| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Laboratorio de Microbiología, Instituto de Investigaciones Biológicas Clemente Estable, Avenida Italia 3318, CP11600 Montevideo, Uruguay
Correspondence
Pablo Zunino
pablo{at}iibce.edu.uy
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
). It shows a predilection for the upper urinary tract where it can cause severe histological damage and it is frequently associated with bladder and kidney stone formation (Mobley & Hausinger, 1989).
Several potential P. mirabilis virulence factors related to UTI have been described, including fimbrial-mediated adherence to the uroepithelium, swarming motility mediated by flagella, outer-membrane protein (OMP) expression, cell invasiveness, urease production, haemolysin production, and iron acquisition (Coker et al., 2000; Rozalski et al., 1997). Some of these virulence factors are crucial for the successful colonization of the urinary tract and progression of infection (Coker et al., 2000), but the specific role and pathogenic significance of other factors is still the subject of debate (Burall et al., 2004). Although the role of specific virulence factors has been associated with colonization of the urinary tract, the existence of P. mirabilis clonal groups related to uropathogenesis has not yet been determined.
E. coli appears as a clear example of the existence of uropathogenic strains that show clonal segregation of specific genes whose expression is associated with pathogenesis (Johnson, 1991). Uropathogenic E. coli (UPEC) differ from non-pathogenic E. coli and from other E. coli pathotypes by the production of specific virulence factors, such as adhesins, toxins, capsules and iron-uptake systems that contribute to virulence and to establishing UTIs (Oelschlaeger et al., 2002a). The presence of different pathogenicity islands containing several genes related to virulence determinants of UPEC strains, and their absence in non-pathogenic E. coli, have been recently reported. The acquisition of these pathogenicity islands creates new pathotypes that are more efficient at establishing a UTI (Oelschlaeger et al., 2002b).
Proteus species are widely distributed in nature and play a significant ecological role. When present in different niches of higher organisms, these species are able to evoke pathological events in diverse regions of the human body (Rozalski et al., 1997). Several authors have investigated P. mirabilis virulence factor distribution among strains from different sources, although data are not extensive or conclusive. Mobley & Chippendale (1990) suggest that P. mirabilis mannose-resistant, Proteus-like (MR/P) haemagglutinin can be significantly more predominant in pyelonephritogenic isolates compared to those from asymptomatic bacteriuria and faecal strains. However, the same authors suggest that expression of urease, a major uropathogenic P. mirabilis virulence factor, is present in all isolates from any source (Mobley & Chippendale, 1990). Gaastra et al. (1996) assessed the presence and expression of genes of MR/P and uroepithelial cell adhesion (UCA) fimbriae, and production of haemolysin in P. mirabilis strains isolated from the urine and faeces of dogs with UTI. These authors observed that all but one of the P. mirabilis strains were haemolytic and that most strains produced fimbriae. They also showed that the UCA fimbrial subunits from dog and human isolates had identical molecular masses and N-terminal sequences, and were immunologically cross-reactive.
In order to improve the comprehension of P. mirabilis UTI pathogenesis, and to determine a possible correlation between origin, virulence factors and in vivo infectivity, in the present study, a collection of clinical and non-clinical P. mirabilis isolates was subjected to several in vitro assays and was used in the experimental ascending UTI model in mice. In addition, the Dienes test and the enterobacterial repetitive intergenic consensus (ERIC)-PCR assay were performed to evaluate the genetic diversity within the P. mirabilis isolate collection.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
. All isolates were stored at –80 °C in Luria–Bertani (LB) broth supplemented with 12 % (v/v) glycerol and grown aerobically at 37 °C. All media were from Difco Laboratories and all chemicals were reagent grade (Sigma). Ovine erythrocytes were from Biokey Laboratory (Uruguay).
|
) and then incubated overnight at 37 °C.
Urease production.
The expression of urease was assessed by placing differentiation disks (Difco Laboratories) aseptically on the surface of cultures grown overnight on LB agar plates and checking the change of colour after 5 min.
Growth rates in urine.
