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Carbon monoxide and nitric oxide as antimicrobial agents: focus on ESBL-producing uropathogenic E.coli
Örebro University, School of Medical Sciences.
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Urinary tract infections (UTI) are common in humans and most often caused by uropathogenic Escherichia coli (E. coli). Extended-spectrum beta-lactamase (ESBL)-producing E. coli are increasing worldwide and they are frequently multidrug-resistant with limited treatment options. The overall aim of this thesis was to study the role of the host-derived factors nitric oxide (NO) and carbon monoxide (CO) as antimicrobial agents against ESBL-producing uropathogenic strains of E. coli (UPEC).

The NO-donor DETA/NO caused a temporary growth inhibition in ESBL-producing UPEC. The antibacterial effect of DETA/NO was improved when DETA/NO was combined with miconazole, a pharmacological inhibitor of NO-protective mechanisms. Combination treatment with DETA/NO, miconazole and polymyxin B nonapeptid prolonged the bacteriostatic effect in the majority of examined isolates. The CO-donor CORM-2 showed a pronounced antibacterial effect in ESBL-producing UPEC with a fast bactericidal effect. Moreover, CORM-2 was shown to reduce the bacterial viability of ESBL-producing UPEC grown under biofilm-like conditions and to decrease the bacterial colonization of human bladder epithelial cells. A microarray analysis was performed to define transcriptomic targets of CORM-2 after a single exposure and after repeated exposure to CORM-2. Many processes were affected by CORM-2, including a downregulation in energy metabolism and biosynthesis pathways and upregulation of the SOS response and DNA repair. Repeated exposure to CORM-2 did not change the gene expression patterns or fold changes and the growth inhibitory response to CORM-2 was not altered after repeated exposure.

In conclusion, NO- and CO-donors have antibacterial effects against ESBL-producing UPEC and may be interesting candidates for development of new antibiotics to treat UTI caused by multidrug-resistant uropathogens.

Abstract [en]

Populärvetenskaplig sammanfattning

Urinvägsinfektioner är vanligt förekommande och orsakas ofta av Escherichia coli (E. coli)-stammar. Runt om i världen märks en tilltagande antibiotikaresistens hos bakterier vilket har ökat behovet av att finna nya behandlingsmetoder. Speciellt svårbehandlade ar urinvägsinfektioner orsakade av E. coli som bildar beta-laktamas enzymer med utvidgat spektrum sa kallade ESBL-bildande E. coli. ESBL-bildande bakterier kan bryta ner de flesta antibiotika ur penicillingruppen, som cefalosporiner, samt är ofta resistenta mot flera andra antibiotika (multiresistenta). Spridning av ESBL-bildande E. coli sker inte enbart i sjukhusmiljö utan även ute i samhället.

Vid urinvägsinfektion spelar immunforsvaret en viktig roll. Förekomst av bakterier i urinblåsan medför att ett komplext vävnadsförsvar aktiveras. Målet for avhandlingen har varit att studera den bakteriehämmande effekten av de kroppsegna faktorerna kväveoxid (NO) och kolmonoxid (CO) och då speciellt mot multiresistenta ESBL-bildande E. coli som orsakar urinvägsinfektion. NO har tidigare visats ge en kortvarig bakteriehämmande effekt på uropatogena E. coli. Bakterier kan bilda enzymet flavohemoglobin som omvandlar toxiskt NO till harmlöst nitrat. I avhandlingsarbetet har effekterna av mikonazol, en substans som hämmar flavohemoglobin, i kombination med NO undersökts. Mikonazol visades forlänga den bakteriehämmande effekten av en NO-donator (DETA/NO).

