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Microbial Adhesion Group, Center for Biomedical Microbiology, BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark
Correspondence
Per Klemm
pkl{at}biocentrum.dtu.dk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The recurrence rate is high and often the infections tend to become chronic with many episodes. UTI usually starts as a bladder infection but often evolves to encompass the kidneys and ultimately can result in renal failure or dissemination to the blood. UTI is the most common infection in patients with a chronic indwelling bladder catheter; bacteriuria is essentially unavoidable in this patient group (Foxman, 2002). UTI is classified into disease categories by the site of infection: cystitis (the bladder), pyelonephritis (the kidney) and bacteriuria (the urine). The colonization of urine in the absence of clinical symptoms is called asymptomatic bacteriuria (ABU). ABU occurs in up to 6 % of healthy individuals and 20 % of elderly individuals. As the name implies, ABU strains generally do not cause symptoms; most patients with ABU do not need treatment and in many cases the colonizing organism actually helps to prevent infection by other more virulent bacteria (Darouiche et al., 2001). Escherichia coli is responsible for more than 80 % of all UTIs and causes both ABU and symptomatic UTI (Ronald, 2003). Generally, such infections are caused by a single bacterial clone and are in effect mono-cultures. Based on the symptoms they produce, UTI E. coli can be divided into ABU E. coli and uropathogenic E. coli (UPEC) strains.
Many bacteria live as sessile communities adhered to surfaces, rather than as planktonic isolated cells. These compact microbial consortia, referred to as biofilms, are commonly associated with many economic and health problems (Costerton et al., 1999). In medicine, biofilm-associated infections have a major impact on permanent and temporary artificial implants placed in the human body, often with devastating consequences. Moreover, biofilms associated with implants often serve as a source for recurrent infections. Many persistent and chronic bacterial infections are now believed to be linked to the formation of biofilms (Costerton et al., 1995, 1999). In the urinary tract, bacterial biofilms can develop on many living surfaces and virtually all artificial implants, producing chronic and often intractable infections. Notable biofilm-associated infections include chronic cystitis, prostatitis and catheter- and stent-associated infections (Warren, 2001). Bacterial biofilms were reported to affect 90 % of indwelling stents in patients (Reid et al., 1992). E. coli is responsible for most infections in patients with indwelling bladder catheters; 10–50 % of patients undergoing short-term catheterization develop UTI and essentially all patients with an indwelling urinary catheter in place for more than 30 days will have a UTI (Warren, 2001). Biofilm-associated bacteria are often hard to eradicate by antibiotics and a common observation is that soon after a course of antibiotic treatment the urinary tract will be readily reinfected by bacteria that survived in catheter biofilms (Warren, 2001). E. coli has also been reported to be able to form intracellular biofilm-like aggregates inside bladder cells making the bacteria hard to reach by both host defence mechanisms and antibiotics (Anderson et al., 2003).
It is a common observation that individuals with ABU due to E. coli have a reduced risk of pyelonephritis if they are left untreated (Hansson et al., 1989). The concept of using non-virulent but niche-dominant bacteria to prevent UTI with other, often virulent, bacteria is an example of bacterial interference. This concept can be used passively when patients infected with an ABU strain are left untreated, to prevent infections by virulent strains, or can be used actively by deliberate inoculations with selected ABU strains. One example is ABU strain 83972, which has successfully been used as a prophylactic agent to prevent infections in individuals prone to infections by harmful uropathogens (Darouiche et al., 2001, 2005; Hull et al., 2000; Trautner et al., 2002). The mechanisms employed by such ABU strains to exclude pathogens are largely unknown. On this background we have studied the biofilm-forming capacity on abiotic surfaces in human urine of a spectrum of ABU strains and compared it with that of UPEC and UTI-associated Klebsiella strains. Notably, we have probed the ability of selected ABU strains to compete with uropathogenic strains during biofilm formation.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. The prototypic ABU E. coli strain 83972 was originally isolated from a young Swedish girl (Lindberg et al., 1975). VR11 and VR12 are E. coli asymptomatic strains isolated from different cases of ABU; the E. coli strain VR10 and all the Klebsiella pneumoniae and Klebsiella oxytoca strains were isolated from different cases of symptomatic UTIs.
