Surface Proteins of Lactobacillus crispatus : Adhesive Properties and Cell Wall Anchoring

Bacterial surface-associated proteins are important in communication with the environment and bacteria-host interactions. In this thesis work, surface molecules of Lactobacillus crispatus important in host interaction were studied. The L. crispatus strains of the study were known from previous studies to be efficient in adhesion to intestinal tract and ECM. L. crispatus JCM 5810 possess an adhesive surface layer (S-layer) protein, whose functions and domain structure was characterized. We cloned two S-layer protein genes (cbsA; collagen-binding S-layer protein A and silent cbsB) and identified the protein region in CbsA important for adhesion to host tissues, for polymerization into a periodic layer as well as for attachment to the bacterial cell surface. The analysis was done by extensive mutation analysis and by testing His6-tagged fusion proteins from recombinant Escherichia coli as well as by expressing truncated CbsA peptides on the surface of Lactobacillus casei. The N-terminal region (31-274) of CbsA showed efficient and specific binding to collagens, laminin and extracellular matrix on tissue sections of chicken intestine. The N-terminal region also contained the information for formation of periodic S-layer polymer. This region is bordered at both ends by a conserved short region rich in valines, whose substitution to leucines drastically affected the periodic polymer structure. The mutated CbsA proteins that failed to form a periodic polymer, did not bind collagens, which indicates that the polymerized structure of CbsA is needed for collagen-binding ability. The C-terminal region, which is highly identical in S-layer proteins of L. crispatus, Lactobacillus acidophilus and Lactobacillus helveticus, was shown to anchor the protein to the bacterial cell wall. The C-terminal CbsA peptide specifically bound to bacterial teichoic acid and lipoteichoic acids. In conclusion, the N-terminal domain of the S-layer protein of L. crispatus is important for polymerization and adhesion to host tissues, whereas the C-terminal domain anchors the protein to bacterial cell-wall teichoic acids. Lactobacilli are fermentative organisms that effectively lower the surrounding pH. While this study was in progress, plasminogen-binding proteins enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were identified in the extracellular proteome of L. crispatus ST1. In this work, the cell-wall association of enolase and GAPDH were shown to rely on pH-reversible binding to the cell-wall lipoteichoic acids. Enolase from L. crispatus was functionally compared with enolase from L. johnsonii as well as from pathogenic streptococci (Streptococcus pneumoniae, Streptococcus pyogenes) and Staphylococcus aureus. His6-enolases from commensal lactobacilli bound human plasminogen and enhanced its activation by human plasminogen activators similarly to, or even better than, the enolases from pathogens. Similarly, the His6-enolases from lactobacilli exhibited adhesive characteristics previously assigned to pathogens. The results call for more detailed analyses of the role of the host plasminogen system in bacterial pathogenesis and commensalism as well of the biological role and potential health risk of the extracellular proteome in lactobacilli.Bakteerien pintaproteiinit ovat tärkeitä bakteerien vuorovaikutuksessa isäntänsä, kuten ihmisen kanssa. Tässä työssä tutkittiin Lactobacillus crispatus bakteerin pintaproteiineja ja niiden merkitystä isäntä-bakteeri vuorovaikutuksessa, kuten bakteerin tarttumisessa suoliston limakalvoille. Laktobasillit kuuluvat maitohappobakteereihin, joita käytetään yleisesti lihan ja kasvisten valmistamisessa käymisreaktiolla sekä meijeriteollisuudessa mm. jogurtin valmistuksessa. Laktobasilleilla on havaittu monia terveyttä edistäviä vaikutuksia, ja niitä käytetäänkin yleisesti probioottisissa eli terveyttä edistävissä valmisteissa. Tutkimuksessani osoitettiin Lactobacillus crispatus bakteerien S-kerros -pintaproteiinin merkitys bakteerin tarttumisessa suolistoon sekä tutkittiin tarkoin proteiinin molekylaarinen rakenne. Proteiinin ensimmäiset 274 aminohappoa muodostavat polymeerisen S-kerros rakenteen ja ne tarvitaan bakteerin sitoutumiseen suoliston pintaan. Sen sijaan proteiinin loppupään 123 aminohappoa sitoutuvat bakteerin soluseinän ainesosiin, lipoteikohappoihin, ja siten kiinnittävät proteiinin bakteerin soluseinään. Laktobasillit tuottavat kasvaessaan maitohappoa, joka laskee ympäristön pH-arvoa. Työssäni osoitettiin, että kaksi muuta Lactobacillus crispatus bakteerin pintaproteiinia, enolaasi ja glyseraldehydi-3-fosfaatti-dehydrogenaasi (GAPDH), ovat sitoutuneet bakteerin soluseinään happamissa olosuhteissa, mutta irtoavat solusta korkeassa pH:ssa. Lisäksi työssä verrattiin enolaasi-proteiineja laktobasilleilla ja tautia aiheuttavilla bakteereilla, ja havaittiin, että enolaasi-proteiineilla on laktobasilleilla samanlaisia ominaisuuksia kuin tautia aiheuttavien bakteerien enolaaseilla. Tämä tutkimus valottaa pintaproteiinien roolia laktobasillien terveyttä edistävissä ominaisuuksissa sekä kuvaa mekanismin, joilla laktobasillit muokkaavat pintarakennettaan ympäristön muuttuessa