CO binder framförallt till hemproteiner och är en effektiv hämmare av bakteriell cellandning. Resultaten i avhandlingen visar att CO-donatorn CORM-2 har en kraftfull bakteriedödande effekt. Effekten av CORM-2 är snabb och inom fyra timmar har en bakteriedödande effekt uppnåtts. Vidare visas att CORM-2 påverkar genuttrycket av vissa virulensfaktorer hos uropatogena E. coli. Uropatogena E. coli har förmåga att bilda skyddande biofilm och dessutom kan de invadera epitelceller i urinvägarna och på så sätt bilda skyddade miljöer inuti cellerna. I avhandlingen visas att CORM-2 minskar antalet levande bakterier i en biofilm och att CORM-2 har en hämmande effekt på bakterier som koloniserat humana epitelceller från urinblåsa. I microarray försök, där förändringar av hela genuttrycket hos bakterier studeras, visas att exponering av CORM-2 generellt leder till nedreglering av energimetabolism och biosyntes samt uppreglering av en SOS stressresponsvag och DNA reparation. Uttryck av gener som har visats ha samband med virulens, biofilm och antibiotikaresistens påverkades också. Den bakteriedödande effekten och känsligheten för CORM-2 kvarstår hos bakterier som exponerats upprepade gånger för CORM-2.

Sammanfattningsvis visar resultaten i avhandlingen att NO- och COdonatorer har bakteriehämmande effekter på ESBL-bildande uropatogener. NO- och CO-donatorer kan vara intressanta att vidareutveckla vid design av framtida antibiotika mot uropatogena multiresistenta ESBL-bildande E. coli.

Place, publisher, year, edition, pages
Örebro: Örebro university , 2016.
Series
Örebro Studies in Medicine, ISSN 1652-4063 ; 136
Keyword [en]
biofilm, carbon monoxide, CORM-2, DETA/NO, extendedspectrum beta-lactamases, nitric oxide, urinary tract infection, uropathogenic Escherichia coli
National Category
Other Basic Medicine
Research subject
Biomedicine
Identifiers
URN: urn:nbn:se:oru:diva-47478ISBN: 978-91-7529-119-2 (print)OAI: oai:DiVA.org:oru-47478DiVA: diva2:895045
Public defence
2016-04-08, Universitetssjukhuset, hörsal C1, Södra Grev Rosengatan, Örebro, 09:00 (English)
Opponent
Supervisors
Available from: 2016-01-18 Created: 2016-01-18 Last updated: 2017-10-17Bibliographically approved
List of papers
1. The antibacterial effect of nitric oxide against ESBL-producing uropathogenic E-coli is improved by combination with miconazole and polymyxin B nonapeptide
Open this publication in new window or tab >>The antibacterial effect of nitric oxide against ESBL-producing uropathogenic E-coli is improved by combination with miconazole and polymyxin B nonapeptide
Show others...
2014 (English)In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 14, 65Article in journal (Refereed) Published
Abstract [en]

Background: Nitric oxide (NO) is produced as part of the host immune response to bacterial infections, including urinary tract infections. The enzyme flavohemoglobin, coded by the hmp gene, is involved in protecting bacterial cells from the toxic effects of NO and represents a potentially interesting target for development of novel treatment concepts against resistant uropathogenic bacteria. The aim of the present study was to investigate if the in vitro antibacterial effects of NO can be enhanced by pharmacological modulation of the enzyme flavohemoglobin.

Results: Four clinical isolates of multidrug-resistant extended-spectrum beta-lactamase (ESBL)-producing uropathogenic E. coli were included in the study. It was shown that the NO-donor substance DETA/NO, but not inactivated DETA/NO, caused an initial growth inhibition with regrowth noted after 8 h of exposure. An hmp-deficient strain showed a prolonged growth inhibition in response to DETA/NO compared to the wild type. The imidazole antibiotic miconazole, that has been shown to inhibit bacterial flavohemoglobin activity, prolonged the DETA/NO-evoked growth inhibition. When miconazole was combined with polymyxin B nonapeptide (PMBN), in order to increase the bacterial wall permeability, DETA/NO caused a prolonged bacteriostatic response that lasted for up to 24 h.

Conclusion: An NO-donor in combination with miconazole and PMBN showed enhanced antimicrobial effects and proved effective against multidrug-resistant ESBL-producing uropathogenic E. coli.

