Sort by: Order: Results:

Now showing items 1-18 of 18
  • 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.
  • Amatya, Sajeen Bahadur; Salmi, Sonja; Kainulainen, Veera; Karihtala, Peeter; Reunanen, Justus (2021)
    Simple Summary:& nbsp;Microbial dysbiosis has been credited as one of the contributing factors to the development and progression of gastrointestinal tract cancer. The altered microbiota influences carcinogenesis through the induction of instability and damage to genetic material, modulation of host metabolic and inflammatory pathways, production of carcinogenic metabolites, and suppression of host antitumor response. These microbes secrete extracellular vesicles that are possibly carrying carcinogenic bioactive metabolites within their cargo. Studies have illustrated the ability of bacterial extracellular vesicles to cross the intestinal epithelial barrier and selectively accumulate near intestinal tumor cells. The purpose of this systemic review was to highlight the possible role of gut bacterial vesicles in the development, progression, and pathogenesis of gastrointestinal tract cancer and their possible involvement in the modulation of the tumor microenvironment. An infinitesimal amount of research has been carried out on the impact of bacterial extracellular vesicles on oncogenesis and tumor progression. This review aimed to encourage more investigations on this subject.Bacterial extracellular vesicles are membrane-enclosed, lipid bi-layer nanostructures that carry different classes of biomolecules, such as nucleic acids, lipids, proteins, and diverse types of small molecular metabolites, as their cargo. Almost all of the bacteria in the gut secrete extracellular vesicles to assist them in competition, survival, material exchange, host immune modulation, infection, and invasion. The role of gut microbiota in the development, progression, and pathogenesis of gastrointestinal tract (GIT) cancer has been well documented. However, the possible involvement of bacterial extracellular vesicles (bEVs) in GIT cancer pathophysiology has not been given due attention. Studies have illustrated the ability of bEVs to cross physiological barriers, selectively accumulate near tumor cells, and possibly alter the tumor microenvironment (TME). A systematic search of original published works related to bacterial extracellular vesicles on gastrointestinal cancer was performed for this review. The current systemic review outlines the possible impact of gut microbiota derived bEVs in GIT cancer in light of present-day understanding. The necessity of using advanced sequencing technologies, such as genetic, proteomic, and metabolomic investigation methodologies, to facilitate an understanding of the interrelationship between cancer-associated bacterial vesicles and gastrointestinal cancer is also emphasized. We further discuss the clinical and pharmaceutical potential of bEVs, along with future efforts needed to understand the mechanism of interaction of bEVs in GIT cancer pathogenesis.
  • Ongey, Elvis Legala; Pflugmacher, Stephan; Neubauer, Peter (2018)
    This review article provides an overview of recent developments in antimicrobial peptides (AMPs), summarizing structural diversity, potential new applications, activity targets and microbial killing responses in general. The use of artificial and natural AMPs as templates for rational design of peptidomimetics are also discussed and some strategies are put forward to curtail cytotoxic effects against eukaryotic cells. Considering the heat-resistant nature, chemical and proteolytic stability of AMPs, we attempt to summarize their molecular targets, examine how these macromolecules may contribute to potential environmental risks vis-a-vis the activities of the peptides. We further point out the evolutional characteristics of the macromolecules and indicate how they can be useful in designing target-specific peptides. Methods are suggested that may help to assess toxic mechanisms of AMPs and possible solutions are discussed to promote the development and application of AMPs in medicine. Even if there is wide exposure to the environment like in the hospital settings, AMPs may instead contribute to prevent healthcare-associated infections so long as ecotoxicological aspects are considered.
  • Mäntynen, Sari; Laanto, Elina; Oksanen, Hanna M.; Poranen, Minna M.; Diaz-Munoz, Samuel L. (2021)
    The canonical lytic-lysogenic binary has been challenged in recent years, as more evidence has emerged on alternative bacteriophage infection strategies. These infection modes are little studied, and yet they appear to be more abundant and ubiquitous in nature than previously recognized, and can play a significant role in the ecology and evolution of their bacterial hosts. In this review, we discuss the extent, causes and consequences of alternative phage lifestyles, and clarify conceptual and terminological confusion to facilitate research progress. We propose distinct definitions for the terms 'pseudolysogeny' and 'productive or non-productive chronic infection', and distinguish them from the carrier state life cycle, which describes a population-level phenomenon. Our review also finds that phages may change their infection modes in response to environmental conditions or the physiological state of the host cell. We outline known molecular mechanisms underlying the alternative phage-host interactions, including specific genetic pathways and their considerable biotechnological potential. Moreover, we discuss potential implications of the alternative phage lifestyles for microbial biology and ecosystem functioning, as well as applied topics such as phage therapy.
