Browsing by Subject "antibiotic resistance"

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  • Fondi, Marco; Karkman, Antti; Tamminen, Manu V.; Bosi, Emanuele; Virta, Marko; Fani, Renato; Alm, Eric; McInerney, James O. (2016)
    The spatial distribution of microbes on our planet is famously formulated in the Baas Becking hypothesis as everything is everywhere but the environment selects." While this hypothesis does not strictly rule out patterns caused by geographical effects on ecology and historical founder effects, it does propose that the remarkable dispersal potential of microbes leads to distributions generally shaped by environmental factors rather than geographical distance. By constructing sequence similarity networks from uncultured environmental samples, we show that microbial gene pool distributions are not influenced nearly as much by geography as ecology, thus extending the Bass Becking hypothesis from whole organisms to microbial genes. We find that gene pools are shaped by their broad ecological niche (such as sea water, fresh water, host, and airborne). We find that freshwater habitats act as a gene exchange bridge between otherwise disconnected habitats. Finally, certain antibiotic resistance genes deviate from the general trend of habitat specificity by exhibiting a high degree of cross-habitat mobility. The strong cross-habitat mobility of antibiotic resistance genes is a cause for concern and provides a paradigmatic example of the rate by which genes colonize new habitats when new selective forces emerge.
  • Stotani, Silvia; Gatta, Viviana; Medda, Federico; Padmanaban, Mohan; Karawajzyk, Anna; Tammela, Päivi Sirpa Marjaana; Giordanetto, Fabrizio; Tzalis, Dimitrios; Collina, Simona (2018)
    Resistance to antibiotics is an increasingly serious threat to global public health and its management translates to significant health care costs. The validation of new Gram-negative antibacterial targets as sources for potential new antibiotics remains a challenge for all the scientists working in this field. The interference with bacterial Quorum Sensing (QS) mechanisms represents a potentially interesting approach to control bacterial growth and pursue the next generation of antimicrobials. In this context, our research is focused on the discovery of novel compounds structurally related to (S)-4,5-dihydroxy-2,3-pentanedione, commonly known as (S)-DPD, a small signaling molecule able to modulate bacterial QS in both Gram-negative and Gram-positive bacteria. In this study, a practical and versatile synthesis of racemic DPD is presented. Compared to previously reported syntheses, the proposed strategy is short and robust: it requires only one purification step and avoids the use of expensive or hazardous starting materials as well as the use of specific equipment. It is therefore well suited to the synthesis of derivatives for pharmaceutical research, as demonstrated by four series of novel DPD-related compounds described herein.
  • Henttonen, Kaisu (Helsingin yliopisto, 2020)
    The human gut is inhabited by gut microbiota, a complex and diverse ecological community of trillions of microbes that affect both the normal human physiology and countless disease states and susceptibilities. Understanding the composition, functions and the causes and effects of changes in the microbiota is invaluable for understanding diseases that are connected to the microbiota and developing better treatments to the diseases. The gut microbiota varies between individuals and keeps changing over time. Behind the variability are e.g. the person’s age, genetics, diet, environment, and especially diseases and the use of antibiotics. When antibiotic use disrupts the gut microbiota, the changes can persist for years. Antibiotic resistance tends to increase after the use of antibiotics. Since antibiotic resistance in bacterial pathogens is considered a major health threat, the characterization of the human gut resistome (the antibiotic resistance genes (ARGs) found in the gut microbiota) is of great medical interest. Next-generation sequencing techniques have enabled studying also those microbe species that cannot be cultured at the moment. Metagenomics provides information on all genetic material collected from a given environment and enables searching for any sequences of interest within it, e.g. ARG sequences. The development of Parkinson’s disease (PD) is suspected to begin in the enteric nervous system and spread from there toward the central nervous system. The use of antibiotics could be linked to PD through their effects on gut microbiota, and since these effects are modified by the gut resistome, the aim of this study was to find gene sequences coding antibiotic resistance in human gut metagenomics data originating from stool samples of PD patients and healthy controls, and to find out potential differences in the occurrence of antibiotic resistance genes in the gut microbes of the two study groups. DeepARG was the chosen method for searching antibiotic resistance gene sequences in the metagenomics data. The statistical data analyses, including alpha diversity, multivariate analyses, and differential abundance analysis, were performed with the R statistical programming language in RStudio. DeepARG found 840 different ARGs in 192 samples. The ARGs belonged to 29 different ARG classes. The alpha diversity analysis showed a small estimated difference between PD and control groups indicating a possible slightly higher ARG diversity in the PD group. Multivariate analysis did not give any strong suggestions of definite biologically meaningful differences between the study groups. 