Urine supplied by healthy volunteers was collected under aseptic conditions, pooled and filtered immediately after collection using a 0.45 µm pore nitrocellulose filter membrane (Millipore). Bacterial suspensions of similar concentration (OD600 0.015) were prepared in PBS from freshly streaked LB agar plates, and 100 µl of these suspensions was transferred to 10 ml fresh urine and incubated at 37 °C with shaking. The capability to grow in urine was monitored by serial viable bacterial counts on nutrient agar (NA). Three independent assays were performed and the following parameters were used to evaluate growth (Carlberg, 1986): doubling time [ln(2tm)/ln(R2/R1)] and specific growth rate [ln(R2/R1)/t] where R1 represents the viable bacterial count at time 1 (4 h), R2 the viable bacterial count at time 2 (7 h), t the interval time in hours, and tm the interval time in minutes.
Iron-dependent growth assays.
To test the ability of P. mirabilis to use different iron sources, the procedure described by Sanders et al. (1994) was used. Diverse iron compounds, including haemin (5 mg ml–1), haemoglobin (5 mg ml–1), ferric citrate (50 mM) and human transferrin (1 mg ml–1), were evaluated using embedded disks of 5 mm diameter. As a negative control, a disk was soaked in sterile PBS. Three independent assays were performed and the diameter of the growth zones was measured after incubation for 24 h at 37 °C.
Haemagglutination.
Isolates were tested for their ability to agglutinate fresh human erythrocytes using the procedure described by Bahrani & Mobley (1994). Bacteria were statically subcultured three times for 48 h at 37 °C in LB broth to enable the expression of MR/P fimbriae (Old & Adegbola, 1982).
Haemolytic activity.
-Haemolytic activity was determined by plating bacteria onto blood agar base medium supplemented with 5 % (v/v) ovine blood. Haemolytic activity was titred using the procedure described by Mobley & Chippendale (1990).
Extraction of OMPs.
OMP extraction was performed according to the procedure described by Piccini et al. (1998).
SDS-PAGE.
Proteins or whole bacteria were suspended in sample buffer, boiled for 10 min and electrophoresed by 12 % SDS-PAGE as described by Sambrook et al. (1989). The proteins were stained with 0.1 % (v/v) Coomasie brilliant blue.
Western blotting.
Whole-cell preparations obtained from 48 h static cultures in LB broth were run on SDS-PAGE and proteins were transferred to nitrocellulose membranes (Bio-Rad) as described by Towbin et al. (1979). Western immunoblots were performed using a 1 : 200 dilution in PBS/Tween 20/1 % (w/v) skimmed milk of rabbit polyclonal immune sera raised against MrpA (Zunino et al., 2001), PmfA (Zunino et al., 2003) and AtfA (Zunino et al., 2000), and mouse polyclonal immune sera raised against UcaA (Scavone et al., 2004). Isogenic fimbrial mrpA, pmfA, atfA–C and ucaA mutants were included as controls in the Western blotting assays (strains MSD2, P2, A4 and UM1 respectively; see Table 1
).
DNA amplification by PCR.
Genomic DNA of the different isolates was used as a template to amplify the mrpA, pmfA and ucaA genes and for ERIC-PCR analysis. For DNA extraction, bacterial suspensions in MilliQ water were boiled for 10 min, centrifuged (12 096 g, 10 min, 4 °C), and supernatants were recovered for PCR reactions.
The primers used in this study were synthesized by Sigma–Genosys and are described in Table 2. Primers used to amplify mrpA were derived from the published mrp fimbrial operon sequence from strain HI4320 (GenBank accession no. Z32686; Bahrani & Mobley, 1994). The cycle used was 94 °C for 3 min, followed by 30 cycles of 94 °C for 30 s, 40 °C for 30 s and 72 °C for 30 s, with a final cycle at 72 °C for 7 min. Primers used to amplify ucaA were derived from the published uca sequence from strain HU1069 (GenBank accession no. U28420; Cook et al., 1995). The cycle used was 94 °C for 3 min, followed by 30 cycles of 94 °C for 30 s, 50 °C for 30 s and 72 °C for 30 s, with a final cycle at 72 °C for 7 min. The primers used for ERIC-PCR analysis were derived from those published by Versalovic et al. (1991) and the PCR programme was 94 °C for 5 min, followed by 40 cycles of 94 °C for 1 min, 40 °C for 2 min and 65 °C for 8 min, with a final cycle at 65 °C for 16 min.
|
Animals and experimental ascending UTI.
All animal experiments were conducted in accordance with procedures authorized by Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Montevideo, Uruguay. Female CD-1 6–8-week-old mice from the breeding facilities at IIBCE were used and provided with food pellets and tap water ad libitum. The ascending UTI model in mice (Zunino et al., 2000) was used to assess the virulence of different P. mirabilis isolates. Groups of 9 to 11 mice were challenged with each isolate.