Clinical use of fungal PCR from deep tissue samples in the diagnosis of invasive fungal diseases : a retrospective observational study

Objectives: To assess the clinical use of panfungal PCR for diagnosis of invasive fungal diseases (IFDs). We focused on the deep tissue samples. Methods: We first described the design of panfungal PCR, which is in clinical use at Helsinki University Hospital. Next we retrospectively evaluated the results of 307 fungal PCR tests performed from 2013 to 2015. Samples were taken from normally sterile tissues and fluids. The patient population was nonselected. We classified the likelihood of IFD according to the criteria of the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG), comparing the fungal PCR results to the likelihood of IFD along with culture and microscopy results. Results: There were 48 positive (16%) and 259 negative (84%) PCR results. The sensitivity and specificity of PCR for diagnosing IFDs were 60.5% and 91.7%, respectively, while the negative predictive value and positive predictive value were 93.4% and 54.2%, respectively. The concordance between the PCR and the culture results was 86% and 87% between PCR and microscopy, respectively. Of the 48 patients with positive PCR results, 23 had a proven or probable IFD. Conclusions: Fungal PCR can be useful for diagnosing IFDs in deep tissue samples. It is beneficial to combine fungal PCR with culture and microscopy. M. Ala-Houhala, Clin Microbiol Infect 2018;24:301 (C) 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.Peer reviewe

Evaluation of three molecular carbapenemase tests : Eazyplex SuperBug complete B, Novodiag CarbaR plus , and Amplidiag CarbaR plus MCR

Background: Carbapenemase-producing Gram-negative bacilli, i.e., Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter, are of increased concern for the public health around the world. There is urgent need for rapid and accurate tests in order to provide correct treatment and to prevent bacterial spread in healthcare settings. Methods: The aim of this study was to evaluate three commercial multiplex carbapenemase tests with CE-IVD marking: Eazyplex SuperBug complete B (AmplexDiagnostics), Novodiag CarbaR+ (Mobidiag), and Amplidiag CarbaR+MCR (Mobidiag). All these tests recognize KPC, NDM, OXA-48/181 group, VIM, OXA-23 group, and OXA-24/40 group, and Novodiag CarbaR+ and Amplidiag CarbaR+MCR additionally recognize IMP, OXA-51 group (with promoter located within ISAbaI), OXA-58 group, and MCR, and Amplidiag CarbaR+MCR further recognizes GES (carbapenemase-type only). Results: The sensitivities and specificities of these tests with bacterial isolates were 100%. The sensitivity directly from clinical samples was 100%, but the specificity was lower, which is simply explained by the higher sensitivity of the molecular methods compared with culture method. Conclusions: Overall, these CE-IVD marked tests provide a good alternative in the detection of carbapenemase-producing organisms.Peer reviewe

Bacterial, viral and parasitic pathogens analysed by qPCR: Findings from a prospective study of travellers' diarrhoea