  • Chen, Wen; Roslund, Kajsa; Fogarty, Christopher L.; Pussinen, Pirkko J.; Halonen, Lauri; Groop, Per-Henrik; Metsala, Markus; Lehto, Markku (2016)
    Hydrogen cyanide (HCN) has been recognized as a potential biomarker for non-invasive diagnosis of Pseudomonas aeruginosa infection in the lung. However, the oral cavity is a dominant production site for exhaled HCN and this contribution can mask the HCN generated in the lung. It is thus important to understand the sources of HCN production in the oral cavity. By screening of oral anaerobes for HCN production, we observed that the genus of Porphyromonas, Prevotella and Fusobacterium generated low levels of HCN in vitro. This is the first study to show that oral anaerobes are capable of producing HCN in vitro. Further investigations were conducted on the species of P. gingivalis and we successfully detected HCN production (0.9-10.9 ppb) in the headspace of three P. gingivalis reference strains (ATCC 33277, W50 and OMG 434) and one clinical isolate. From P. gingivalis ATCC 33277 and W50, a strong correlation between HCN and CO2 concentrations (r(s) = 0.89, p <0.001) was observed, indicating that the HCN production of P. gingivalis might be connected with the bacterial metabolic activity. These results indicate that our setup could be widely applied to the screening of in vitro HCN production by both aerobic and anaerobic bacteria.
  • He, Wei; Zhang, Zhaoyu; Chen, Jing; Zheng, Yudong; Xie, Yajie; Liu, Wenbo; Wu, Jian; Mosselhy, Dina A. (2021)
    Chronic wounds are a serious worldwide problem, which are often accompanied by wound infections. In this study, bacterial cellulose (BC)-based composites introduced with tannic acid (TA) and magnesium chloride (BC-TA-Mg) were fabricated for anti-biofilm activities. The prepared composites' surface properties, mechanical capacity, thermal stability, water absorption and retention property, releasing behavior, anti-biofilm activities and potential cytotoxicity were tested. Results showed that TA and MgCl2 particles closely adhered to the nanofibers of BC membranes, thus increasing surface roughness and hydrophobicity of the membranes. While the introduction of TA and MgCl2 did not influence the transparency of the membranes, making it beneficial for wound inspection. BC-TA and BC-TA-Mg composites displayed increased tensile strength and elongation at break compared to pure BC. Moreover, BC-TA-Mg exhibited higher water absorption and retention capacity than BC and BC-TA, suitable for the absorption of wound exudates. BC-TA-Mg demonstrated controlled release of TA and good inhibitory effect on both singly cultured Staphylococcus aureus and Pseudomonas aeruginosa biofilm and co-cultured biofilm of S. aureus and P. aeruginosa. Furthermore, the cytotoxicity grade of BC-TA-6Mg membrane was eligible based on standard toxicity classifications. These indicated that BC-TA-Mg is potential to be used as wound dressings combating biofilms in chronic wounds.
  • Hussain, Nazar; Tariq, Muhammad; Saris, Per Erik Joakim; Zaidi, Arsalan (2021)
    Introduction: Probiotic and postbiotic potential of thirty-two strains of lactic acid bacteria (LAB), obtained earlier from artisanal dairy sources in Pakistan, have been investigated against major multi-drug resistant (MDR) and food borne pathogenic bacteria. Methodology: LAB strains were identified by 16S rRNA gene sequencing and their antibacterial activity was assessed by the microdilution method. Four LAB isolates, Weissella confusa PL6, Enterococcus faecium PL7, and Lactobacillus delbrueckii PL11 and PL13 were shortlisted. Their ability to degrade lactose and safety for human consumption in terms of hemolysis and antibiotic susceptibility were assessed in vitro. The antibacterial components in the cell-free supernatants (CFSs) of isolate cultures were characterized biochemically by HPLC. Results: Acid neutralization but not protease treatment abolished the antibacterial activity of CFSs. Lactic, acetic and propionic acids were the main acids in the CFSs, and acid production peaked in the stationary phase of growth. The antibacterial activity of the LAB cultures resulted from secretion of organic acids that lowered the pH. The strains exhibited variable ability to degrade lactose and were non-hemolytic and susceptible to the most common antibiotics. Conclusions: These LAB strains are probiotic candidates for further investigation of their postbiotic role in naturally preserving processed foods and for attenuation of lactose intolerance.