16 ARGs were deemed differentially abundant in the study groups. BepE, cmeA, cmlv, dfrE, mefC, msrB, opcM, oprM and RbpA seemed to have increased abundance, and arnC, BN537_02049, dfrK, mgrA, murA, tet35 and tetT were suggested to have decreased abundance in PD patients compared to the healthy controls. These ARGs do not appear interconnected in any other way except for some sharing antibiotic types to which they offer resistance, and some having similar resistance mechanisms. In the light of an ongoing, unpublished epidemiological study of the connection between PD and the use of antibiotics it would seem that only three ARGs (msrB, mefC and dfrE) might be somehow relevant in PD development, but their effects, if any, are most likely minor. Eight ARG classes were shown to have differential abundance between PD patients and healthy controls. Bacitracin, fosfomycin and polymyxin classes showed decrease and chloramphenicol, fosmidomycin, puromycin, rifampin and sulfonamide classes showed increase in abundance in PD compared to controls. The change in the abundance of a certain ARG could reflect change in the abundance of the bacteria carrying that resistance gene. If so, the follow-up questions would be how much change in the abundance of bacteria is due to the use of certain antibiotics and how much is caused by environmental factors. It also remains to be studied whether specific antibiotics associated with the ARGs that in this study showed differential abundance in PD patients and healthy controls might have an actual role in PD development. The results of this thesis study are later to be combined with and further studied alongside information coming from ongoing studies on antibiotics use in general population and in PD patients. While this study did not concentrate its efforts into finding novel ARGs, the metagenomics dataset could also in the future be applied for that purpose.
  • Topp, Edward; Larsson, D. G. Joakim; Miller, Daniel N.; Van den Eede, Chris; Virta, Marko P. J. (2018)
    A roundtable discussion held at the fourth International Symposium on the Environmental Dimension of Antibiotic Resistance (EDAR4) considered key issues concerning the impact on the environment of antibiotic use in agriculture and aquaculture, and emissions from antibiotic manufacturing. The critical control points for reducing emissions of antibiotics from agriculture are antibiotic stewardship and the pre-treatment of manure and sludge to abate antibiotic-resistant bacteria. Antibiotics are sometimes added to fish and shellfish production sites via the feed, representing a direct route of contamination of the aquatic environment. Vaccination reduces the need for antibiotic use in high value (e.g. salmon) production systems. Consumer and regulatory pressure will over time contribute to reducing the emission of very high concentrations of antibiotics from manufacturing. Research priorities include the development of technologies, practices and incentives that will allow effective reduction in antibiotic use, together with evidence-based standards for antibiotic residues in effluents. All relevant stakeholders need to be aware of the threat of antimicrobial resistance and apply best practice in agriculture, aquaculture and pharmaceutical manufacturing in order to mitigate antibiotic resistance development. Research and policy development on antimicrobial resistance mitigation must be cognizant of the varied challenges facing high and low income countries.
  • Cairns, Johannes; Koskinen, Katariina; Penttinen, Reetta; Patinen, Tommi; Hartikainen, Anna; Jokela, Roosa; Ruusulehto, Liisa; Viitamäki, Sirja; Mattila, Sari; Hiltunen, Teppo; Jalasvuori, Matti (2018)
    Mobile genetic elements such as conjugative plasmids are responsible for antibiotic resistance phenotypes in many bacterial pathogens. The ability to conjugate, the presence of antibiotics, and ecological interactions all have a notable role in the persistence of plasmids in bacterial populations. Here, we set out to investigate the contribution of these factors when the conjugation network was disturbed by a plasmid-dependent bacteriophage. Phage alone effectively caused the population to lose plasmids, thus rendering them susceptible to antibiotics. Leakiness of the antibiotic resistance mechanism allowing Black Queen evolution (i.e. a "race to the bottom") was a more significant factor than the antibiotic concentration (lethal vs sublethal) in determining plasmid prevalence. Interestingly, plasmid loss was also prevented by protozoan predation. These results show that outcomes of attempts to resensitize bacterial communities by disrupting the conjugation network are highly dependent on ecological factors and resistance mechanisms. IMPORTANCE Bacterial antibiotic resistance is often a part of mobile genetic elements that move from one bacterium to another. By interfering with the horizontal movement and the maintenance of these elements, it is possible to remove the resistance from the population. Here, we show that a so-called plasmid-dependent bacteriophage causes the initially resistant bacterial population to become susceptible to antibiotics. However, this effect is efficiently countered when the system also contains a predator that feeds on bacteria. Moreover, when the environment contains antibiotics, the survival of resistance is dependent on the resistance mechanism. When bacteria can help their contemporaries to degrade antibiotics, resistance is maintained by only a fraction of the community. On the other hand, when bacteria cannot help others, then all bacteria remain resistant. The concentration of the antibiotic played a less notable role than the antibiotic used. This report shows that the survival of antibiotic resistance in bacterial communities represents a complex process where many factors present in real-life systems define whether or not resistance is actually lost.
