Browsing by Subject "Escherichia coli"

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  • Horesh, Gal; Blackwell, Grace A.; Tonkin-Hill, Gerry; Corander, Jukka; Heinz, Eva; Thomson, Nicholas R. (2021)
    Escherichia coli is a highly diverse organism that includes a range of commensal and pathogenic variants found across a range of niches and worldwide. In addition to causing severe intestinal and extraintestinal disease, E. coli is considered a priority pathogen due to high levels of observed drug resistance. The diversity in the E. coli population is driven by high genome plasticity and a very large gene pool. All these have made E. coli one of the most well- studied organisms, as well as a commonly used laboratory strain. Today, there are thousands of sequenced E. coli genomes stored in public databases. While data is widely available, accessing the information in order to perform analyses can still be a challenge. Collecting relevant available data requires accessing different sources, where data may be stored in a range of formats, and often requires further manipulation and processing to apply various analyses and extract useful information. In this study, we collated and intensely curated a collection of over 10 000 E. coli and Shigella genomes to provide a single, uniform, high- quality dataset. Shigella were included as they are considered specialized pathovars of E. coli. We provide these data in a number of easily accessible formats that can be used as the foundation for future studies addressing the biological differences between E. coli lineages and the distribution and flow of genes in the E. coli population at a high resolution. The analysis we present emphasizes our lack of understanding of the true diversity of the E. coli species, and the biased nature of our current understanding of the genetic diversity of such a key pathogen.
  • Kneis, David; Hiltunen, Teppo; Hess, Stefanie (2019)
    Horizontal gene transfer is an essential component of bacterial evolution. Quantitative information on transfer rates is particularly useful to better understand and possibly predict the spread of antimicrobial resistance. A variety of methods has been proposed to estimate the rates of plasmid-mediated gene transfer all of which require substantial labor input or financial resources. A cheap but reliable method with high-throughput capabilities is yet to be developed in order to better capture the variability of plasmid transfer rates, e.g. among strains or in response to environmental cues. We explored a new approach to the culture-based estimation of plasmid transfer rates in liquid media allowing for a large number of parallel experiments. It deviates from established approaches in the fact that it exploits data on the absence/presence of transconjugant cells in the wells of a well plate observed over time. Specifically, the binary observations are compared to the probability of transconjugant detection as predicted by a dynamic model. The bulk transfer rate is found as the best-fit value of a designated model parameter. The feasibility of the approach is demonstrated on mating experiments where the RP4 plasmid is transfered from Serratia marcescens to several Escherichia coil recipients. The methods uncertainty is explored via split sampling and virtual experiments.
  • Yrjänheikki, Ulla (Helsingfors universitet, 2019)
    Background: The World Health Organization (WHO) outlined in their report published in 2014 that antimicrobial resistance (AMR) is a real public health threat worldwide and the actions against it should be taken. Otherwise, the post-antibiotic era where common community-acquired infections can lead to death, could hypothetically become true. The discovery and development of novel antibiotics (ATBs) against Gram-negative bacteria (GNB)-related infections is difficult due to a dual defence mechanism: the extra protection barrier called the outer membrane and efflux pumps which GNB utilize to protect themselves against external noxious compounds. Efflux pumps are expressed at the basal level in GNB, such as E. coli, but when exposed to sub-inhibitory concentrations of ATBs and the intrinsic extruding capacity is exceeded, GNB start overexpressing these “so-called” multi-drug resistance (MDR) efflux pumps. The most abundant and studied MDR efflux pump in E. coli is a tripartite protein complex AcrAB-TolC which traverse through the bacterial cell envelope and is capable of extruding a broad range of structurally unrelated compounds, thus leading to cross-resistance against several classes of ATBs. It has been suggested that antibacterial activity of existing ATBs could be restored again by inhibiting increased efflux activity through efflux pump inhibitors (EPIs). Objectives: Define the optimal assay conditions and a positive control (EPI) to be used in high throughput screening (HTS) of novel EPIs. The assay consists of one E. coli strain of clinical relevance with high intrinsic efflux activity, one ATB and one EPI, both of them at specific concentrations defined during this study. Methods: The intrinsic efflux activities of seven E. coli strains were studied by Hoechst 33342 (H33342) accumulation assay, both in the absence and presence of five commercially available EPIs. The same assay was used in the dose-response studies in which an optimal concentration of EPIs was identified for further to be utilized in the checkerboard assays. The minimum inhibitory concentrations (MICs) were determined by broth microdilution method according to Clinical and Laboratory Standards Institute. The synergistic effects of ATB and EPI in terms of decreasing the intrinsic MIC value of the ATB were determined in the checkerboard assays partially performed by the Biomek i7 Automated Workstation. The data was analysed by using Microsoft Excel and IBM SPSS Statistics, version 25. Results and discussion: E. coli ATCC 25922 had statistically significantly the highest efflux activity of all wild-type pathogenic and non-pathogenic E. coli strains. However, when H33342 accumulation assay was carried out in conjunction with EPIs, E. coli BAA1161 (uropathogenic strain) had the highest median increase in the intracellular level of H33342. Mefloquine showed to be the most potent of all EPIs at the tested concentrations. However, mefloquine increased the intracellular H33342 accumulation even in efflux-deficient E. coli JW5503 (ΔtolC), thus possible additional modes of action or inhibitory activity towards other efflux pumps might exist. Dose-response studies carried out in ΔtolC E. coli JW5503 suggested that CCCP at 1.25 g/ml and mefloquine at 0.5 g/ml were the optimal concentrations. However, for mefloquine, when tested at 0.5 g/ml, the intracellular level of H33342 was not increased in six remaining E. coli strains. Therefore higher concentrations up to ½ MIC were tested in the checkerboard assays. In the antibacterial susceptibility testing, E. coli BAA1161 was the only strain showing resistance to tetracycline and piperacillin, resulting in MIC ratios (MIC wild-type/MIC mutant) of 512 to 2048. Piperacillin and ofloxacin, which showed a MIC ratio of 4 in two E. coli strains, were chosen to the checkerboard assays in which mefloquine reduced the intrinsic MIC of piperacillin by 16-fold and CCCP by 32-fold in E. coli BAA1161. Conclusions: E. coli BAA1161 was chosen to be used as a model strain in HTS due to the highest median increase in intracellular H33342 accumulation and also for being the only strain with resistance towards the ATBs tested. Mefloquine (16 g/ml) was the EPI of choice for the positive control in HTS because the synergistic effects observed between piperacillin and mefloquine were most probably explained by efflux pump inhibition and not by antibacterial activity of mefloquine itself. Piperacillin (256 g/ml) was selected to be used as an ATB in HTS because it was the only ATB which was potentiated by the tested EPIs.