  • Chen, W.; Metsala, M.; Vaittinen, Olavi; Halonen, L. (2014)
  • Paino, Annamari; Ahlstrand, Tuuli; Nuutila, Jari; Navickaite, Indre; Lahti, Maria; Tuominen, Heidi; Välimaa, Hannamari; Lamminmaki, Urpo; Pollanen, Marja T.; Ihalin, Riikka (2013)
  • Ahlstrand, Tuuli; Torittu, Annamari; Elovaara, Heli; Välimaa, Hannamari; Pöllänen, Marja T.; Kasvandik, Sergo; Högbom, Martin; Ihalin, Riikka (2018)
    Naturally competent bacteria acquire DNA from their surroundings to survive in nutrient-poor environments and incorporate DNA into their genomes as new genes for improved survival. The secretin HofQ from the oral pathogen Aggregatibacter actinomycetemcomitans has been associated with DNA uptake. Cytokine sequestering is a potential virulence mechanism in various bacteria and may modulate both host defense and bacterial physiology. The objective of this study was to elucidate a possible connection between natural competence and cytokine uptake in A. actinomycetemcomitans. The extramembranous domain of HofQ (emHofQ) was shown to interact with various cytokines, of which IL-8 exhibited the strongest interaction. The dissociation constant between emHofQ and IL-8 was 43nM in static settings and 2.4M in dynamic settings. The moderate binding affinity is consistent with the hypothesis that emHofQ recognizes cytokines before transporting them into the cells. The interaction site was identified via crosslinking and mutational analysis. By structural comparison, relateda type I KH domain with a similar interaction site was detected in the Neisseria meningitidis secretin PilQ, which has been shown to participate in IL-8 uptake. Deletion of hofQ from the A. actinomycetemcomitans genome decreased the overall biofilm formation of this organism, abolished the response to cytokines, i.e., decreased eDNA levels in the presence of cytokines, and increased the susceptibility of the biofilm to tested -lactams. Moreover, we showed that recombinant IL-8 interacted with DNA. These results can be used in further studies on the specific role of cytokine uptake in bacterial virulence without interfering with natural-competence-related DNA uptake.
  • Falgenhauer, Linda; Schwengers, Oliver; Schmiedel, Judith; Baars, Christian; Lambrecht, Oda; Hess, Stefanie; Berendonk, Thomas U.; Falgenhauer, Jane; Chakraborty, Trinad; Imirzalioglu, Can (2019)
    Water is considered to play a role in the dissemination of antibiotic-resistant Gram-negative bacteria including those encoding Extended-spectrum beta-lactamases (ESBL) and carbapenemases. To investigate the role of water for their spread in more detail, we characterized ESBL/Carbapenemase-producing bacteria from surface water and sediment samples using phenotypic and genotypic approaches. ESBL/Carbapenemase-producing isolates were obtained from water/sediment samples. Species and antibiotic resistance were determined. A subset of these isolates (n = 33) was whole-genome-sequenced and analyzed for the presence of antibiotic resistance genes and virulence determinants. Their relatedness to isolates associated with human infections was investigated using multilocus sequence type and cgMLST-based analysis. Eighty-nine percent of the isolates comprised of clinically relevant species. Fifty-eight percent exhibited a multidrug-resistance phenotype. Two isolates harbored the mobile colistin resistance gene mcr-1. One carbapenemase-producing isolate identified as Enterobacter kobei harbored bla(VIM-)(1). Two Escherichia coli isolates had sequence types (ST) associated with human infections (ST131 and ST1485) and a Klebsiella pneumoniae isolate was classified as hypervirulent. A multidrug-resistant (MDR) Pseudomonas aeruginosa isolate encoding known virulence genes associated with severe lung infections in cystic fibrosis patients was also detected. The presence of MDR and clinically relevant isolates in recreational and surface water underlines the role of aquatic environments as both reservoirs and hot spots for MDR bacteria. Future assessment of water quality should include the examination of the multidrug resistance of clinically relevant bacterial species and thus provide an important link regarding the spread of MDR bacteria in a One Health context.