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as described previously (Diederich et al., 1992). Briefly, the NotI fragment of pSM2361 and pSM2362, carrying the cfp and yfp genes, respectively, was ligated into the bla-attP-containing NotI fragment of pLDR11 and transformed into E. coli cells expressing -Int from pLDR8. Transformants were selected on ampicillin-containing plates and checked for their ability to fluoresce. The proper insertion of the fragment into the attachment site was confirmed by PCR using primers 681 (5'-CGGTTTGATCAGAAGGACG-3'), 682 (5'-TTGATGTCGATGAAGGTGCC-3'), 683 (5'-TGGCCATGGAACAGGTAGT-3') and 684 (5'-ACCACATGGTCCTTCTTGAG-3').
Growth media.
All cultivations were performed in LB or pooled human urine. For each growth experiment, human urine was collected from three or four healthy volunteers who had no history of UTI or antibiotic use in the prior 2 months. The urine was pooled, filter-sterilized, stored at 4 °C and used within the following 2–3 days. When appropriate, antibiotics were added to the medium at the following concentrations: 100 µg ampicillin (Ap) ml–1, 50 µg kanamycin (Km) ml–1, 50 µg nalidixic acid (Nal) ml–1 and 100 µg streptomycin (Sm) ml–1.
Biofilm formation in microtitre plates.
Cells were grown overnight in pooled human urine and 10 µl was used to inoculate 1 ml urine in 24-well flat-bottom microplates (Iwaki). The microplates were incubated statically at 37 °C overnight. Unbound cells were removed by inversion of the microplate and tapping on absorbent paper; adhered cells were then stained with 0.1 % crystal violet for 15 min. Excess stain was removed by washing with PBS. The crystal violet was then solubilized by the addition of ethanol and A590 was measured (Ultrospec III, Pharmacia). Each strain was assayed in four wells on each plate and the whole experiment was repeated at least three times in different batches of urine. In each plate four wells were used as blanks containing sterile urine, and strains 83972 and CFT073 were inoculated in four wells each for reference.
Competition during biofilm formation in microtitre plates.
The wells of a 24-well flat-bottom microtitre plate were filled with 1 ml human urine and inoculated with an equal number of cells of the two strains in competition to a final OD600 of 0.01. After incubation at 37 °C for 24 h, the planktonic cells were removed and the wells were washed three times with 1 ml PBS. Adhered cells were carefully scraped from the wells with a pipette tip and resuspended in 300 µl PBS, and the mixture was vortexed vigorously for 30 s to disperse cell aggregates. The detachment of cells was confirmed by staining the well with crystal violet after this treatment. The number of c.f.u. of each strain in the final biofilm population was determined by plating serial dilutions of the cell suspension onto plain LB-agar plates or LB-agar plates supplemented with the appropriate antibiotic. The initial 1 : 1 ratio was confirmed in the same way. As most of the Klebsiella strains tested did not have any antibiotic resistance, the difference in colony morphology was used to distinguish between Klebsiella spp. (white opaque colonies) and ABU E. coli strains (translucent colonies).
Competition during biofilm formation in flow-cell chambers.