  • Vanic, Zeljka; Rukavina, Zora; Manner, Suvi; Fallarero, Adyary; Uzelac, Lidija; Kralj, Marijeta; Klaric, Daniela Amidzic; Bogdanov, Anita; Raffai, Timea; Virok, Dezso Peter; Filipovic-Grcic, Jelena; Skalko-Basnet, Natasa (2019)
    Background: Efficient localized cervicovaginal antibacterial therapy, enabling the delivery of antibiotic to the site of action at lower doses while escaping systemic drug effects and reducing the risk of developing microbial resistance, is attracting considerable attention. Liposomes have been shown to allow sustained drug release into vaginal mucosa and improve delivery of antibiotics to bacterial cells and biofilms Azithromycin (AZI), a potent broad-spectrum macrolide antibiotic, has not yet been investigated for localized therapy of cervicovaginal infections, although it is administered orally for the treatment of sexually transmitted diseases. Encapsulation of AZI in liposomes could improve its solubility, antibacterial activity, and allow the prolonged drug release in the cervicovaginal tissue, while avoiding systemic side effects. Purpose: The objective of this study was to develop AZI-liposomes and explore their potentials for treating cervicovaginal infections. Methods: AZI-liposomes that differed in bilayer elasticity/rigidity and surface charge were prepared and evaluated under simulated cervicovaginal conditions to yield optimized liposomes, which were assessed for antibacterial activity against several planktonic and biofilm-forming Escherichia coli strains and intracellular Chlamydia trachomatis, ex vivo AZI vaginal deposition/penetration, and in vitro cytotoxicity toward cervical cells. Results: Negatively charged liposomes with rigid bilayers (CL-3), propylene glycol liposomes (PGL-2) and deformable propylene glycol liposomes (DPGL-2) were efficient against planktonic E. coli ATCC 700928 and K-12. CL-3 was superior for preventing the formation of E. coli ATCC 700928 and K-12 biofilms, with IC50 values (concentrations that inhibit biofilm viability by 50%) up to 8-fold lower than those of the control (free AZI). DPGL-2 was the most promising for eradication of already formed E. coli biofilms and for treating C. trachomatis infections. All AZI-liposomes were biocompatible with cervical cells and improved localization of the drug inside vaginal tissue compared with the control. Conclusion: The performed studies confirm the potentials of AZI-liposomes for localized cervicovaginal therapy.
  • Claassens, Nico J.; Finger-Bou, Max; Scholten, Bart; Muis, Frederieke; de Groot, Jonas J.; de Gier, Jan-Willem; de Vos, Willem M.; van der Oost, John (2019)
    Escherichia coli has been widely used as a platform microorganism for both membrane protein production and cell factory engineering. The current methods to produce membrane proteins in this organism require the induction of target gene expression and often result in unstable, low yields. Here, we present a method combining a constitutive promoter with a library of bicistronic design (BCD) elements, which enables inducer-free, tuned translation initiation for optimal protein production. Our system mediates stable, constitutive production of bacterial membrane proteins at yields that outperform those obtained with E. coli Lemo21(DE3), the current gold standard for bacterial membrane protein production. We envisage that the continuous, fine-tunable, and high-level production of membrane proteins by our method will greatly facilitate their study and their utilization in engineering cell factories.
  • Dusa, Filip; Chen, Wen; Witos, Joanna; Wiedmer, Susanne Kristina (2019)
    The importance of using biomimicking membranes for various biological applications is rising, as such models are relevant for imitating real organisms. In addition, biomimicking membranes are usually much more repeatable in preparation and easier to handle during analysis than real organisms or biological membranes. In this work, we developed a method for the adsorption of intact small unilamellar Escherichia coli (E. cols) vesicles (Z-average size of 73 nm) on SiO2 substrate material. We describe the adsorption process based on the use of two surface sensitive techniques, i.e., nanoplasmonic sensing (NPS) and quartz crystal microbalance (QCM). The acquired data show that the adsorption follows a two-step process. The first step is a slow adsorption of E coil vesicle aggregates held together by 5 mM of calcium (Z-average size of 531 nm). The Z-average of the aggregates decreased almost three times when the calcium concentration was decreased to 0.1 mM. This suggests that the aggregates were disassembling to some extent when calcium was removed from the system. With both techniques, i.e., NPS and QCM, we observed a second rapid adsorption step after the solution was changed to deionized water. In this second step, the aggregates started to fall apart as the calcium concentration dropped, and the released vesicles started to adsorb onto unoccupied spots at the SiO2 surface of the sensors. Extensive release of mass from the surface was confirmed by QCM, where it was reflected by a sharp increase of frequency, while NPS, due to its lower sensing depth of a few tens of nanometers, did not record such a change. Taken together, we have developed a protocol to form a supported vesicle layer (SVL) of E coli vesicles on SiO2 surface using sodium 4-(2-hydroxyethyppiperazine-1-ethanesulfonate buffer, thus enabling the preparation of E coli biomimicking SVLs for interaction studies of compounds of interest. The immobilization happens via a two-step adsorption process.