Dienes test.
The Dienes test was performed as described by Bale & Hollis (1992). Each isolate was tested against all other isolates within the collection and the assays were performed in duplicate.
Data analysis.
The results of doubling time and specific growth rate were assessed using one-way analysis of variance (ANOVA). Numbers of c.f.u. per organ were compared by Kruskal–Wallis non-parametric analysis. In all cases, the differences were considered significant at P<0.05. For ERIC-PCR analysis, each amplified product revealed by electrophoresis was recorded as binary data presence (1) and absence (0), and an initial matrix of 0 and 1 was constructed. A dendrogram was created by using the Pearson product–moment correlation coefficient and the unweighted pair-group method with arithmetic means (UPGMA) algorithm.
|
RESULTS AND DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
; Williams & Schwarzhoff, 1978). Some reports have indicated that P. mirabilis swarming motility occurs in vivo, promoting UTI (Mobley et al., 1996; Allison et al., 1994; Pazin & Braude, 1974). However, other studies indicate that this property is not essential in P. mirabilis UTI (Jansen et al., 2003; Legnani-Fajardo et al., 1996; Zunino et al., 1994). In the present study, typical P. mirabilis swarming motility was exhibited by all isolates when grown on agar plates.
Urease production
Urease, a nickel metalloenzyme that catalyses the hydrolysis of urea, resulting in an elevation of pH, contributes to the formation of bladder and kidney stones in P. mirabilis UTI (Mobley & Hausinger, 1989). In this study, all P. mirabilis isolates showed strong production of urease. However, although urease can be associated with stone formation and serious kidney damage, it cannot be used as an index of virulence, since nearly all P. mirabilis strains produce this enzyme (Mobley & Chippendale, 1990).
Growth in urine
Although urine may prevent bacterial growth, not only by its continuous flow, but also because of its osmolarity, urea concentration and low iron concentration (Robledo et al., 1990), uropathogenic micro-organisms have developed the ability to grow in this medium (Russo et al., 1996). Gordon & Riley (1992) suggest that E. coli strains isolated from the urinary tract have significantly higher in vitro growth rates in urine than strains isolated from other areas. Therefore, we tested the ability of P. mirabilis isolates to grow in human urine, assessing the doubling time and the specific growth rate (Table 3). All but two P. mirabilis isolates (EMI2 and SAF1) were able to grow and showed no significant differences in doubling time or in specific growth rate (P>0.05). Six isolates showed a large standard deviation in growth rate, which may suggest a lack of significant differences between bacterial generation times.
|
suggest that P. mirabilis expresses iron-regulated OMPs that could be involved in haem uptake and may have a role in pathogenesis. When iron-dependent growth was assessed, all P. mirabilis isolates were able to use ferric citrate, haemin and haemoglobin as unique iron sources when cultured on iron-free solid media [NA supplemented with ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDHA)]. Diameters of growth zones around the disks were similar between every isolate (21–31 mm, 19–25 mm and 11–21 mm for ferric citrate, haemin and haemoglobin, respectively). However, none of the isolates was able to grow on NA/EDDHA supplemented only with transferrin.
OMP patterns
OMPs are potential virulence factors with a wide range of possible functions related to the development of UTI (Moayeri et al., 1991). When OMP profile was analysed using SDS-PAGE, all isolates showed similar patterns. A predominant 39 kDa band that corresponded to OmpA and a 36 kDa band that may have represented a peptidoglycan-associated matrix protein were observed. Both proteins have been described by Bub et al. (1980) in P. mirabilis strains.
Haemolysin production
Haemolysin production by uropathogens has usually been correlated with in vitro cytotoxicity (Peerbooms et al., 1983). In this study, haemolysin expression by P. mirabilis isolates was initially evaluated on blood agar base medium plates supplemented with ovine blood. All isolates except OPS, SEC1 and SAF1 generated -haemolysis haloes around bacterial growth areas. When haemolytic activity was titred, we observed that titres varied between isolates, but all produced measurable haemolytic activity that peaked during the late-exponential phase of growth. However, differences of more than twofold were found in haemolytic titres between different isolates (Table 3). No correlation between haemolytic activity and origin of isolates could be established. Our results are in accordance with those reported by Mobley & Chippendale (1990), who did not find significant differences between the haemolytic activity of P. mirabilis strains isolated from pyelonephrithis, and that of catheter-associated and faecal strains.
