To Örebro University

Urinary tract infection (UTI) is one of the most common bacterial infections in humans, and the majority are caused by uropathogenic Escherichia coli (UPEC). The rising antibiotic resistance among UPEC and the frequent failure of antibiotics to effectively treat recurrent UTI and catheter-associated UTI motivate research on alternative ways of managing UTI. Abundant evidence indicates that the toxic radical nitric oxide (NO), formed by activation of the inducible nitric oxide synthase, plays an important role in host defence to bacterial infections, including UTI. The major source of NO production during UTI is from inflammatory cells, especially neutrophils, and from the uroepithelial cells that are known to orchestrate the innate immune response during UTI. NO and reactive nitrogen species have a wide range of antibacterial targets, including DNA, heme proteins, iron-sulfur clusters, and protein thiol groups. However, UPEC have acquired a variety of defence mechanisms for protection against NO, such as the NO-detoxifying enzyme flavohemoglobin and the NO-tolerant cytochrome bd-I respiratory oxidase. The cytotoxicity of NO-derived intermediates is nonspecific and may be detrimental to host cells, and a balanced NO production is crucial to maintain the tissue integrity of the urinary tract. In this review, we will give an overview of how NO production from host cells in the urinary tract is activated and regulated, the effect of NO on UPEC growth and colonization, and the ability of UPEC to protect themselves against NO. We also discuss the attempts that have been made to develop NO-based therapeutics for UTI treatment.

Treatment of urinary tract infections is today a challenge due to the increasing prevalence of multidrug-resistant ESBL-producing uropathogenic Escherichia coli (UPEC). There is an urgent need for new treatment strategies for multidrug-resistant UPEC and preferably with targets that have low potential for development of resistance. Carbon monoxide-releasing molecules (CORMs) are novel and potent antibacterial agents. The present study examines the transcriptomic targets of CORM-2 in a multidrug-resistant ESBL-producing UPEC isolate in response to a single exposure to CORM-2 and after repeated exposure to CORM-2. The bacterial viability and minimal inhibitory concentration (MIC) were also examined after repeated exposure to CORM-2. Microarray analysis revealed that a wide range of processes were affected by CORM-2, including a general trend of down-regulation in energy metabolism and biosynthesis pathways and up-regulation of the SOS response and DNA repair. Several genes involved in virulence (ibpB), antibiotic resistance (marAB, mdtABC) and biofilm formation (bhsA, yfgF) were up-regulated, while some genes involved in virulence (kpsC, fepCEG, entABE), antibiotic resistance (evgA) and biofilm formation (artIP) were down-regulated. Repeated exposure to CORM-2 did not alter the gene expression patterns, the growth inhibitory response to CORM-2 or the MIC values for CORM-2, cefotaxime, ciprofloxacin and trimethoprim. This study identifies several enriched gene ontologies, modified pathways and single genes that are targeted by CORM-2 in a multidrug-resistant UPEC isolate. Repeated exposure to CORM-2 did not change the gene expression patterns or fold changes and the susceptibility to CORM-2 remained after repeated exposure.

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.

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.

Increased resistance to antimicrobial agents is a characteristic of many bacteria growing in biofilms on for example indwelling urinary catheters or in intracellular bacterial reservoirs. Biofilm-related infections caused by multidrug-resistant bacteria, such as extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae, are a major challenge. The aim of this study was to investigate if a carbon monoxide-releasing molecule (CORM-2) has antibacterial effects against ESBL-producing uropathogenic E. coli (UPEC) in the biofilm mode of growth and following colonization of host bladder epithelial cells.

Results

The effect of CORM-2 was examined on bacteria grown within an established biofilm (biofilm formed for 24 h on plastic surface) by a live/dead viability staining assay. CORM-2 (500 μM) exposure for 24 h killed approximately 60 % of the ESBL-producing UPEC isolate. A non-ESBL-producing UPEC isolate and the E. coli K-12 strain TG1 were also sensitive to CORM-2 exposure when grown in biofilms. The antibacterial effect of CORM-2 on planktonic bacteria was reduced and delayed in the stationary growth phase compared to the exponential growth phase. In human bladder epithelial cell colonization experiments, CORM-2 exposure for 4 h significantly reduced the bacterial counts of an ESBL-producing UPEC isolate.

Conclusion

This study shows that CORM-2 has antibacterial properties against multidrug-resistant UPEC under biofilm-like conditions and following host cell colonization, which motivate further studies of its therapeutic potential.

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.

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.