  • Kapp, Karmen; Püssa, Tõnu; Orav, Anne; Roasto, Mati; Raal, Ain; Vuorela, Pia; Vuorela, Heikki; Tammela, Päivi (2020)
    Mentha spp. are used in the food and pharmaceutical industry; the plants are characterized by natural interspecies hybridization. In this study, knowledge of the chemical composition of Mentha spp. was broadened by focusing on plants grown in a geographically small region of Estonia. The antibacterial activity of Mentha spp. essential oils and water extracts was evaluated. Polyphenolic water extracts of M. x villosa Huds., M. x suaveolens Ehrh., and M. x gracilis Sole were tested for the first time on Escberichia coli and Staphylococcus aura's. Leaves of cultivated and wild-grown plants (n = 33) were collected. The microdistilled essential oil composition reflected the diversity within the genus Mentha. Determined by gas chromatography-mass spectrometry (MS), major compounds were cis-piperitone oxide, carvone, linalool, menthol, and menthofuran. Based on high-performance liquid chromatographyultraviolet-MS/MS analyses of the water extracts, no species-specific polyphenolic compounds could be proposed. Abundant polyphenols were rosmarinic acid, salvianolic acid B, and eriocitrin. Essential oils exhibited antibacterial activity on E. coli and S. aureus by the broth dilution method. Water extracts showed activity only against S. aureus. This study supports the use of Mentha spp. as health-promoting ingredients in food. However, further studies are still needed to widen the knowledge of the chemical composition of these plants.
  • Fu, Y; Qiao, W; Zhu, D; Wang, X; Liu, F; Xu, H; Saris, Per Erik Joakim; Kuipers, Osacar; Qiao, Mingqiang (2018)
    Nisin, an important bacteriocin from Lactococcus lactis subsp., is primarily active against various Gram-positive bacteria. Leucocin C, produced by Leuconostoc carnosum 4010, is a class IIa bacteriocin used to inhibit the growth of Listeria monocytogenes. Because two bacteriocins have different modes of action, the combined use of them could be a potential strategy for effective inhibition of foodborne pathogens. In this study, L. lactis N8-r-lecCI (N8 harboring lecCI gene) coexpressing nisin–leucocin C was constructed based on the food-grade carrier L. lactis N8. Production of both bacteriocins was stably maintained. Antimicrobial measurements showed that the recombinant strain is effectively against Listeria monocytogenes and Staphylococcus aureus and moderately against Salmonella enterica serovar Enteritidis and Escherichia coli because of its stronger antibacterial activity than the parental strain, this result first demonstrated that the co-expression of nisin and leucocin C results in highly efficient antimicrobial activity. The checkerboard assay showed that the antibacterial activity of L. lactis N8-r-lecCI supernatant was enhanced in the presence of low concentration of EDTA. Analysis of the scanning electron microscope image showed the biggest cellular morphology change in L. monocytogenes treated with a mixture of EDTA and L. lactis N8-r-lecCI supernatant. The practical effect was verified in pasteurized milk through time-kill assay. The L. lactis N8-r-lecCI strain expressing both nisin and leucocin C has a promising application prospect in pasteurized milk processing and preservation because of its strong antibacterial activity.
  • Kögler, Martin; Paul, Andrea; Anane, Emmanuel; Birkholz, Mario; Bunker, Alex; Viitala, Tapani; Maiwald, Michael; Junne, Stefan; Neubauer, Peter (2018)
    The application of Raman spectroscopy as a monitoring technique for bioprocesses is severely limited by a large background signal originating from fluorescing compounds in the culture media. Here, we compare time-gated Raman (TG-Raman)-, continuous wave NIR-process Raman (NIR-Raman), and continuous wave micro-Raman (micro-Raman) approaches in combination with surface enhanced Raman spectroscopy (SERS) for their potential to overcome this limit. For that purpose, we monitored metabolite concentrations of Escherichia coli bioreactor cultivations in cell-free supernatant samples. We investigated concentration transients of glucose, acetate, AMP, and cAMP at alternating substrate availability, from deficiency to excess. Raman and SERS signals were compared to off-line metabolite analysis of carbohydrates, carboxylic acids, and nucleotides. Results demonstrate that SERS, in almost all cases, led to a higher number of identifiable signals and better resolved spectra. Spectra derived from the TG-Raman were comparable to those of micro-Raman resulting in well-discernable Raman peaks, which allowed for the identification of a higher number of compounds. In contrast, NIR-Raman provided a superior performance for the quantitative evaluation of analytes, both with and without SERS nanoparticles when using multivariate data analysis. (c) 2018 American Institute of Chemical Engineers
  • Kapp, Karmen; Orav, Anne; Roasto, Mati; Raal, Ain; Püssa, Tõnu; Vuorela, Heikki; Tammela, Päivi; Vuorela, Pia (2020)
    Mint flavorings are widely used in confections, beverages, and dairy products. For the first time, mint flavoring composition of mint candies and food supplements (n=45), originating from 16 countries, as well as their antibacterial properties, was analyzed. The flavorings were isolated by Marcussons type micro-apparatus and analyzed by GC-MS. The total content of the mint flavoring hydrodistilled extracts was in the range of 0.01-0.9%. The most abundant compounds identified in the extracts were limonene, 1,8-cineole, menthone, menthofuran, isomenthone, menthol and its isomers, menthyl acetate. The antimicrobial activity of 13 reference substances and 10 selected mint flavoring hydrodistilled extracts was tested on Escherichia coli and Staphylococcus aureus by broth dilution method. Linalool acetate and (-)-carvone, as most active against both bacteria, had the lowest MIC (90) values. (+)-Menthyl acetate, (-)-menthyl acetate, and limonene showed no antimicrobial activity. Three of the tested extracts had antimicrobial activity against E. coli and 8 extracts against S. aureus . Their summary antimicrobial activity was not always in concordance with the activities of respective reference substances.