Place, publisher, year, edition, pages
London: BioMed Central, 2014
Keyword
Uropathogenic E. coli, Extended-spectrum beta-lactamase, Nitric oxide, Polymyxin B nonapeptide
National Category
Microbiology Medical and Health Sciences
Research subject
Microbiology
Identifiers
urn:nbn:se:oru:diva-35214 (URN)10.1186/1471-2180-14-65 (DOI)000335406100001 ()24629000 (PubMedID)2-s2.0-84899073270 (Scopus ID)
Note

Funding Agencies:

Swedish Council for Working Life and Social Research (FAS)

Nyckelfonden at Örebro University Hospital

Örebro University

Available from: 2014-06-03 Created: 2014-06-02 Last updated: 2017-03-14Bibliographically approved
2. Multiresistant uropathogenic extended-spectrum β-lactamase (ESBL)-producing Escherichia coli are susceptible to the carbon monoxide releasing molecule-2 (CORM-2).
Open this publication in new window or tab >>Multiresistant uropathogenic extended-spectrum β-lactamase (ESBL)-producing Escherichia coli are susceptible to the carbon monoxide releasing molecule-2 (CORM-2).
Show others...
2014 (English)In: Microbial Pathogenesis, ISSN 0882-4010, E-ISSN 1096-1208, Vol. 66, 29-35 p.Article in journal (Refereed) Published
Abstract [en]

Carbon monoxide (CO) releasing molecules (CO-RMs) have been shown to inhibit growth of commensal Escherichia coli (E. coli). In the present study we examined the effect of CORM-2 on uropathogenic E. coli (UPEC) that produces extended-spectrum β-lactamase (ESBL). Viability experiments showed that CORM-2 inhibited the growth of several different ESBL-producing UPEC isolates and that 500 μM CORM-2 had a bactericidal effect within 4 h. The bactericidal effect of CORM-2 was significantly more pronounced than the effect of the antibiotic nitrofurantoin. CORM-2 demonstrated a low level of cytotoxicity in eukaryotic cells (human bladder epithelial cell line 5637) at the concentrations and time-points where the antibacterial effect was obtained. Real-time RT-PCR studies of different virulence genes showed that the expression of capsule group II kpsMT II and serum resistance traT was reduced and that some genes encoding iron acquisition systems were altered by CORM-2. Our results demonstrate that CORM-2 has a fast bactericidal effect against multiresistant ESBL-producing UPEC isolates, and also identify some putative UPEC virulence factors as targets for CORM-2. CO-RMs may be candidate drugs for further studies in the field of finding new therapeutic approaches for treatment of uropathogenic ESBLproducing E. coli.

Place, publisher, year, edition, pages
London: Elsevier, 2014
Keyword
Carbon monoxide; CORM-2; Extended-spectrum β-lactamases; Uropathogenic E. coli; CTX-M
National Category
Microbiology in the medical area Immunology in the medical area
Identifiers
urn:nbn:se:oru:diva-33229 (URN)10.1016/j.micpath.2013.12.003 (DOI)000331853700005 ()24361394 (PubMedID)2-s2.0-84891068559 (Scopus ID)
Note

Funding Agency:

Swedish Council for Working Life and Social Research, from the Faculty of Medicine and Health at Orebro University and Nyckelfonden at Orebro University Hospital

Available from: 2014-01-22 Created: 2014-01-22 Last updated: 2017-03-14Bibliographically approved
3. Carbon monoxide releasing molecule-2 (CORM-2) inhibits growth of multidrug-resistant uropathogenic Escherichia coli in biofilm and following host cell colonization
Open this publication in new window or tab >>Carbon monoxide releasing molecule-2 (CORM-2) inhibits growth of multidrug-resistant uropathogenic Escherichia coli in biofilm and following host cell colonization
(English)Article in journal (Other academic) Submitted
National Category
Other Basic Medicine
Identifiers
urn:nbn:se:oru:diva-49071 (URN)
Available from: 2016-03-10 Created: 2016-03-10 Last updated: 2017-10-17Bibliographically approved
4. Global gene expression profiling and antibiotic susceptibility after repeated exposure to the carbon monoxide-releasing molecule-2 (CORM-2) in multidrug-resistant ESBL-producing uropathogenic Escherichia coli
Open this publication in new window or tab >>Global gene expression profiling and antibiotic susceptibility after repeated exposure to the carbon monoxide-releasing molecule-2 (CORM-2) in multidrug-resistant ESBL-producing uropathogenic Escherichia coli
(English)Manuscript (preprint) (Other academic)
National Category
Other Basic Medicine
Research subject
Biomedicine
Identifiers
urn:nbn:se:oru:diva-49073 (URN)
Available from: 2016-03-10 Created: 2016-03-10 Last updated: 2017-10-17Bibliographically approved

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