  • Chamlagain, Bhawani Shankar (Helsingfors universitet, 2011)
    Dynamics of raw milk associated bacteria during cold storage of raw milk and their antibiotic resistance was reviewed, with focus on psychrotrophic bacteria. This study aimed to investigate the significance of cold storage of raw milk on antibiotic-resistant bacterial population and analyse the antibiotic resistance of the Gram-negative antibiotic-resistant psychrotrophic bacteria isolated from the cold-stored raw milk samples. Twenty-four raw milk samples, six at a time, were obtained from lorries that collected milk from Finnish farms and were stored at 4°C/4 d, 6°C/3 d and 6°C/4 d. Antibiotics representing four classes of antibiotics (gentamicin, ceftazidime, levofloxacin and trimethoprim-sulfamethoxazole) were used to determine the antibiotic resistance of mesophilic and psychrotrophic bacteria during the storage period. A representative number of antibiotic-resistant Gram-negative isolates retrieved from the cold-stored raw milk samples were identified by the phenotypic API 20 NE system and a few isolates by the 16S rDNA gene sequencing. Some of the isolates were further evaluated for their antibiotic resistance by the ATB PSE 5 and HiComb system. The initial average mesophilic counts were found below 105 CFU/mL, suggesting that the raw milk samples were of good quality. However, the mesophilic and psychrotrophic population increased when stored at 4°C/4 d, 6°C/3 d and 6°C/4 d. Gentamicin- and levofloxacin-resistant bacteria increased moderately (P < 0.05) while there was a considerable rise (P < 0.05) of ceftazidime- and trimethoprim-sulfamethoxazole-resistant population during the cold storage. Of the 50.9 % (28) of resistant isolates (total 55) identified by API 20 NE, the majority were Sphingomonas paucimobilis (8), Pseudomonas putida (5), Sphingobacterium spiritivorum (3) and Acinetobacter baumanii (2). The analysis by ATB PSE 5 system suggested that 57.1% of the isolates (total 49) were multiresistant. This study showed that the dairy environment harbours multidrug-resistant Gramnegative psychrotrophic bacteria and the cold chain of raw milk storage amplifies the antibioticresistant psychrotrophic bacterial population.
  • Hietikko, Alli (Helsingin yliopisto, 2019)
    Antibiotic-resistant bacteria are an increasing threat to global health, caused by the excessive use of antibiotics and the lack of new antimicrobial agents being introduced to the market. New approaches to prevent and cure bacterial infections are needed to halt the growing crisis. One of the most promising alternatives is phage therapy which utilizes bacteriophages to target and kill pathogens with specificity. Pseudomonas aeruginosa is a common opportunistic pathogen that is intrinsically resistant to antibiotics, making it one of the most heavily studied targets of phage therapy. In this study, I characterized four P. aeruginosa phages, fHo-Pae01, PA1P1, PA8P1 and PA11P1, and evaluate their potency in therapeutic applications. Bioinformatic analysis of the genomes revealed the phages to be genetically highly similar and belonging to the Pbunavirus genus of the Myoviridae family. No genes encoding harmful toxins, antibiotic-resistance, or lysogeny were predicted. On the other hand, many of the predicted genes had unknown functions. The host ranges of the phages were assessed using 47 clinical P. aeruginosa strains and predicted host receptor binding tail proteins were compared. Some correlation between the host ranges and mutations in the tail proteins were observed but this alone was not sufficient to explain the differences in the host ranges. The recently isolated vB_PaeM_fHoPae01 (fHo-Pae01) phage was further characterized by a one-step growth curve and imaged with a promising atomic force microscopy method that had not been used before in the Skurnik group. Though the imaging results failed to provide any further knowledge of the phage, the 70-minute-long latent period of infection could be determined from the growth curve. Anion- exchange chromatography was found inefficient in purifying the fHo-Pae01 phage, so alternative methods such as endotoxin columns should be used when purifying these phages for patient use. In conclusion, all four phages appeared to be safe for therapeutic use based on current knowledge, and PA1P1 and PA11P1 were the most promising candidates due to their broad host ranges.