Haemagglutination assay
Haemagglutination, a property that reveals specific adhesin-receptor interactions, is associated with expression of P. mirabilis MR/P fimbriae (Old & Adegbola, 1985, 1982; Adegbola et al., 1983). When haemagglutination was evaluated within our isolate collection, it was observed that every tested isolate was able to agglutinate fresh human erythrocytes. As expected, the reaction was not inhibited by 50 mM mannose.
PCR amplification and expression of fimbrial genes
P. mirabilis represents a particular case in which various types of fimbriae can be expressed simultaneously by the same cell (Mobley & Belas, 1995). Several authors suggest that P. mirabilis fimbriae are implicated in the colonization of the urinary tract (Coker et al., 2000; Rozalski et al., 1997).
In order to detect mrpA, pmfA and ucaA fimbrial genes, PCR was performed using specific primers and genomic DNA from each isolate. PCR amplification of fimbrial genes indicated that all isolates carried mrpA and pmfA. However, ucaA was not detected when genomic DNA of isolates BZ3, XT1 and XT2 was tested. In all cases, P. mirabilis mrpA, pmfA and ucaA fimbrial genes amplified by PCR exhibited the predicted sizes (565, 563 and 580 bp, respectively).
The ability of the different isolates to synthesize MR/P, UCA, PMF and ATF fimbriae was assessed by Western blotting using immune polyclonal antisera. We determined that all isolates expressed MR/P and PMF fimbriae. These two particular types of fimbriae are frequently related to P. mirabilis UTI pathogenesis (Zunino et al., 2001, 2003; Bahrani et al., 1994; Massad et al., 1994).
A different situation was observed when expression of UCA and ATF fimbriae was assessed, since these types of fimbriae were not expressed by all isolates. Nineteen isolates were able to express UCA fimbriae, except XT1, XT2 and BZ3, which also lacked the ucaA gene, as observed after PCR amplification (Table 3). The isolates that expressed UCA fimbriae exhibited bands whose molecular masses ranged from 23 to 29 kDa. These results confirm those reported by Tolson et al. (1995), who suggest that the molecular mass of the structural subunit of P. mirabilis non-agglutinating fimbriae (NAF, an alternative name proposed for UCA fimbriae) is also in this range. Interestingly, we also observed that the two isolates Pr783 and 43071 showed two bands of 
23 and 26 kDa, respectively, which specifically reacted with the anti-UcaA immune serum. This could be explained by fimbrial glycosylation or the existence of fimbrial antigenic variation, among other hypotheses (Power & Jennings, 2003).
Finally, 18 isolates expressed ATF fimbriae, while no reactive bands were seen in the lanes corresponding to Pr6515, OPS, 4149 and SAF1 when Western blotting was performed. We could not observe any correlation between expression of UCA or ATF fimbriae and isolate sources.
Experimental ascending UTI in mice
Although the experimental ascending UTI model in mice has been extensively used to evaluate the role of different P. mirabilis virulence factors (Zunino et al., 2000, 2001, 2003; Legnani-Fajardo et al., 1996; Bahrani et al., 1994; Massad et al., 1994; Jones et al., 1990; Swihart & Welch, 1990), so far, it has not been used to assess the potential infectivity of P. mirabilis isolates from diverse sources. Different groups of mice were transurethrally challenged with each isolate. After sacrifice, the bladder and kidneys were homogenized and viable bacterial counts were performed. The numbers of c.f.u. recovered from bladders and kidneys of the different groups of mice are shown in Fig. 1. All isolates from our collection were able to infect the urinary tract of mice and no significant differences were observed among the number of bacteria recovered from the kidneys and bladders of the different groups of animals (P>0.05).
|
). If two strains are related or identical, there is no mutual inhibition and no lines of demarcation are formed. This assay was highly effective for differentiating the diverse isolates, and we could determine that all isolates tested, except BZ1-BZ2 and XT1-XT2, were genetically unrelated (Dienes lines were present).