  • Sarfraz, Sonia; Mäntynen, Pilvi-Helinä; Laurila, Marisa; Suojanen, Juho; Saarnio, Juha; Rossi, Sami; Horelli, Jani; Kaakinen, Mika; Leikola, Junnu; Reunanen, Justus (2022)
    The aim of this study was to assess the biofilm formation of Streptococcus mutans, Staphylococcus aureus, Enterococcus faecalis, and Escherichia coli on titanium implants with CAD-CAM tooling techniques. Twenty specimens of titanium were studied: Titanium grade 2 tooled with a Planmeca CAD-CAM milling device (TiGrade 2), Ti6Al4V grade 5 as it comes from CAD-DMLS device (computer aided design-direct metal laser sintering device) (TiGrade 5), Ti6Al4V grade 23 as it comes from a CAD-CAM milling device (TiGrade 23), and CAD-DMLS TiGrade 5 polished with an abrasive disc (TiGrade 5 polished). Bacterial adhesion on the implants was completed with and without saliva treatment to mimic both extraoral and intraoral surgical methods of implant placement. Five specimens/implant types were used in the bacterial adhesion experiments. Autoclaved implant specimens were placed in petri plates and immersed in saliva solution for 30 min at room temperature and then washed 3x with 1x PBS. Bacterial suspensions of each strain were made and added to the specimens after saliva treatment. Biofilm was allowed to form for 24 h at 37 degrees C and the adhered bacteria was calculated. Tooling techniques had an insignificant effect on the bacterial adhesion by all the bacterial strains studied. However, there was a significant difference in biofilm formation between the saliva-treated and non-saliva-treated implants. Saliva contamination enhanced S. mutans, S. aureus, and E. faecalis adhesion in all material types studied. S. aureus was found to be the most adherent strain in the saliva-treated group, whereas E. coli was the most adherent strain in the non-saliva-treated group. In conclusion, CAD-CAM tooling techniques have little effect on bacterial adhesion. Saliva coating enhances the biofilm formation; therefore, saliva contamination of the implant must be minimized during implant placement. Further extensive studies are needed to evaluate the effects of surface treatments of the titanium implant on soft tissue response and to prevent the factors causing implant infection and failure.
  • Sorokina, Dina (Helsingfors universitet, 2015)
    Lactic acid bacteria (LAB) are generally recognized as safe micro-organisms and used in food preservation and as health promoting probiotics. Beside lactic acid, LAB produce several antimicrobial compounds of which especially bacteriocins provide new potential applications for food and pharmaceutical industries. Bacteriocins are ribosomally synthetized proteins or peptides with antimicrobial activity usually against closely related species. Whole genome sequencing project of lactic acid bacterium Lactococcus lactis N8 has revealed a new bacteriocin operon which consists of a bacteriocin gene and ABC transporter genes. Similar operon has been also found in several other L.lactis strains including IL1403. Peptides expressed by these bacteriocin genes belong to lactococcin 972 protein family according to their amino acid sequences. In this master’s thesis, these novel bacteriocin genes from L. lactis N8 and IL1403 were cloned into Escherichia coli with plasmid vectors. New bacteriocins were named encacin A and B. Strong inducible promoters were chosen to achieve high bacteriocin production. Encacins were expressed in cytosolic and periplasmic spaces to compare the effect of localization on antimicrobial activity of peptides. The prevalence of encacin genes among different L. lactis strains was also studied. Four of ten E. coli recombinant strains constructed during this study were shown to produce bacteriocins. Two of them, which produced encacins into periplasmic space, also appeared to be weakly active against L. lactis MG1614 strain. Therefore it seems that localization of encacins in E. coli bacterial cell has an impact on the bioactivity of peptides. Screening of bacteriocins genes showed that over 90 % of L. lactis stains bear encacin genes, from which encacin B is the more frequent form. More precise characterization of encacin genes and peptides may help to gain new information about qualities and mode of action of these novel potential bacteriocins.