  • Dirks, Anna (Helsingin yliopisto, 2021)
    Antibiotic resistance is an increasing, terrible threat to human health, leading to a growing need for alternative therapies. Phage therapy, using bacterial viruses to fight infections, is a promising alternative to antibiotic therapy. However, several obstacles need to be overcome. Regrettably, phage therapy remains inaccessible to many laboratories worldwide due to the need for expensive machinery to establish sensitivity of bacteria to phage. Moreover, shipping phages between laboratories remains challenging. In the current study a device-free bacteriophage typing PhagoGramAssay was developed. In the assay bacteria suspended in soft agar were poured onto a 60-well Terasaki plate containing phages suspended in fibrillated nanocellulose separated from the bacteria by a seal. Phages were released into the bacterial agar layer by puncturing the seal to test for sensitivity observable with the naked eye. Contrast between lysis zone and bacterial lawn was enhanced using 2,3,5-triphenyltetrazolium chloride. Optimized parameters included the amount of bacteria and phage added, volume of phage suspension, agar percentage and thickness and puncturing tool size. In addition, a prototype of such a puncturing tool was developed. The optimized PhagoGramAssay was tested using several bacteria-phage combinations. Moreover, infectivity and stability of phages stored on Terasaki plates was followed over the course of 4 weeks. The optimal bacterial amount added was found to be a 1:300 dilution in soft agar taken from a OD600 = 1 culture. Phage suspensions used in the assay were found to need to have a titer of at least 108 PFU/ml in the original lysate, with 8 µl of 1:10 dilution in fibrillated nanocellulose present in the wells. Optimal agar conditions were found to be 0.4% – 0.5% (w/v) with a thickness of 2 mm – 3 mm. The optimal puncturing tool shape was found to be a slit with a thickness of 0.5 mm. When using these conditions sensitivity could be established for a vast number of bacteria-phage combinations. All phages remained stable and infective over the course of 4 weeks . The newly developed PhagoGramAssay can be further developed into a kit-like phage typing assay that would enable laboratories to test for sensitivity on site whenever a multi-drug resistant bacterial strain is isolated from a patient sample, effectively making phage therapy accessible to laboratories that cannot afford expensive machinery. Additionally, the use of fibrillated nanocellulose should enable laboratories to exchange phages. The final form of such a kit, however, is dependent on manufacturers and investors and may need to be adjusted accordingly.
  • Spruit, Cindy; Wicklund, Anu; Wan, Xing; Skurnik, Mikael; Pajunen, Maria (2020)
    The lytic phage, fHe-Kpn01 was isolated from sewage water using an extended-spectrum beta-lactamase-producing strain of Klebsiella pneumoniae as a host. The genome is 43,329 bp in size and contains direct terminal repeats of 222 bp. The genome contains 56 predicted genes, of which proteomics analysis detected 29 different proteins in purified phage particles. Comparison of fHe-Kpn01 to other phages, both morphologically and genetically, indicated that the phage belongs to the family Podoviridae and genus Drulisvirus. Because fHe-Kpn01 is strictly lytic and does not carry any known resistance or virulence genes, it is suitable for phage therapy. It has, however, a narrow host range since it infected only three of the 72 tested K. pneumoniae strains, two of which were of capsule type KL62. After annotation of the predicted genes based on the similarity to genes of known function and proteomics results on the virion-associated proteins, 22 gene products remained annotated as hypothetical proteins of unknown function (HPUF). These fHe-Kpn01 HPUFs were screened for their toxicity in Escherichia coli. Three of the HPUFs, encoded by the genes g10, g22, and g38, were confirmed to be toxic.
