanger sequencing of the regulatory gene cpsB of the capsulation locus of S. pneumoniae, using a modification of an earlier protocol (Leung et. al. 2012). To increase the coverage of the 1,000 bp sequence length of the cpsB gene, two additional sequencing primers in the central region of the gene were designed. The sequences were compared to available reference sequences, using the NCBI BLAST function. The results were compared to those obtained by multiplex qPCR, modified from a protocol published by the U.S. Centres of Disease Control and Prevention, and with the results obtained by gel diffusion and/or Quellung reactions, performed by the Public Health Agency of Sweden. Results: In total, 22 different serotypes/serotype groups were acquired by sequetyping of the 46 isolates. Contrary to the 74% of isolates that were serotyped/serotype grouped by qPCR, sequetyping could identify the serotype/serotype group in 98% of the isolates. Of the serotypes correctly identified by sequetyping, 32% were identified to a single serotype, whereas in 68% of the isolates, a single serotype could not be assigned. The serotype groups that could not be differentiated further included 6A/6B/6C/6D and 9A/9V. Conclusion: Sequetyping is a sensitive technique for serotype identification. However, in many cases, the difference between serotypes is detected in only one or two base-pairs, or the sequences are identical, making a definitive serotype identification difficult. To obtain serotype results in these cases, additional PCRs for differentiating particular serotypes/groups are required.
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| 31. |
- Tamire, Mulugeta, et al.
(författare)
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Household fuel use and its association with potential respiratory pathogens among healthy mothers and children in Ethiopia
- 2022
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 17:11
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Tidskriftsartikel (refereegranskat)abstract
- Background Over 90% of Ethiopians still rely on solid fuels for cooking food. The pollution from the burning process causes adverse respiratory outcomes including respiratory infections. This study aimed to assess the association of the pollution with nasopharyngeal occurrence of potential pathogens. Methods We conducted a comparative cross-sectional study in urban and rural settings in Ethiopia in 2016. Questionnaire-based data were collected from 168 mothers and 175 children aged below two years. Multiplex real-time PCR assays were performed on nasopharyngeal secretions for detection of bacteria and viruses and for the identification of pneumococcal serotypes/groups. Results High rates of bacteria and viruses in the nasopharynx were detected by PCR among both the children and the mothers. Among the detected viruses, enterovirus was more commonly detected among rural children than among children from urban areas. Streptococcus pneumoniae and Haemophilus influenzae were both more prevalent among children and mothers from rural areas compared with urban groups and among those using solid fuels compared with cleaner fuel users. Children from rural households using solid fuels and children whose mothers had educational status below high school had four times higher odds for detection of S. pneumoniae compared with those households using cleaner energy or those children having mothers with a higher educational status, respectively. One or more serotype/serogroup was identified in about 40% of the samples that were positive for pneumococci. Out of all identified serotypes/serogroups, 43% in the children and 45% in the mothers belonged to PCV13, indicating the larger majority of detected pneumococci being non-PCV13 serotypes. Conclusion This study presented a high carriage rate of S. pneumoniae and H. influenzae among both children and their mothers, especially in rural areas and among solid fuel users. Thus, interventions should target cleaner energy sources to the public and promote maternal education.
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