  • Munsch-Alatossava, Patricia; Alatossava, Tapani (2020)
    Worldwide, the dairy sector remains of vital importance for food production despite severe environmental constraints. The production and handling conditions of milk, a rich medium, promote inevitably the entrance of microbial contaminants, with notable impact on the quality and safety of raw milk and dairy products. Moreover, the persistence of high concentrations of microorganisms (especially bacteria and bacterial spores) in biofilms (BFs) present on dairy equipment or environments constitutes an additional major source of milk contamination from pre- to post-processing stages: in dairies, BFs represent a major concern regarding the risks of disease outbreaks and are often associated with significant economic losses. One consumption trend toward "raw or low-processed foods" combined with current trends in food production systems, which tend to have more automation and longer processing runs with simultaneously more stringent microbiological requirements, necessitate the implementation of new and obligatory sustainable strategies to respond to new challenges regarding food safety. Here, in light of studies, performed mainly with raw milk, that considered dominant "planktonic" conditions, we reexamine the changes triggered by cold storage alone or combined with nitrogen gas (N-2) flushing on bacterial populations and discuss how the observed benefits of the treatment could also contribute to limiting BF formation in dairies.
  • 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.
  • Yung, Yeni P.; Wickramasinghe, Raveendra; Vaikkinen, Anu; Kauppila, Tiina J.; Veryovkin, Igor V.; Hanley, Luke (2017)
    A hand-held diode laser is implemented for solid sampling in portable ambient mass spectrometry (MS). Specifically, a pseudocontinuous wave battery powered surgical laser diode is employed for portable laser diode thermal desorption (LDTD) at 940 nm and compared with nanosecond pulsed laser ablation at 2940 nm. Postionization is achieved in both cases using atmospheric pressure photoionization (APPI). The laser ablation atmospheric pressure photoionization (LAAPPI) and LDTD-APPI mass spectra of sage leaves (Salvia officinalis) using a field-deployable quadrupole ion trap MS display many similar ion peaks, as do the mass spectra of membrane grown biofilms of Pseudomonas aeruginosa: These results indicate that LDTD-APPI method should be useful for in-field sampling of plant and microbial communities, for example, by portable ambient MS. The feasibility of many portable MS applications is facilitated by the availability of relatively low cost, portable, battery-powered diode lasers. LDTD could also be coupled with plasma- or electrospray-based ionization for the analysis of a variety of solid-samples.
  • Durcik, Martina; Tammela, Päivi Sirpa Marjaana; Barančoková, Michaela; Tomašič, Tihomir; Ilaš, Janez; Kikelj, Danijel; Zidar, Nace (2018)
    ATP-competitive inhibitors of DNA gyrase and topoisomerase IV are among the most interesting classes of antibacterial drugs that are unrepresented in the antibacterial pipeline. We developed 32 new N-phenylpyrrolamides and evaluated them against DNA gyrase and topoisomerase IV from E.coli and Staphylococcus aureus. Antibacterial activities were studied against Gram-positive and Gram-negative bacterial strains. The most potent compound displayed an IC50 of 47 nm against E.coli DNA gyrase, and a minimum inhibitory concentration (MIC) of 12.5 mu m against the Gram-positive Enterococcus faecalis. Some compounds displayed good antibacterial activities against an efflux-pump-deficient E.coli strain (MIC=6.25 mu m) and against wild-type E.coli in the presence of efflux pump inhibitor PA beta N (MIC=3.13 mu m). Here we describe new findings regarding the structure-activity relationships of N-phenylpyrrolamide DNA gyrase B inhibitors and investigate the factors that are important for the antibacterial activity of this class of compounds.
  • Salih, E.Y.A.; Kanninen, M.; Sipi, M.; Luukkanen, O.; Hiltunen, R.; Vuorela, H.; Julkunen-Tiitto, R.; Fyhrqvist, P. (2017)
    Terminalia laxiflora, Terminalia brownii and Anogeissus leiocarpus are used as decoctions, macerations, infusions and fumigations in East and West African traditional medicine for treatment of infectious diseases and their symptoms. Using this ethnopharmacological information as a guideline for our research and owing to the fact that these species have not been subjected to in depth antibacterial and phytochemical studies, thirty-nine extracts of various polarities of the stem bark, stem wood and roots were studied for growth inhibitory effects against the human pathogenic bacteria Staphylococcus epidermidis, Staphylococcus aureus, Micrococcus luteus and Pseudomonas aeruginosa. Our results indicate that the studied species contain antibacterial compounds of a wide range of polarities. All polar root extracts of T. laxiflora and various polar extracts of T. brownii roots, including hot water decoctions, gave broad-spectrum antibacterial effects and low MIC values of 39 mu g/ml. The main ellagitannins in an ethyl acetate extract of the root of T. laxiflora were found to be corilagin and its derivative and punicalagin. A methanol extract of the roots of T. brownii contained methyl-(S)-flavogallonate and its derivative as the main identified ellagitannins. Moreover, both Terminalia species were found to contain ellagic acid xylopyranoside and methyl ellagic acid xyloside and pure ellagic acid was present in T. brownii. Pure punicalagin did not give as low MIC as an ethyl acetate extract of the roots of T. laxiflora, containing punicalagin as one of its main compounds, although this ellagitannin totally inhibited the growth of S. aureus at 125 mu g/ml and P. aeruginosa at 500 mu g/ml. Similarly, pure ellagic and gallic acid gave higher MIC values than the methanolic root extract of T. brownii against S. aureus and P. aeruginosa. Moreover, a Sephadex LH-20 fraction of the methanolic extract of the roots of T. brownii, enrichedwithmethyl-(S)-flavogallonate and its isomer, gave higher MIC values than the crude methanolic extract. These results suggest that the polyphenols in the extracts might act synergistically with each other. A methanolic soxhlet extract of the roots of A. leiocarpus, containing ampelopsin, aromadendrin, taxifolin, pinosylvin and 4'-methylpinosylvin gave a low MIC value of 39 mu g/ml against all bacterial strains used in this investigation. Our results demonstrate that the roots, stem bark and stem wood of T. brownii, T. laxiflora and A. leiocarpus are rich sources of (new) antimicrobial compounds and justify the uses of these plants for treatment of infections in African traditional medicine.