Secondly, we analysed the genetic profile of the isolates using ERIC-PCR. ERIC sequences, 126 bp elements which contain a highly conserved, central inverted repeat and are located in extragenic regions of the bacterial genome, have been successfully used for DNA typing (Olive & Bean, 1999). All P. mirabilis isolates from our collection were typable by ERIC-PCR. Fingerprints consisted of 4–17 amplification bands, ranging in size from 400 to 4000 bp (Fig. 2). All isolates showed unique ERIC-PCR patterns, even the BZ1–BZ2 and XT1–XT2 pairs that were not differentiated by the Dienes test.
|
Conclusions
P. mirabilis has been described as an aetiological agent of a wide range of infections, both in humans and in diverse animal species. Moreover, P. mirabilis is a ubiquitous organism that can be found in different environments outside the host, such as soil, water and sewage (Rozalski et al., 1997).
In the present study, we used in vitro and in vivo assays to characterize a collection of P. mirabilis isolates recovered from clinical (urinary and non-urinary) and non-clinical sources. The main goal of our investigation was to assess a possible correlation between origin, presence of potential urovirulence factors, and infectivity of the different isolates, using the experimental ascending UTI model in mice. For this purpose, we characterized a collection of P. mirabilis isolates from different sources with regard to several potential urovirulence factors, infectivity in mice and genetic diversity.
Our results support the hypothesis that clinical and non-clinical P. mirabilis isolates represent overlapping populations that show similar capacities for causing UTI in mice. The distribution of virulence factors between strains of different origin may reflect the opportunistic nature of the organism, and also indicates that P. mirabilis strains cannot be distributed into clonal pathogenic groups.
Our data also indicate that uropathogenesis of P. mirabilis may be a multifactorial process in which different virulence factors can act in a concerted or even redundant fashion, since absence of different factors did not abolish the colonization of the lower and upper urinary tract in mice (Zunino et al., 2000, 2001, 2003; Legnani-Fajardo et al., 1996; Bahrani et al., 1994; Massad et al., 1994).
The considerable similarities between virulence features of clinical and non-clinical P. mirabilis isolates have potentially important implications for disease prevention, antibiotic resistance avoidance and pathogenesis studies.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
Allison, C., Emody, L., Coleman, N. & Hughes, C. (1994). The role of swarm cell differentiation and multicellular migration in the uropathogenicity of Proteus mirabilis. J Infect Dis 169, 1155–1158.[Medline]
Bahrani, F. K. & Mobley, H. L. (1994). Proteus mirabilis MR/P fimbrial operon: genetic organization, nucleotide sequence, and conditions for expression. J Bacteriol 176, 3412–3419.
Bahrani, F. K., Massad, G., Lockatell, C. V., Johnson, D. E., Russell, R. G., Warren, J. W. & Mobley, H. L. (1994). Construction of an MR/P fimbrial mutant of Proteus mirabilis: role in virulence in a mouse model of ascending urinary tract infection. Infect Immun 62, 3363–3371.
Bale, M. & Hollis, J. (1992). Characterization of organisms for epidemiologic purposes: serotyping, pyocin typing, and Dienes test. In Clinical Microbiology Procedures Handbook, pp 11.14.1–11.14.2. Edited by H. D. Isenberg. Washington DC: American Society for Microbiology.
Belas, R., Erskine, D. & Flaherty, D. (1991). Proteus mirabilis mutants defective in swarming cells differentiation and multicellular behaviour. J Bacteriol 173, 6279–6288.
Bub, F., Bieker, P., Martin, H. H. & Nixdorff, K. (1980). Immunological characterization of two major proteins isolated from the outer membrane of Proteus mirabilis. Infect Immun 27, 315–321.
Burall, L. S., Harro, J. M., Li, X., Lockatell, C. V., Himpsl, S. D., Hebel, J. R., Johnson, D. E. & Mobley, H. L. (2004). Proteus mirabilis genes that contribute to pathogenesis of urinary tract infection: identification of 25 signature-tagged mutants attenuated at least 100-fold. Infect Immun 72, 2922–2938.
Carlberg, D. M. (1986). Determining the effects of antibiotics on bacterial growth by optical and electrical methods. In Antibiotics in Laboratory Medicine, pp. 64–92. Edited by V. Lorian. Baltimore: Williams & Wilkins.