Flow-chamber experiments were performed at 37 °C in human urine, essentially as described previously (Christensen et al., 1999). Briefly, biofilms were grown on a microscope glass coverslip (Knittel 24x50 mm st1; Knittel Gläser) in three-channel chambers (1x4x40 mm). Each channel was inoculated with 250 µl of a mix (final OD600 0·05) of the two competing strains grown overnight in human urine. The cells were allowed to attach to the substratum for 1 h before the flow (3 ml urine h–1) was turned on. Biofilm formation was monitored over time by scanning confocal laser microscopy (SCLM; Zeiss LSM510 microscope) using a 40x/1.3 Plan-Neofluar oil objective. Acquired pictures were processed for display using the IMARIS software (Bitplane). Each competition experiment in flow chambers was performed in duplicate channels and repeated at least twice in different batches of urine.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Welch et al., 2002), were assessed in microtitre plates by quantitative crystal violet staining; human urine was used as growth medium to better mimic in vivo conditions. The ability to form a biofilm is often considered to be a virulence-associated trait. However, it transpired that the UPEC strains performed poorly compared with the ABU strains (Fig. 1). The six UPEC strains tested formed 72–87 % less biofilm than the prototypic ABU strain 83972. Furthermore, all the ABU strains formed significantly more biofilm than the reference UPEC strain CFT073 (paired two-tailed t test, P<0.001). Taken together, the data suggest that among our UTI E. coli strain set biofilm formation is primarily an ABU-linked phenotype.
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, 2003). Arguably, biofilm formation could be invoked to explain this phenomenon. To probe whether the good biofilm performance of strain 83972 would confer any advantage during mixed-strain biofilm competition in urine, equal amounts of ABU strain 83972 and each of the four prototypic UPEC strains, NU14, J96, 536 and CFT073cfp, were inoculated in microtitre plates. The fluorescent derivative of CFT073, CFT073cfp, was used in place of the wild-type strain to facilitate selection; CFT073cfp did not show any advantage/disadvantage during biofilm formation when competed against an equal number of the CFT073 parent (48±4 % of CFT073cfp was present in CFT073cfp-CFT073 mixed biofilms). When competed against the UPEC strains it transpired that after 24 h strain 83972 accounted for more than 50 % of the total bacterial population present in each one of the four mixed biofilms (Fig. 2). The fraction of 83972 ranged from over 90 % in the case of NU14 and J96 competitors down to about 60 % in the case of strain 536. The results suggest that strain 83972 has a significant advantage over the UPEC strains when it comes to biofilm formation in urine and is able to out-perform them in this mode of growth.
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). Interestingly, the best ABU biofilm former strain, viz. VR89, appeared to be the less competitive strain against NU14; furthermore, its performance against NU14 was highly variable (Fig. 3). This suggests that the ability to form a monoclonal biofilm on abiotic surfaces does not directly correlate with the ability to out-compete other strains in mixed populations. Nevertheless, the data indicate that the ability to out-perform UPEC strains during biofilm formation is not a unique quality of strain 83972, but seems to be common among ABU strains.
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). In fact, only a few CFT073cfp cells can be observed in the 83972yfp-CFT073cfp (1 : 1) mixed biofilm, where the yellow-fluorescent tagged strain, 83972yfp, strongly dominates and colonizes the whole surface (Fig. 4a). A similar observation was made when the strains were tagged in the reverse way, i.e. 83972cfp and CFT073yfp; in this case, the predominance of 83972cfp in the biofilm resulted in an essentially blue-coloured biofilm (Fig. 4c). This confirmed that the fluorescent protein used for the tagging does not affect the competitive behaviour of the strain and that 83972 out-performs CFT073 efficiently during biofilm formation in the flow-chamber system. The ability of a second ABU strain, VR50, to compete with CFT073 during biofilm formation in flow chambers was investigated. VR50 was tagged with yfp and the resulting VR50yfp strain was mixed in a 1 : 1 ratio with CFT073cfp. The proportion of each strain in the VR50yfp-CFT073cfp mixed biofilm was then analysed at different time points by SCLM (Fig. 4d). After 16 h, both strains had colonized the surface in similar proportions. However, after 24 h and 40 h, VR50yfp had clearly taken the advantage over CFT073cfp: VR50yfp covered most of the surface, while CFT073cfp developed small patches only. Taken together, these results demonstrate that both 83972 and VR50 ABU strains out-compete the prototypic UPEC strain CFT073 under hydrodynamic urine flow conditions, mimicking those encountered in the urinary tract and UT-associated catheters.