  • Mostowy, Rafal; Croucher, Nicholas J.; Andam, Cheryl P.; Corander, Jukka; Hanage, William P.; Marttinen, Pekka (2017)
    Prokaryotic evolution is affected by horizontal transfer of genetic material through recombination. Inference of an evolutionary tree of bacteria thus relies on accurate identification of the population genetic structure and recombination-derived mosaicism. Rapidly growing databases represent a challenge for computational methods to detect recombinations in bacterial genomes. We introduce a novel algorithm called fastGEAR which identifies lineages in diverse microbial alignments, and recombinations between them and from external origins. The algorithm detects both recent recombinations (affecting a few isolates) and ancestral recombinations between detected lineages (affecting entire lineages), thus providing insight into recombinations affecting deep branches of the phylogenetic tree. In simulations, fastGEAR had comparable power to detect recent recombinations and outstanding power to detect the ancestral ones, compared with state-of-the-art methods, often with a fraction of computational cost. We demonstrate the utility of the method by analyzing a collection of 616 whole-genomes of a recombinogenic pathogen Streptococcus pneumoniae, for which the method provided a high-resolution view of recombination across the genome. We examined in detail the penicillin-binding genes across the Streptococcus genus, demonstrating previously undetected genetic exchanges between different species at these three loci. Hence, fastGEAR can be readily applied to investigate mosaicism in bacterial genes across multiple species. Finally, fastGEAR correctly identified many known recombination hotspots and pointed to potential new ones. Matlab code and Linux/Windows executables are available at https://users.ics.aalto.fi/similar to pemartti/fastGEAR/ (last accessed February 6, 2017).
  • Prieto, Ana M. Guzman; van Schaik, Willem; Rogers, Malbert R. C.; Coque, Teresa M.; Baquero, Fernando; Corander, Jukka; Willems, Rob J. L. (2016)
    Enterococci are Gram-positive bacteria that are found in plants, soil and as commensals of the gastrointestinal tract of humans, mammals, and insects. Despite their commensal nature, they have also become globally important nosocomial pathogens. Within the genus Enterococcus. Enterococcus faecium, and Enterococcus faecalis are clinically most relevant. In this review, we will discuss how E. faecium and E. faecalis have evolved to become a globally disseminated nosocomial pathogen. E faecium has a defined sub-population that is associated with hospitalized patients and is rarely encountered in community settings. These hospital associated clones are characterized by the acquisition of adaptive genetic elements, including genes involved in metabolism, biofilm formation, and antibiotic resistance. In contrast to E. faecium, clones of E. faecalis isolated from hospitalized patients, including strains causing clinical infections, are not exclusively found in hospitals but are also present in healthy individuals and animals. This observation suggests that the division between commensals and hospital-adapted lineages is less clear for E. faecalis than for E. faecium. In addition, genes that are reported to be associated with virulence of E. faecalis are often not unique to clinical isolates, but are also found in strains that originate from commensal niches. As a reflection of more ancient association of E. faecalis with different hosts, these determinants Thus, they may not represent genuine virulence genes but may act as host adaptive functions that are useful in a variety of intestinal environments. The scope of the review is to summarize recent trends in the emergence of antibiotic resistance and explore recent developments in the molecular epidemiology, population structure and mechanisms of adaptation of E. faecium and E faecalls.
  • Hultman, Jenni; Tamminen, Manu; Pärnänen, Katariina; Cairns, Johannes; Karkman, Antti; Virta, Marko (2018)
    Wastewater treatment plants (WWTPs) collect wastewater from various sources for a multi-step treatment process. By mixing a large variety of bacteria and promoting their proximity, WWTPs constitute potential hotspots for the emergence of antibiotic resistant bacteria. Concerns have been expressed regarding the potential of WWTPs to spread antibiotic resistance genes (ARGs) from environmental reservoirs to human pathogens. We utilized epicPCR (Emulsion, Paired Isolation and Concatenation PCR) to detect the bacterial hosts of ARGs in two WWTPs. We identified the host distribution of four resistance-associated genes (tetM, int1, qacE Delta 1 and bla(OXA-58)) in influent and effluent. The bacterial hosts of these resistance genes varied between the WWTP influent and effluent, with a generally decreasing host range in the effluent. Through 16S rRNA gene sequencing, it was determined that the resistance gene carrying bacteria include both abundant and rare taxa. Our results suggest that the studied WWTPs mostly succeed in decreasing the host range of the resistance genes during the treatment process. Still, there were instances where effluent contained resistance genes in bacterial groups not carrying these genes in the influent. By permitting exhaustive profiling of resistance-associated gene hosts in WWTP bacterial communities, the application of epicPCR provides a new level of precision to our resistance gene risk estimates.