Coker, C., Poore, C. A., Li, X. & Mobley, H. L. (2000). Pathogenesis of Proteus mirabilis urinary tract infection. Microbes Infect 2, 1497–1509.[CrossRef][Medline]
Cook, S. W., Mody, N., Valle, J. & Hull, R. (1995). Molecular cloning of Proteus mirabilis uroepithelial cell adherence (uca) genes. Infect Immun 63, 2082–2086.[Abstract]
Gaastra, W., van Oosterom, R. A., Pieters, E. W., Bergmans, H. E., van Dijk, L., Agnes, A. & ter Huurne, H. M. (1996). Isolation and characterisation of dog uropathogenic Proteus mirabilis strains. Vet Microbiol 48, 57–71.[CrossRef][Medline]
Gordon, D. M. & Riley, M. A. (1992). A theoretical and experimental analysis of bacterial growth in the bladder. Mol Microbiol 6, 555–562.[Medline]
Jansen, A. M., Lockatell, C. V., Johnson, D. E. & Mobley, H. L. (2003). Visualization of Proteus mirabilis morphotypes in the urinary tract: the elongated swarmer cell is rarely observed in ascending urinary tract infection. Infect Immun 71, 3607–3613.
Johnson, J. R. (1991). Virulence factors in Escherichia coli urinary tract infections. Clin Microbiol Rev 4, 80–128.
Jones, B. D., Lockatell, C. V., Johnson, D. E., Warren, J. W. & Mobley, H. L. (1990). Construction of a urease-negative mutant of Proteus mirabilis: analysis of virulence in a mouse model of ascending urinary tract infection. Infect Immun 58, 1120–1123.
Legnani-Fajardo, C., Zunino, P., Piccini, C., Allen, A. & Maskell, D. (1996). Defined mutants of Proteus mirabilis lacking flagella cause ascending urinary tract infection in mice. Microb Pathog 21, 395–405.[CrossRef][Medline]
Massad, G., Lockatell, C. V., Johnson, D. E. & Mobley, H. L. (1994). Proteus mirabilis fimbriae: construction of an isogenic pmfA mutant and analysis of virulence in a CBA mouse model of ascending urinary tract infection. Infect Immun 62, 536–542.
Moayeri, N., Collins, C. M. & O'Hanley, P. (1991). Efficacy of a Proteus mirabilis outer membrane protein vaccine in preventing experimental pyelonephritis in a BALB-c mouse model. Infect Immun 59, 3778–3786.
Mobley, H. L. T. & Hausinger, R. P. (1989). Microbial ureases: significance, regulation, and molecular characterization. Microbiol Rev 53, 85–108.
Mobley, H. L. & Chippendale, G. R. (1990). Hemagglutinin, urease, and hemolysin production by Proteus mirabilis from clinical sources. J Infect Dis 161, 525–530.[Medline]
Mobley, H. L. T. & Belas, R. (1995). Swarming and pathogenicity of Proteus mirabilis in the urinary tract. Trends Microbiol 3, 280–284.[CrossRef][Medline]
Mobley, H. L., Belas, R., Lockatell, V., Chippendale, G., Trifillis, A. L., Johnson, D. E. & Warren, J. W. (1996). Construction of a flagellum-negative mutant of Proteus mirabilis: effect on internalization by human renal epithelial cells and virulence in a mouse model of ascending urinary tract infection. Infect Immun 64, 5332–5340.[Abstract]
Oelschlaeger, T. A., Dobrindt, U. & Hacker, J. (2002a). Pathogenicity islands of uropathogenic Escherichia coli and the evolution of virulence. Int J Antimicrob Agents 19, 517–521.[CrossRef][Medline]
Oelschlaeger, T. A., Dobrindt, U. & Hacker, J. (2002b). Virulence factors of uropathogens. Curr Opin Urol 12, 33–38.[CrossRef][Medline]
Old, D. C. & Adegbola, R. A. (1982). Haemagglutinins and fimbriae of Morganella, Proteus and Providencia. J Med Microbiol 15, 551–564.[Abstract]
Old, D. C. & Adegbola, R. A. (1985). Antigenic relationships among type-3 fimbriae of Enterobacteriaceae revealed by immunoelectronmicroscopy. J Med Microbiol 20, 113–121.[Abstract]
Olive, D. M. & Bean, P. (1999). Principles and applications of methods for DNA-based typing of microbial organisms. J Clin Microbiol 37, 1661–1669.