Background: The diagnostics of travellers' diarrhoea (TD) has been revolutionised by multiplex qPCR assays. While mostly of bacterial aetiology, viruses and parasites account for the disease among 10-20% of travellers. Despite this, prospective studies applying qPCR assays remain scarce that cover not only bacteria, such as the various diarrhoeagenic Escherichia coli (DEC), but also viral and parasitic pathogens. Method: We analysed by qPCR pre- and post-travel stool samples of 146 Finnish travellers for bacterial, viral and parasitic pathogens: enteropathogenic (EPEC), enteroaggregative (EAEC), enterotoxigenic (ETEC), enterohaemorrhagic (EHEC), and enteroinvasive (EIEC) E. coli; Shigella, Campylobacter, Salmonella, Yersinia and Vibrio cholerae; norovirus G1 and G2, rotavirus, enteroviruses, and sapovirus; and Giardia lamblia, Entamoeba histolytica, and Cryptosporidium. Symptoms and medication data during travel were collected by questionnaires. Results: We detected bacterial pathogens in 102/146 samples (69.9%; EAEC, EPEC, ETEC most common), viral ones in 13 (8.9%; norovirus most common), and parasitic ones in one (0.7%; Giardia). Noroviruses were associated with severe symptoms (23.5% versus non-severe 4.9%). In the TD group, 41.7% (5/12) of those with viral pathogens (vs. 13.3%; 11/83 without) took antibiotics. Conclusion: Viral pathogens, particularly noroviruses, prevail in severe TD. The symptoms of viral disease are often severe and lead to unwarranted use of antibiotics.Peer reviewe

Multiplex PCR detection of Cryptosporidium sp, Giardia lamblia and Entamoeba histolytica directly from dried stool samples from Guinea-Bissauan children with diarrhoea

Background: In developing countries, diarrhoea is the most common cause of death for children under five years of age, with Giardia lamblia, Cryptosporidium and Entamoeba histolytica as the most frequent pathogenic parasites. Traditional microscopy for stool parasites has poor sensitivity and specificity, while new molecular methods may provide more accurate diagnostics. In poor regions with sample storage hampered by uncertain electricity supply, research would benefit from a method capable of analysing dried stools. Methods: A real-time multiplex PCR method with internal inhibition control was developed for detecting Giardia lamblia, Cryptosporidium hominis/parvum and Entamoeba histolytica directly from stool specimens. Applicability to dried samples was checked by comparing with fresh ones in a small test material. Finally, the assay was applied to dried specimens collected from Guinea-Bissauan children with diarrhoea. Results: The PCR's analytical sensitivity limit was 0.1 ng/ml for G. lamblia DNA, 0.01 ng/ml for E. histolytica DNA and 0.1 ng/ml for Cryptosporidium sp. In the test material, the assay performed similarly with fresh and dried stools. Of the 52 Guinea-Bissauan samples, local microscopy revealed a parasite in 15%, while PCR detected 62% positive for at least one parasite: 44% of the dried samples had Giardia, 23% Cryptosporidium and 0% E. histolytica. Conclusions: Our new multiplex real-time PCR for protozoa presents a sensitive method applicable to dried samples. As proof of concept, it worked well on stools collected from Guinea-Bissauan children with diarrhoea. It provides an epidemiological tool for analysing dried specimens from regions poor in resources.Peer reviewe

Impact of pre-operative antimicrobial treatment on microbiological findings from endocardial specimens in infective endocarditis