  • Jääskeläinen, Susanna (Helsingfors universitet, 2016)
    The cold storage, utilized to increase the shelf life of raw milk, favors the growth of psychrotrophic bacteria. Psychrotrophs are considered as problematic because they produce heat-stable spoilage enzymes. In addition, some pathogenic bacteria are capable of growing at cold storage temperatures and it has been observed in previous studies that the psychrotrophs in raw milk can be resistant to several antibiotics. In the literature review section of this study, the microbial composition of raw milk and the effect of cold storage, nitrogen gas (N2) treatment and activation of the lactoperoxidase system (LPS) on the raw milk bacteria was reviewed; moreover, the evolution, spread and mechanisms of bacterial antibiotic resistance and the antibiotic resistance on dairy farms and of raw milk bacteria was reviewed. Also the methods to study raw milk bacteria and their antibiotic resistance were documented. The aim of the experimental part was to study the effects of cold storage, N2 gas treatment and LPS treatment on the levels of antibiotic resistant bacteria in raw milk. Untreated, N2-treated and LPS-treated raw milk samples were stored at 6 °C and the counts of bacteria resistant to gentamicin, ceftatzidime, levofloxacin and trimethoprim-sulfamethoxazole were determined in the beginning of the experiments and after three and seven days of cold storage. The antibiotic resistance and spoilage features of bacterial isolates selected from different plates were compared. In addition, DNA fingerprints of selected isolates were obtained by rep-PCR method and for a couple of isolates 16S rRNA gene partial sequencing was performed. The presence of certain antibiotic resistance genes for the isolates partially identified was investigated using a PCR-based method. The length of cold storage affected the proportions of antibiotic resistant bacteria in raw milk. The proportions of antibiotic resistant bacteria in the control milk sample were generally, with the exception of TS-resistant bacteria in some cases, at their lowest after seven days of cold storage when the milk was, though, microbiologically unacceptable. There was a lot of variation in the prevalence of bacterial antibiotic resistance between the milk samples treated differently. The proportions of resistant bacteria were on average clearly higher in LPS samples than in control samples, whereas the effect of the N2 treatment on the proportions of resistant bacteria varied between the experiments. The bacterial isolates resistant to all the considered antibiotics produced less frequently protease and phospholipase than the isolates resistant to fewer antibiotics. The isolates from LPS samples were more frequently resistant to all the antibiotics studied and produced less frequently protease and phospholipase than the isolates from control and N2-treated samples. On the basis of the rep-PCR fingerprints, there were both similar and different strains among the bacterial isolates. Based on the 16S rRNA partial gene sequence, two isolates were identified as Pseudomonas spp. and two isolates as Stenotrophomonas spp. The targeted antibiotic resistance genes were not detected among the considered isolates. Possible reasons can be variations in the gene sequences, suboptimal PCR conditions or that the isolates lacked the genes studied. A PCR product was obtained for one Pseudomonas isolate using mexA-F/R primers, but according to the database the gene sequence did not show homology with antibiotic resistance genes. Further analyses would be required to confirm if the gene studied is linked to antibiotic resistance.
  • Markkanen, Melina (Helsingin yliopisto, 2020)
    Constantly increasing level of bacteria becoming resistant to clinically relevant antibiotics challenges the modern medical achievements made over the past century. In global scale, one of the most significant information gaps concerning the occurrence of resistant bacteria is located in West African countries. Klebsiella pneumoniae and Escherichia coli strains resistant to 3rd generation cephalosporins and carbapenems are a major risk to public health through infections with limited or no available treatment options. The resistance to these antibiotics among Enterobacteriaceae is mainly mediated by hydrolyzing enzymes such as extended-spectrum beta-lactamases (ESBL). The focus of this thesis is to study the genes encoding these enzymes and other resistance factors found in K. pneumoniae and E. coli isolated from human stool and waste water samples in Burkina Faso and Mali. Tree Enterobacteriaceae isolates were selected for whole genome sequence (WGS) analysis based on their phenotypic resistance profiles defined by disk diffusion method. Reads were assembled to draft genomes and the genomes were studied for their antibiotic resistance genes, virulence genes and their associations to mobile genetic elements found in these isolates’ genomes. Additionally a pan-genome was created to investigate species specific features of K. pneumoniae and their role in heavy load of antibiotic resistance genes among these isolates. Pan-genome consisted of two genomes sequenced in this study and 12 genomes from the publically available database. 