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) and ranking second in certain groups of the population worldwide (Gales et al., 2000; Gupta et al., 2001; Yuksel et al., 2006). Since little information on the biofilm-forming capacity of UTI-related klebsiellae is available, we tested this feature in a set of Klebsiella spp. strains isolated from symptomatic urinary tract infections. For this purpose, six K. pneumoniae and four K. oxytoca strains were grown in human urine in microtitre plates and the amount of attached cells was determined by crystal violet staining after 24 h. It turned out that the ability to form biofilm was very heterogeneous in this group: four of the Klebsiella strains (C105, i228-86, i239-86 and i129-96) appeared to be poor biofilm formers and did not perform significantly better than the worst performing UPEC strain CFT073 (paired two-tailed t test, P>0.05); on the other hand, three Klebsiella strains (i222-86, i3-89 and i113-96) appeared to be excellent biofilm formers, performing up to four times better than the prototypic ABU strain 83972 (Fig. 1
). Given the ability of 83972 to out-compete UPEC strains during biofilm formation, we tested whether this feature could be extended to another uropathogenic genus such as Klebsiella by competing 83972 against seven selected Klebsiella strains in mixed biofilm grown in human urine. In spite of the excellent biofilm-forming capacity of some of these uropathogenic Klebsiella strains, they were all efficiently excluded from the biofilms by strain 83972 and a majority of them constituted approximately 10 % of the population after 24 h (median=8 %) (Fig. 5a). Interestingly, the best Klebsiella biofilm former (i222-86) constituted only 15 % of the final biofilm population, suggesting an absence of correlation between the monoclonal biofilm-forming capacity of a strain and its ability to compete with 83972 in the biofilm growth mode. Finally, we challenged i222-86 with the three good ABU biofilm formers out-competing CFT073, i.e. VR50, VR89 and VR92. In line with the results obtained in the ABU-UPEC co-cultures, we found that all of the ABU strains tested were able to out-number the Klebsiella isolate i222-86 in the biofilm population (Fig. 5b). Taken together, our data indicate that selected ABU strains can efficiently compete against UPEC and Klebsiella spp. strains during biofilm formation.
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) could be due to different growth rates in human urine. We have recently demonstrated that the model ABU strain 83972 as well as several other ABU strains are able to out-compete UPEC strains during planktonic growth in urine (Roos et al., 2006a, b) and that the ability to compete in this growth mode was correlated with the growth rate (Roos et al., 2006a). Growth characteristics in urine might therefore be invoked to explain the performance of the strains in biofilm formation. However, it transpired that there was no correlation between the growth rates of our strains and their biofilm-forming capacities (Fig. 6). Comparing the growth rates of the strains competed against each other further revealed that the ability of a strain to compete during biofilm growth was not directly due to the fitness of the strain in urine; e.g. Klebsiella strain i222-86 displayed a growth rate similar to that of VR50 and higher than that of VR89 and VR92 although it was out-competed by them all. Also, nearly all Klebsiella strains reached a higher cell density in urine than 83972; C105 and i3-89 showed final cell densities over 70 % higher than 83972 but were both out-competed in the biofilm. The data suggest that fast growth is not of prime importance for biofilm formation or competitiveness of urinary tract-colonizing E. coli and Klebsiella, and that growth rate and biofilm formation seem to be unrelated phenotypes.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). E. coli and Klebsiella spp. are among the most common aetiological agents involved in UTI, accounting for up to 80 % and 15 %, respectively, of UTIs (Ronald, 2003; Wilson & Gaido, 2004). It seems that uropathogenic E. coli and Klebsiella are rapidly adapting to the antibiotics employed to treat UTIs. β-Lactams such as ampicillin and trimethoprim-sulfamethoxazole (TMP-SMX) combination have for decades been considered as drugs of choice against UTIs. However, the proportion of E. coli and Klebsiella spp. strains resistant to these groups of antibiotics have increased dramatically worldwide during the 1990s (for review: Gupta et al., 2001); the proportion of extended-spectrum β-lactamases (ESBL)-producing strains, principally E. coli and K. pneumoniae, increased