Treatment of infective endocarditis (IE) should be initiated promptly. This might hamper the chances to identify the causative organism in blood cultures. Microbiological sampling of infected valve in patients undergoing surgery might identify the causative organism. The impact of pre-operative antimicrobial treatment on the yield of valve samples is not known. This study evaluated the impact of the duration of the pre-operative antibiotic treatment on valve culture and 16S rRNA PCR findings from resected endocardial samples. Patients meeting the modified Duke criteria of definite or possible IE and undergoing valve surgery due to IE during 2011–2016 were included from Southern Finland. Eighty-seven patients were included. In patients with shorter than 2 weeks of pre-operative antimicrobial treatment, PCR was positive in 91% (n = 42/46) and valve culture in 41% (n = 19/46) of cases. However, in patients who had 2 weeks or longer therapy before operation, PCR was positive in 53% (n = 18/34) and all valve cultures were negative. In 14% of patients, PCR had a diagnostic impact. In blood-culture negative cases (n = 13), PCR could detect the causative organism in ten patients (77%). These included five cases of Bartonella quintana, one Tropheryma whipplei, and one Coxiella burnetii. Long pre-operative antimicrobial treatment was shown to have a negative impact on microbiological tests done on resected endocardial material. After 2 weeks of therapy, all valve cultures were negative, but PCR was positive in half of the cases. PCR aided in diagnostic work-up, especially in blood culture negative cases.Peer reviewe

Effects of multidomain lifestyle intervention on frailty among older men and women - a secondary analysis of a randomized clinical trial

Abstract Background Frailty is a common geriatric syndrome associated with poor clinical outcomes. Effectiveness of lifestyle intervention programmes among frail older people has been examined earlier, but effects of interventions on prevention of frailty have been rarely studied. The aim of this study was to investigate to what extent the multidomain lifestyle intervention in the Finnish Geriatric Intervention Study to Prevent Cognitive Impairment and Disability (FINGER) affected changes in frailty status among older men and women at risk of cognitive disorders. Methods The 2-year multidomain lifestyle intervention trial including simultaneous nutritional counseling, physical exercise, cognitive training and social activity, and management of metabolic and vascular risk factors, was conducted among 1259 older people (mean age 68.9 years). A modified Fried’s frailty phenotype (weight loss, exhaustion, weakness, slowness, and low physical activity) was used to assess frailty at baseline and after the 2-year intervention. Participants with one or more components of the frailty phenotype were classified as pre-frail or frail. A multinomial regression model was applied to investigate efficacy of the intervention on frailty. Results We observed a favorable trend in reversing frailty among older men with the intervention. Pre-frail or frail men in the intervention group had higher probability of being non-frail after the intervention (44%) than pre-frail or frail men in the control group (30%) (p = 0.040). Among men, the intervention was especially beneficial in terms of increasing physical activity. Among women, multidomain lifestyle intervention did not affect the frailty status. Conclusion Modifying lifestyle-related factors may have potential to reverse first signs of frailty among older men. However, the intervention lasted only two years, therefore, research with longer follow-up is needed to see possible long-term effects of lifestyle management on the development of frailty.Abstract Background Frailty is a common geriatric syndrome associated with poor clinical outcomes. Effectiveness of lifestyle intervention programmes among frail older people has been examined earlier, but effects of interventions on prevention of frailty have been rarely studied. The aim of this study was to investigate to what extent the multidomain lifestyle intervention in the Finnish Geriatric Intervention Study to Prevent Cognitive Impairment and Disability (FINGER) affected changes in frailty status among older men and women at risk of cognitive disorders. Methods The 2-year multidomain lifestyle intervention trial including simultaneous nutritional counseling, physical exercise, cognitive training and social activity, and management of metabolic and vascular risk factors, was conducted among 1259 older people (mean age 68.9 years). A modified Fried’s frailty phenotype (weight loss, exhaustion, weakness, slowness, and low physical activity) was used to assess frailty at baseline and after the 2-year intervention. Participants with one or more components of the frailty phenotype were classified as pre-frail or frail. A multinomial regression model was applied to investigate efficacy of the intervention on frailty. Results We observed a favorable trend in reversing frailty among older men with the intervention. Pre-frail or frail men in the intervention group had higher probability of being non-frail after the intervention (44%) than pre-frail or frail men in the control group (30%) (p = 0.040). Among men, the intervention was especially beneficial in terms of increasing physical activity. Among women, multidomain lifestyle intervention did not affect the frailty status. Conclusion Modifying lifestyle-related factors may have potential to reverse first signs of frailty among older men. However, the intervention lasted only two years, therefore, research with longer follow-up is needed to see possible long-term effects of lifestyle management on the development of frailty