16-month old Burkinabe child was a carrier of one ESBL-producing K. pneumoniae (isolate Burkina_1) and one ESBL-positive E. coli along with the resistance to multiple other antibiotics. With genome wide analysis the K. pneumoniae strain could be described as sequence type (ST) 45 representing, multidrug resistant and ESBL-gene CTX-M-15 carrying strain with highly similar virulence gene profile to strains previously described as pathogenic K. pneumoniae causing neonatal sepsis. K. pneumoniae isolated from the stool sample of an adult living in Burkina Faso was found to be multidrug resistant, though non-ESBL-producer strain (isolate Burkina_2). The isolate showed no similarity to any previously described sequence type. CTX-M-15 encoding E. coli of ST38 (isolate Mali_1) carried by Malian child showed resistance to five different classes of antibiotics in addition to the 3rd generation cephalosporins. At the same time the isolate showed hybrid virulence gene profile with virulence genes associated to many different E. coli pathotypes including neonatal meningitis causing E. coli (NMEC). The exceptional plasticity of K. pneumoniae genome could be recognized as one of the putative explanations for the high number of resistance genes found among the isolates studied in this work. Antibiotic resistance genes were found to be associated to mobile genetic elements (MGE) and as the genetic plasticity is caused by the acquisition of external genetic material via MGEs such as plasmids, this can lead to indirect accumulation of resistance genes in these genomes. The results in this thesis work show alarming examples of pathogens that potentially cause severe infections, have extremely narrow or no treatment options and are carried by infants. These findings are in line with the few data about the level of faecal carriage of ESBL-producing strains by people in Burkina Faso and Mali reported previously.
  • Ruotsalainen, Pilvi; Penttinen, Reetta; Mattila, Sari; Jalasvuori, Matti (2019)
    ABSTRACTThe possibility to modify gut bacterial flora has become an important goal, and various approaches are used to achieve desirable communities. However, the genetic engineering of existing microbes in the gut, which are already compatible with the rest of the community and host immune system, has not received much attention. Here, we discuss and experimentally evaluate the possibility to use modified and mobilizable CRISPR-Cas9-endocing plasmid as a tool to induce changes in bacterial communities. This plasmid system (briefly midbiotic) is delivered from bacterial vector into target bacteria via conjugation. Compared to, for example, bacteriophage-based applications, the benefits of conjugative plasmids include their independence of any particular receptor(s) on host bacteria and their relative immunity to bacterial defense mechanisms (such as restriction-modification systems) due to the synthesis of the complementary strand with host-specific epigenetic modifications. We show that conjugative plasmid in association with a mobilizable antibiotic resistance gene targeting CRISPR-plasmid efficiently causes ESBL-positive transconjugants to lose their resistance, and multiple gene types can be targeted simultaneously by introducing several CRISPR RNA encoding segments into the transferred plasmids. In the rare cases where the midbiotic plasmids failed to resensitize bacteria to antibiotics, the CRISPR spacer(s) and their adjacent repeats or larger regions were found to be lost. Results also revealed potential caveats in the design of conjugative engineering systems as well as workarounds to minimize these risks.
  • Arredondo-Alonso, Sergio; Rogers, Malbert R. C.; Braat, Johanna C.; Verschuuren, Tess D.; Top, Janetta; Corander, Jukka; Willems, Rob J. L.; Schürch, Anita C. (2018)
    Assembly of bacterial short-read whole-genome sequencing data frequently results in hundreds of contigs for which the origin, plasmid or chromosome, is unclear. Complete genomes resolved by long-read sequencing can be used to generate and label short-read contigs. These were used to train several popular machine learning methods to classify the origin of contigs from Enterococcus faecium, Klebsiella pneumoniae and Escherichia colt using pentamer frequencies. We selected support-vector machine (SVM) models as the best classifier for all three bacterial species (Fl-score E. faecium=0.92, F1-score K. pneumoniae=0.90, F1-score E. coli=0.76), which outperformed other existing plasmid prediction tools using a benchmarking set of isolates. We demonstrated the scalability of our models by accurately predicting the plasmidome of a large collection of 1644 E. faecium isolates and illustrate its applicability by predicting the location of antibiotic-resistance genes in all three species. The SVM classifiers are publicly available as an R package and graphical-user interface called 'mlplasmids'. We anticipate that this tool may significantly facilitate research on the dissemination of plasmids encoding antibiotic resistance and/or contributing to host adaptation.