  • Hiekkaranta, Milla; Styrman, Miia (Helsingfors universitet, 1996)
    Tutkimuksen tarkoituksena oli selvittää mikrobilääkehoidon vaikutusta E. coli-bakteerin aiheuttamassa utaretulehduksessa vastapoikineilla lehmillä. Useissa tutkimuksissa on osoitettu, ettei antibioottilääkitys olisi paranemiselle välttämätöntä. Sairauden oireet johtuvat pääosin elimistön tulehdusvasteesta bakteerinsoluseinämän endotoksiinimolekyyliä vastaan, eivätkä bakteerin ominaisuuksista. Ei-steroidi-kipulääkkeet(esim. fluniksiini) ovat osoittautuneet erittäin tehokkaiksi elimistön puolustusreaktion hillinnässä, joten niiden käyttö koliutaretulehduksen hoidossa on yleisesti hyväksyttyä. Varhaislypsykaudella olevien lehmien puolustuskyky on heikentynyt, joten niiden koliformitulehdukset saattavat kehittyä vakaviksi oireisiksi. Koliformeihin bakteereihin in vitro tehoavia sekä farmakokineettisesti lypsylehmälle soveltuvia antibiootteja on vähän. Tutkimusten perusteella fluorokinoloni-ryhmäin lukeutuva antibiootti enrofloksasiini on osoittautunut soveltuvan näiltä osin käytettäväksi E. coli -utaretulehdusten hoitoon. Kirjallisuuskatsauksessa käsitellään koliformeja bakteereja utaretulehduksen aiheuttajina sekä koliutaretulehduksen hoitoa ja mikrobilääkkeitä. Kokeessa kuusi lehmää tartutettiin kahdesti E. colt -bakteerikannalla, joka oli eristetty kliinisestä utaretulehduksesta. Hoitomuotoja oli kaksi: fluniksiini ja tiheä lypsy sekä nämä yhdistettynä enrofloksasiiniin. Toisella tartutuskerralla lehmien hoidot vaihdettiin, joten kukin yksilö toimi omana kontrollinaan. Taudinkulkua valvottiin seuraamalla oireita sekä ottamalla maito- ja verinäytteitä. Yleis- ja paikallisoireet, maidon tulehdusparametrit ja bakteerimäärät näyttivät muuttuvan nopeammin normaaleiksi enrofloksasiinilla hoidettujen ryhmässä, mutta tilastollisesti tulos ei ollut merkitsevä. Vähäisempi maitotuotoksen lasku enrofloksasiinilla hoidetuilla lehmillä oli ainoa tilastollisesti merkitsevä tulos. Kahden pelkästään fluniksiinilla hoidetun yksilön sairastuminen vakavaan endotoksemiaan kertoo mahdollisesta antibiootin tarpeellisuudesta varhaisen lypsykauden koliutaretulehduksen hoidossa.
  • Näsi, Päivi (Helsingfors universitet, 2007)
    Lisensiaatin tutkielma koostuu kirjallisuuskatsauksesta ja tutkimusosiosta. Kokeellinen osuus kuuluu osana ELL Leena Suojalan väitöskirjaprojektiin, jossa tuloksia käsitellään laajemmin. Tässä tutkielmassa keskitytään eläinten kliiniseen vasteeseen ja bakteriologiseen paranemiseen. Lypsylehmät saavat Escherichia coli–utaretulehduksen yleensä lähellä poikimista tai alkulypsykaudella. Tartunnan vakavuuteen vaikuttaa lehmän oma immuunivaste sekä bakteereiden määrä. Kolimastiitin mikrobilääkehoidon tekee ongelmalliseksi se, että endotoksiinivaste eli elimistön omat sytokiinit ja muut välittäjäaineet aiheuttavat kliiniset oireet. Kolimastiitin hoitoon on yleensä käytetty laajakirjoisia antibiootteja, vaikka joissakin tutkimuksissa on havaittu, että mikrobilääkehoito ei välttämättä paranna hoitotulosta. Lisääntynyt antibioottiresistenssi ja yleinen pyrkimys vähentää antibioottien käyttöä ovatkin asettaneet kyseenalaiseksi kolimastiitin rutiininomaisen antibioottihoidon. Kokeen tarkoituksena oli selvittää mikrobilääke- (enrofloksasiini) ja tukihoidon tai pelkän tukihoidon hoitovastetta kliinisen E. coli–utaretulehduksen hoidossa. Koe suoritettiin kenttäkokeena ja aineistoa lehmien hoitovasteesta kerättiin kolmen vuoden ajan. Kokeeseen osallistui yhteensä 183 lehmää. Eläinten bakteriologinen parantuminen arvioitiin kolmen viikon kuluttua hoidon aloituksesta otetusta maitonäytteestä. Tulosten mukaan ne eläimet, jotka saivat tukihoidon lisäksi myös antibioottia, paranivat hieman paremmin kuin pelkkää tukihoitoa saaneet eläimet. Antibioottihoidetuista lehmistä bakteriologisesti parantui 89,1 %, pelkkää tukihoitoa saaneista parantui 81,2 %. Kliinisen paranemisen tulos oli parempi pelkkää tukihoitoa saaneella ryhmällä, joista 57,3 % oli parantunut kliinisesti kolmen viikon kuluttua hoidon aloittamisesta. Antibiootti- ja tukihoitoa saaneesta ryhmästä kliinisesti parani 43,3 %. Tämän tutkimuksen perusteella näyttäisi siltä, että kolimastiitin hoitoon ei kannata käyttää antibioottihoitoa, vaan pelkkä tukihoito olisi riittävä. Antibioottihoito paransi hieman bakteriologisen paranemisen tulosta, mutta käytännön eläinlääkinnän kannalta paljon merkittävämpää on kliinisen paranemisen tulos, joka oli parempi pelkää tukihoitoa saaneella hoitoryhmällä. Molempien hoitoryhmien kliinisen paranemisen tulos oli kuitenkin varsin heikko, mikä kertoo kolimastiitin vakavuudesta; lehmä jää usein soluttamaan pitkäksikin aikaa ja maidontuotos voi laskea pysyvästi.