between 1999 and 2003 among hospitalized patients in Italy (Luzzaro et al., 2006); furthermore, two independent studies reported an increase of TMP-SMX resistance among UPEC isolates between 1991 and 1997 (Dyer et al., 1998) and between 1992 and 1996 (Gupta et al., 1999). To complicate the picture, an increasing fraction of infections are caused by multidrug-resistant strains, as highlighted in two recent studies on E. coli-inflicted UTI in which 22 % and 41 % of the strains were found to be multidrug-resistant (Manges et al., 2001; Vranes et al., 2003). Therefore, other complementary approaches are needed in order to counter this emerging problem. One interesting possibility is to use bacterial interference. Bacterial interference is based on the concept that one bacterial strain can interfere with the ability of another to colonize and infect the host. The use of commensal-type bacteria to inhibit pathogens has a large potential because such bacteria are often natural competitors of pathogens and they are easy to administer. Applications of commensal-type bacteria as probiotics have been shown to reduce the risk of infection in the gastro-intestinal system and the urinary tract (Reid et al., 2001). However, the mode of action employed by the commensals to out-compete the pathogens is often unknown. Here we demonstrate that biofilm competition between selected commensal-type E. coli and two top uropathogens, viz. UPEC strains and uropathogenic Klebsiella spp., might be invoked to account for the well-known ability of some ABU strains to out-compete uropathogens and to keep these out of the urinary tract (Darouiche et al., 2001, 2005). The dominance of selected ABU strains during mixed-strain biofilm competition might also be reflected in the respective proportion of the strains in the planktonic phase of the culture. Indeed, in the case of some strain combinations, the percentage of the ABU strain in the planktonic population correlated with the percentage of the strain in the biofilm population. However, this was not the general rule and in mixed biofilms consisting of VR50 plus NU14, 83972 plus i239-86 and 83972 plus i3-89 the planktonic phase contained less of the ABU strain than did the biofilm phase (data not shown), again supporting the notion that mechanisms specific to biofilm formation are involved in the ability of the ABU strains to exclude uropathogens during biofilm formation.
Implanted prosthetic devices constitute particularly attractive surfaces for bacterial colonization and biofilm formation as they have none of the protective mechanisms of mucosal surfaces, e.g. exfoliation of epithelial cells. Urinary catheters and stents are used extensively in medical settings. Between 15 and 25 % of patients in general hospitals will have a urinary catheter in place for longer or shorter periods of time sometime during their stay (Warren, 2001). A large proportion of these patients will get symptomatic UTI because of catheter-associated bacterial biofilm formation by UPEC strains and uropathogenic Klebsiella. As previously mentioned, many of these strains are multidrug-resistant; however, the problem is further aggravated by the inherently high tolerance of biofilm-dwelling bacteria to antibiotics. As the name implies, ABU E. coli cause no or few symptoms and can coexist harmlessly with the patient for months and even years (Lindberg et al., 1978). ABU strains such as the prototypic 83972 strain have been extensively used for their probiotic qualities as prophylactic agents in patients and have been found to be safe (Darouiche et al., 2001; Hull et al., 2000). We found that strain 83972 can out-compete a range of uropathogenic E. coli and Klebsiella strains during biofilm formation on abiotic surfaces in human urine. Arguably, this experimental scenario mimics conditions in urinary catheters and stents and might explain why deliberate inoculation of catheterized patients with 83972 can prevent subsequent infections with uropathogens. Also, several others of our ABU strains (but not all) performed excellently under these conditions when pitted against uropathogens in competitive biofilm formation. We believe that our results on the biofilm-forming qualities of selected ABU strains underline their potential as alternative treatment agents against recalcitrant uropathogens when antibiotics fail.
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ACKNOWLEDGEMENTS |
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Edited by: I. R. Henderson
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 29 November 2006;
revised 24 January 2007;
accepted 7 February 2007.
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