  • Johansson, Julia (Helsingin yliopisto, 2021)
    This project focuses on development of novel split intein systems for selection of biological activities utilizing protein splicing. Protein splicing is phenomenon that occurs naturally inside the cell and the reaction is catalyzed by inteins, which connect C-terminal and N-terminal exteins with a peptide bond. The activity of the interrupted protein, consisting the exteins, can be restored after the intein is excised and the peptide bond links the exteins together. This occurrence can be used for selection of cells based on different activities including antibiotic resistance. The project aims to insert an intein in antibiotic resistance gene which could allow controlling the protein activity of the antibiotic resistance gene by protein splitting. This method is based on inserting an intein to the antibiotic resistance conferring enzyme which makes the protein inactive. Creating two separate plasmids that include the intein sequence can be transformed into bacterial cells. Other plasmid includes a deletion in the intein sequence and the cells that include this plasmid only, are not able to survive in the presence of an antibiotic. This is due to inactivity of the intein and thus the inactivity of the enzyme that confers the resistance. Incorporating a second plasmid that includes the corresponding sequence to the deletion, the intein activity can be recovered and thus the protein activity. By this method with cotransformation, both plasmids are transformed simultaneously which recovers the intein activity and further the antibiotic resistance. This could be used for the cell selection since only the cells that harbor both of the two complementary plasmids could restore the antibiotic resistance.
  • Colclough, Abigail; Corander, Jukka; Sheppard, Samuel K.; Bayliss, Sion C.; Vos, Michiel (2019)
    Bacteria interact with a multitude of other organisms, many of which produce antimicrobials. Selection for resistance to these antimicrobials has the potential to result in resistance to clinical antibiotics when active compounds target the same bacterial pathways. The possibility of such cross-resistance between natural antimicrobials and antibiotics has to our knowledge received very little attention. The antimicrobial activity of extracts from seaweeds, known to be prolific producers of antimicrobials, is here tested against Staphylococcus aureus isolates with varied clinical antibiotic resistance profiles. An overall effect consistent with cross-resistance is demonstrated, with multidrug-resistant S. aureus strains being on average more resistant to seaweed extracts. This pattern could potentially indicate that evolution of resistance to antimicrobials in the natural environment could lead to resistance against clinical antibiotics. However, patterns of antimicrobial activity of individual seaweed extracts vary considerably and include collateral sensitivity, where increased resistance to a particular antibiotic is associated with decreased resistance to a particular seaweed extract. Our correlation-based methods allow the identification of antimicrobial extracts bearing most promise for downstream active compound identification and pharmacological testing.
  • Pirnay, Jean-Paul; Blasdel, Bob G.; Bretaudeau, Laurent; Buckling, Angus; Chanishvili, Nina; Clark, Jason R.; Corte-Real, Sofia; Debarbieux, Laurent; Dublanchet, Alain; De Vos, Daniel; Gabard, Jerome; Garcia, Miguel; Goderdzishvili, Marina; Gorski, Andrzej; Hardcastle, John; Huys, Isabelle; Kutter, Elizabeth; Lavigne, Rob; Merabishvili, Maia; Olchawa, Ewa; Parikka, Kaarle J.; Patey, Olivier; Pouilot, Flavie; Resch, Gregory; Rohde, Christine; Scheres, Jacques; Skurnik, Mikael; Vaneechoutte, Mario; Van Parys, Luc; Verbeken, Gilbert; Zizi, Martin; Van den Eede, Guy (2015)
    The worldwide antibiotic crisis has led to a renewed interest in phage therapy. Since time immemorial phages control bacterial populations on Earth. Potent lytic phages against bacterial pathogens can be isolated from the environment or selected from a collection in a matter of days. In addition, phages have the capacity to rapidly overcome bacterial resistances, which will inevitably emerge. To maximally exploit these advantage phages have over conventional drugs such as antibiotics, it is important that sustainable phage products are not submitted to the conventional long medicinal product development and licensing pathway. There is a need for an adapted framework, including realistic production and quality and safety requirements, that allowsa timely supplying of phage therapy products for 'personalized therapy' or for public health or medical emergencies. This paper enumerates all phage therapy product related quality and safety risks known to the authors, as well as the tests that can be performed to minimize these risks, only to the extent needed to protect the patients and to allow and advance responsible phage therapy and research.
  • de Been, Mark; van Schaik, Willem; Cheng, Lu; Corander, Jukka; Willems, Rob J. (2013)