  • Järvinen, Hanna (Helsingfors universitet, 2006)
    Escherichia coli aiheuttaa usein vakavaoireisen utaretulehduksen. Tulehduksen voimakkuus riippuu lehmän oman puolustusjärjestelmän tilasta ja tartutusannoksesta. Vastapoikineet ja sairaat lehmät sairastuvat usein vakavasti, koska niiden immuunijärjestelmän teho on laskenut. Lehmän yleisoireet aiheutuvat endotoksiinin, gram-negatiivisten bakteerien soluseinän osan, käynnistämästä tulehdusvasteesta. Lehmälle nousee kuume, pötsin toiminta hidastuu ja maito muuttuu vetiseksi. E. coli –bakteerin hävittäminen utareesta ei yleensä ole lehmän puolustusjärjestelmälle ongelma. Mikrobilääkkeen käyttö koliutaretulehduksen hoitoon on edelleen kiistanalaista. Mikrobilääkehoito voi kuitenkin olla perusteltua puolustusjärjestelmältään heikkojen yksilöiden kohdalla. Intramammaarien käyttö ei tule kysymykseen voimakkaiden paikallisoireiden vuoksi. Yleishoitona käytettävistä mikrobilääkkeistä enrofloksasiini sopii parhaiten kolimastiitin hoitoon farmakokinetiikkansa ja -dynamiikkansa perusteella. Antibiootin käyttö tulisi kuitenkin tapauskohtaisesti punnita. Tulehduskipulääkkeiden käyttöä koliutaretulehduksen hoidossa on tutkittu runsaasti. Useilla lääkkeillä on havaittu kuumetta ja muita oireita lieventäviä vaikutuksia. Nestehoidosta on tehty vain pari tutkimusta, eikä selviä positiivisia vaikutuksia ole havaittu. Tiheää lypsyä yhdistettynä oksitosiiniin on jo pitkään käytetty kentällä kolitulehdusten hoidossa. Hoito perustuu oletettuun bakteeri- ja endotoksiinipitoisen maidon poistamiseen utareesta. Tutkimustuloksia aiheesta on kuitenkin vähän ja tulokset ovat osin ristiriitaisia. Tiheän lypsyn tehokkuutta ei ole tieteellisissä tutkimuksissa todistettu. Kun sairasta neljännestä lypsetään, maidon laskeutuminen käynnistyy kaikissa neljänneksissä. Tämä voi joissakin olosuhteissa altistaa muut neljännekset utaretulehdukselle, mikä on huomioitava tätä hoitotapaa suositeltaessa. Näiden syventävien opintojen kokeellinen osuus oli avustaminen pilottikokeessa, jossa tutkittiin tiheän lypsyn vaikutusta kokeellisessa E. coli -utaretulehduksessa kuudella Holstein-Friisiläisellä lehmällä. Osakokeitten toteutusjärjestys oli arvottu. Toisessa osakokeessa lehmän tartutettu neljännes lypsettiin tiheästi ja toinen osakoe toimi verrokkina. Kaikki eläimet hoidettiin fluniksiinimeglumiinilla. Tartutuksesta 12 tunnin kuluttua kaikilla lehmillä oli havaittavissa yleisoireita, jotka olivat toisella tartutuskerralla lievempiä. Tiheä lypsy toteutettiin lypsämällä lehmiä kahden tunnin välein 12 tunnin ajan alkaen oireiden havaitsemisesta. Verrokki-ryhmä lypsettiin normaalisti kahdesti päivässä. Kliinisen seurannan lisäksi eläimistä otettiin maito- ja verinäytteitä, joista mitattiin tulehdusvastetta. Kokeesta saatuja tuloksia on vaikea tulkita, koska maidontuotos ehtyi tulehtuneesta neljänneksestä niin paljon, että tiheää lypsyä ei voitu kunnolla toteuttaa. Kirjallisuuden perusteella tulehduskipulääkkeiden käyttöä koliutaretulehduksen hoidossa voi suositella. Vaikka nestehoidosta tehosta ei ole näyttöä, sitä voi vakavissa tapauksissa käyttää, kuten muidenkin vakavien tulehdussairauksien hoidossa. Tieteellistä näyttöä tiheän lypsyn edullisesta vaikutuksesta ei saatu, vaikka kokemusperäisesti sitä pidetään hyödyllisenä. Tärkeää on myös sairaan lehmän hyvä yleishoito.