Pazin, G. J. & Braude, A. I. (1974). Immobilizing antibodies in urine. II. Prevention of ascending spread of Proteus mirabilis. Invest Urol 12, 129–133.[Medline]
Peerbooms, P. G., Verweij, A. M. & MacLaren, D. M. (1983). Investigation of the haemolytic activity of Proteus mirabilis strains. Antonie Van Leeuwenhoek 49, 1–11.[CrossRef][Medline]
Pfaller, M. A., Mujeeb, I., Hollis, R. J., Jones, R. N. & Doern, G. V. (2000). Evaluation of the discriminatory powers of the Dienes test and ribotyping as typing methods for Proteus mirabilis. J Clin Microbiol 38, 1077–1080.
Piccini, C. D., Barbé, F. M. & Legnani-Fajardo, C. L. (1998). Identification of iron-regulated outer membrane proteins in uropathogenic Proteus mirabilis and its relationship with heme uptake. FEMS Microbiol Lett 166, 243–248.[CrossRef][Medline]
Power, P. M. & Jennings, M. P. (2003). The genetics of glycosylation in Gram-negative bacteria. FEMS Microbiol Lett 218, 211–222.[CrossRef][Medline]
Robledo, J. A., Serrano, A. & Domingue, G. J. (1990). Outer membrane proteins of Escherichia coli in the host-pathogen interaction in urinary tract infection. J Urol 143, 386–391.[Medline]
Rozalski, A., Sidorcyk, Z. & Kotelko, K. (1997). Potential virulence factors of Proteus bacilli. Microbiol Mol Biol Rev 61, 65–89.[Abstract]
Russo, T. A., Jodush, S. T., Brown, J. J. & Johnson, J. R. (1996). Identification of two previously unrecognized genes (guaA and argC) important for uropathogenesis. Mol Microbiol 22, 217–229.[CrossRef][Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sanders, J. D., Cope, L. D. & Hansen, E. J. (1994). Identification of a locus involved in the utilization of iron by Haemophilus influenzae. Infect Immun 62, 4515–4525.
Scavone, P., Sosa, V., Pellegrino, R., Galvalisi, U. & Zunino, P. (2004). Mucosal vaccination of mice with recombinant Proteus mirabilis structural fimbrial proteins. Microbes Infect 6, 853–860.[CrossRef][Medline]
Swihart, K. G. & Welch, R. A. (1990). Cytotoxic activity of the Proteus hemolysin HpmA. Infect Immun 58, 1861–1869.
Tolson, D. L., Barrigar, D. L., Malean, R. J. C. & Altman, E. (1995). Expression of a nonagglutinating fimbria by Proteus mirabilis. Infect Immun 63, 1127–1129.[Abstract]
Towbin, H., Staehelin, T. & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76, 4350–4354.
Versalovic, J., Koeuth, T. & Lupski, J. R. (1991). Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res 19, 6823–6831.
Warren, J. W., Tenney, J. H., Hoopes, J. M. & Kass, E. H. (1982). A prospective microbiologic study of bacteriuria in patients with chronic indwelling urethral catheters. J Infect Dis 146, 719–723.[Medline]
Williams, F. D. & Schwarzhoff, R. H. (1978). Nature of the swarming phenomenon in Proteus. Annu Rev Microbiol 32, 101–122.[CrossRef][Medline]
Zunino, P., Piccini, C. & Legnani-Fajardo, C. (1994). Flagellate and non-flagellate Proteus mirabilis in the development of experimental urinary tract infection. Microb Pathog 16, 379–385.[CrossRef][Medline]
Zunino, P., Geymonat, L., Allen, A. G., Legnani-Fajardo, C. & Maskell, D. J. (2000). Virulence of a Proteus mirabilis ATF isogenic mutant is not impaired in a mouse model of ascending urinary tract infection. FEMS Immunol Med Microbiol 29, 137–143.[CrossRef][Medline]
Zunino, P., Geymonat, L., Allen, A. G., Preston, A., Sosa, V. & Maskell, D. J. (2001). New aspects of the role of MR/P fimbriae in Proteus mirabilis urinary tract infection. FEMS Immunol Med Microbiol 31, 113–120.[CrossRef][Medline]
Zunino, P., Sosa, V., Allen, A. G., Preston, A., Schlapp, G. & Maskell, D. J. (2003). Proteus mirabilis fimbriae (PMF) are important for both bladder and kidney colonization in mice. Microbiology 149, 3231–3237.
Received 13 January 2006;
revised 5 April 2006;
accepted 6 April 2006.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