  • Nicol, Marion; Alexandre, Stephane; Luizet, Jean-Baptiste; Skogman, Malena; Jouenne, Thierry; Salcedo, Suzana P.; De, Emmanuelle (2018)
    The increasing threat of Acinetobacter baumannii as a nosocomial pathogen is mainly due to the occurrence of multidrug-resistant strains that are associated with the real problem of its eradication from hospital wards. The particular ability of this pathogen to form biofilms contributes to its persistence, increases antibiotic resistance, and promotes persistent/device-related infections. We previously demonstrated that virstatin, which is a small organic compound known to decrease virulence of Vibrio cholera via an inhibition of T4-pili expression, displayed very promising activity to prevent A. baumannii biofilm development. Here, we examined the antibiofilm activity of mono-unsaturated chain fatty acids, palmitoleic (PoA), and myristoleic (MoA) acids, presenting similar action on V. cholerae virulence. We demonstrated that PoA and MoA (at 0.02 mg/mL) were able to decrease A. baumannii ATCC 17978 biofilm formation up to 38% and 24%, respectively, presented a biofilm dispersing effect and drastically reduced motility. We highlighted that these fatty acids decreased the expression of the regulator abaR from the LuxIR-type quorum sensing (QS) communication system AbaIR and consequently reduced the N-acyl-homoserine lactone production (AHL). This effect can be countered by addition of exogenous AHLs. Besides, fatty acids may have additional non-targeted effects, independent from QS. Atomic force microscopy experiments probed indeed that PoA and MoA could also act on the initial adhesion process in modifying the material interface properties. Evaluation of fatty acids effect on 22 clinical isolates showed a strain-dependent antibiofilm activity, which was not correlated to hydrophobicity or pellicle formation ability of the tested strains, and suggested a real diversity in cell-to-cell communication systems involved in A. baumannii biofilm formation.
  • Mikonranta, Lauri; Mappes, Johanna; Laakso, Jouni; Ketola, Tarmo (2015)
    Background: Pathogens evolve in a close antagonistic relationship with their hosts. The conventional theory proposes that evolution of virulence is highly dependent on the efficiency of direct host-to-host transmission. Many opportunistic pathogens, however, are not strictly dependent on the hosts due to their ability to reproduce in the free-living environment. Therefore it is likely that conflicting selection pressures for growth and survival outside versus within the host, rather than transmission potential, shape the evolution of virulence in opportunists. We tested the role of within-host selection in evolution of virulence by letting a pathogen Serratia marcescens db11 sequentially infect Drosophila melanogaster hosts and then compared the virulence to strains that evolved only in the outside-host environment. Results: We found that the pathogen adapted to both Drosophila melanogaster host and novel outside-host environment, leading to rapid evolutionary changes in the bacterial life-history traits including motility, in vitro growth rate, biomass yield, and secretion of extracellular proteases. Most significantly, selection within the host led to decreased virulence without decreased bacterial load while the selection lines in the outside-host environment maintained the same level of virulence with ancestral bacteria. Conclusions: This experimental evidence supports the idea that increased virulence is not an inevitable consequence of within-host adaptation even when the epidemiological restrictions are removed. Evolution of attenuated virulence could occur because of immune evasion within the host. Alternatively, rapid fluctuation between outside-host and within-host environments, which is typical for the life cycle of opportunistic bacterial pathogens, could lead to trade-offs that lower pathogen virulence.