  • Fraktman, Leea (Helsingfors universitet, 2016)
    Työn tarkoituksena on kuvata fasaanien kasvatusta, metsästystä ja käyttöä erityisesti Suomessa ja esittää olemassa olevaa tutkimustietoa zoonoottisten bakteerien esiintymistä fasaaneissa eri puolilla maailmaa. Työn kokeellisessa osuudessa selvitetään laboratorioanalyysein zoonoottisten bakteerien esiintymistä suomalaisissa tarhassa kasvatetuissa fasaaneissa. Tutkimus tuo esille uutta tietoa, koska Suomessa tapahtuvasta fasaanien kasvatuksesta ja lintujen tai kasvatustilojen lukumäärästä ei ole julkaistua ajantasaista tietoa saatavilla. Fasaanien kasvatukseen ja tarhaukseen ei myöskään ole virallista ohjetta. Patogeenisten bakteerien esiintyvyydestä ssuomalaisten fasaanien suolistossa ei ole aiempaa tutkimustietoa saatavilla. Zoonoottisten bakteerien kantajina fasaanit voivat aiheuttaa potentiaalisen riskin ympäristön kontaminoitumiselle ja ihmisten tautitartunnoille. Kokeet tehtiin fasaanien ulostenäytteistä, jotka otettiin 100 luonnosta metsästetyn tarhakasvatetun fasaanin suolistosta. Näytteistä tutkittiin yleisimpien elintarvikevälitteisten patogeenisten bakteerien esiintymistä. Tutkimuksen kohteena olivat shigatoksiinigeeniä (stx) kantava eli stx-positiivinen Escherichia coli -bakteeri (STEC), Salmonella spp. -bakteerit, Listeria spp. -bakteerit, Campylobacter spp. -bakteerit ja Yersinia spp. -bakteerit. Fasaanien ulostenäytteistä Salmonella- ja Yersinia-bakteerit osoitettiin sekä viljely- että reaaliaikaisella PCR-menetelmällä, Campylobacter- ja Listeria-bakteerit määritettiin vain viljelymenetelmällä ja STEC vain reaaliaikaisella polymeraasiketjureaktio eli PCR-menetelmällä. Näytteissä todettiin Salmonella spp. -, stx1-positiivisia Escherichia coli (STEC)-, Campylobacter jejuni -, Listeria monocytogenes -, Yersinia enterocolitica - ja Yersinia pseudotuberculosis -bakteereita. Eniten näytteissä esiintyi Campylobacter jejuni - ja Listeria monocytogenes -bakteereita, joiden esiintyvyys oli 10 %. Yersinia-suvun ail-positiivisia eli taudinaiheuttamiskykyisiä bakteereita todettiin 6 % näytteistä. Salmonella spp. -bakteerien prevalenssi oli 4 % ja stx1-positiivisten STEC-bakteerien 3 %. Tässä tutkimuksessa suomalaisten tarhassa kasvatettujen fasaanien ulostenäytteissä todettiin esiintyvän kaikkia tutkittuja zoonoottisia elintarvikevälitteisiä bakteerisukuja tai niiden DNA:ta (PCRmenetelmä). Koska fasaanin lihaa käytetään ihmisravinnoksi omaan käyttöön ja sitä voidaan myydä tarkastamattomana eteenpäin, on patogeenien kulkeutuminen ihmiseen ja zoonoottisen infektion puhkeaminen teoriassa mahdollista. Jatkotutkimuksia voitaisi tehdä fasaanien kanssa työskentelevien altistumisen ja sairastuvuuden selvittämiseksi ympäristön kontaminaation ja muiden eläinlajien tautiriskien kartoittamiseksi.
  • Murphy, Robert; Palm, Martin; Mustonen, Ville; Warringer, Jonas; Farewell, Anne; Parts, Leopold; Moradigaravand, Danesh (2021)
    Escherichia coli is a common bacterial species in the gastrointestinal tracts of warm-blooded animals and humans. Pathogenicity and antimicrobial resistance in E. coli may emerge via host switching from animal reservoirs. Despite its potential clinical importance, knowledge of the population structure of commensal E. coli within wild hosts and the epidemiological links between E. coli in nonhuman hosts and E. coli in humans is still scarce. In this study, we analyzed the whole-genome sequencing data of a collection of 119 commensal E. coli strains recovered from the guts of 55 mammal and bird species in Mexico and Venezuela in the 1990s. We observed low concordance between the population structures of E. coli isolates colonizing wild animals and the phylogeny, taxonomy, and ecological and physiological attributes of the host species, with distantly related E. coli strains often colonizing the same or similar host species and distantly related host species often hosting closely related E. coli strains. We found no evidence for recent transmission of E. coli genomes from wild animals to either domesticated animals or humans. However, multiple livestockand human-related virulence factor genes were present in E. coli of wild animals, including virulence factors characteristic of Shiga toxin-producing E. coli (STEC) and atypical enteropathogenic E. coli (aEPEC), where several isolates from wild hosts harbored the locus of enterocyte effacement (LEE) pathogenicity island. Moreover, E. coli isolates from wild animal hosts often harbored known antibiotic resistance determinants, including those against ciprofloxacin, aminoglycosides, tetracyclines, and beta-lactams, with some determinants present in multiple, distantly related E. coli lineages colonizing very different host animals. We conclude that genome pools of E. coli colonizing the guts of wild animals and humans share virulence and antibiotic resistance genes, underscoring the idea that wild animals could serve as reservoirs for E. coli pathogenicity in human and livestock infections. IMPORTANCE Escherichia coli is a clinically important bacterial species implicated in humanand livestock-associated infections worldwide. The bacterium is known to reside in the guts of humans, livestock, and wild animals. Although wild animals are recognized as potential reservoirs for pathogenic E. coli strains, the knowledge of the population structure of E. coli in wild hosts is still scarce. In this study, we used fine resolution of whole-genome sequencing to provide novel insights into the evolution of E. coli genomes from a small yet diverse collection of strains recovered within a broad range of wild animal species (including mammals and birds), the coevolution of E. coli strains with their hosts, and the genetics of pathogenicity of E. coli strains in wild hosts in Mexico. Our results provide evidence for the clinical importance of wild animals as reservoirs for pathogenic strains and highlight the need to include nonhuman hosts in the surveillance programs for E. coli infections.
  • Sassetti, Elisa; Cruz, Cristina Durante; Tammela, Päivi; Winterhalter, Mathias; Augustyns, Koen; Gribbon, Philip; Windshügel, Björn (2019)
    The serine protease Caseinolytic protease subunit P (ClpP) plays an important role for protein homeostasis in bacteria and contributes to various developmental processes, as well as virulence. Therefore, ClpP is considered as a potential drug target in Gram-positive and Gram-negative bacteria. In this study, we utilized a biochemical assay to screen several small molecule libraries of approved and investigational drugs for Escherichia coli ClpP inhibitors. The approved drugs bortezomib, cefmetazole, cisplatin, as well as the investigational drug cDPCP, and the protease inhibitor 3,4-dichloroisocoumarin (3,4-DIC) emerged as ClpP inhibitors with IC50 values ranging between 0.04 and 31 mu M. Compound profiling of the inhibitors revealed cefmetazole and cisplatin not to inhibit the serine protease bovine -chymotrypsin, and for cefmetazole no cytotoxicity against three human cell lines was detected. Surface plasmon resonance studies demonstrated all novel ClpP inhibitors to bind covalently to ClpP. Investigation of the potential binding mode for cefmetazole using molecular docking suggested a dual covalent binding to Ser97 and Thr168. While only the antibiotic cefmetazole demonstrated an intrinsic antibacterial effect, cDPCP clearly delayed the bacterial growth recovery time upon chemically induced nitric oxide stress in a ClpP-dependent manner.
  • Karonen, Taru (Helsingin yliopisto, 2019)
    Kolistiini on polymyksiineihin kuuluva antibiootti, joka kuuluu kriittisen tärkeisiin reserviantibiootteihin. Sen käyttö tulisi rajata erityisesti ihmisille ainoastaan todennettuun tarkoitukseen, esimerkiksi muille antibiooteille vastaamattomien, moniresistenttien bakteeri-infektioiden hoitoon. Syksyllä 2017 Evira löysi ensimmäisen kerran Suomeen Venäjältä tuoduista löytökoirista kolistiinille resistenttejä, ESBL-positiivisia Escherihia coli -bakteereja. Näistä bakteereista kolistiiniresistenssi todettiin referenssimenetelmällä eli liemilaimennosmenetelmällä ja kolistiinireisstenssiyden tuova mcr-1-geeni PCR-menetelmällä. Helsingin yliopiston eläinlääketieteellisen tiedekunnan kliinisen mikrobiologian laboratoriossa on seulottu alati kasvavasta potilasnäytemäärästä kolistiiniresistenssiä polymyksiini B -kiekkoherkyysmenetelmällä, koska sen on oletettu tuovan esille kolistiinille resistentit kannat polymyksiineille ominaisen ristiresistenssin vuoksi. Menetelmässä on myös jouduttu turvautumaan Pseudomonas aeruginosa -bakteerin herkkyysraja-arvoihin, koska enterobakteereille ei ole määritelty erikseen herkkyysraja-arvoja. Ei kuitenkaan tiedetä, kuinka luotettavasti ja herkästi tämä herkkyysmenetelmä soveltuu kolistiiniherkkyyden seulontaan. Työ koostuu kirjallisuuskatsauksesta ja kokeellisesta osasta. Kokeellisen osan tarkoituksena oli vertailla kolmea eri herkkyysmenetelmää kolistiiniresistenssin testaamiseksi ja pohtia P. aeruginosan raja-arvojen soveltumista enterobakteereille. Samalla tutkittiin, oliko potilasaineistossa mahdollisesti jäänyt havaitsematta aiemmassa polymyksiini B -kiekkotestissä kolistiinille resistenttejä, mcr-1- positiivisia kantoja. Työn hypoteesina oli, ettei polymyksiini B -kiekkotesti ole tarpeeksi herkkä ja spesifinen menetelmä kolistiiniherkkyyden testaamiseen. Tutkittavina potilaskantoina oli 31 kpl ESBL-positiivisiksi osoittautunutta E. coli -kantaa. Tutkittavat kannat oli eristetty Suomeen Venäjältä (26 kpl), Romaniasta (4 kpl) tai Latviasta (1 kpl) tuoduista, eri-ikäisistä koirista vuosina 2017-2018. Negatiivisina kontrollikantoina käytettiin kolistiinille herkkää E. coli ATCC 25922 -kantaa sekä P. aeruginosa ATCC 27852 -kantaa. Positiivisina kontrolleina (mcr-1 geeni) käytettiin E. coli NCTC 13846 -kantaa sekä kolmea Evirasta saatua mcr-1 –positiivista kantaa. Testattavina menetelminä olivat kiekkoherkkyysmenetelmä (polymyksiini B- ja kolistiinikiekot 10 ,25 ja 50 μg), E-testi (kolistiini), sekä referenssimenetelmänä liemilaimennosmenetelmä (kolistiini). Herkkyysmenetelmissä kolistiinille resistenteiksi osoittautuneet potilaskannat tutkittiin Evirassa mcr-1-geenin osalta PCR-menetelmällä. Työssä osoittautui, ettei polymyksiini B -kiekkotesti sovellu kolistiiniherkkyyden seulontaan eivätkä P. aeruginosan raja-arvot sovellu enterobakteereille. Kaikki kannat, mukaan lukien resistentit kannat, olivat tämän testin perustella herkkiä kolistiinille. Kolistiinikiekkoherkkyystesti (10 μg kiekko), E-testi ja liemilaimennosmenetelmä erottivat herkät ja resistentit kannat toisistaan. PCR-tutkimuksessa 2/31 potilaskantaa kantoivat mcr-1-geeniä, eli geenin esiintyvyys oli 6,5 %. Tämän työn perusteella polymyksiini B-kiekkotesti tulisi korvata toisella herkkyysmenetelmällä, esimerkiksi 10 μg kolistiinikiekkotestillä tai E-testillä. Liemilaimennosmenetelmän vaativuuden ja kalleuden vuoksi se soveltuisi tulosten varmentamiseen PCR-menetelmän ohella. Lisätutkimuksia kuitenkin vaaditaan mm. herkkyysraja-arvojen määrittämiseen ja menetelmien optimoimiseen. Koska työssä löydettiin jo toisen kerran kolistiinille reisistenttejä bakteereja tuontikoirista, vahvistaa työ tarvetta seuloa erityisesti tuontikoiria kolistiiniresistenssin suhteen.