Molecular analysis of type 3 fimbrial genes from Escherichia coli, Klebsiella and Citrobacter species
© Ong et al; licensee BioMed Central Ltd. 2010
Received: 5 February 2010
Accepted: 24 June 2010
Published: 24 June 2010
Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in the United States and is caused by a range of uropathogens. Biofilm formation by uropathogens that cause CAUTI is often mediated by cell surface structures such as fimbriae. In this study, we characterised the genes encoding type 3 fimbriae from CAUTI strains of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter koseri and Citrobacter freundii.
Phylogenetic analysis of the type 3 fimbrial genes (mrkABCD) from 39 strains revealed they clustered into five distinct clades (A-E) ranging from one to twenty-three members. The majority of sequences grouped in clade A, which was represented by the mrk gene cluster from the genome sequenced K. pneumoniae MGH78578. The E. coli and K. pneumoniae mrkABCD gene sequences clustered together in two distinct clades, supporting previous evidence for the occurrence of inter-genera lateral gene transfer. All of the strains examined caused type 3 fimbriae mediated agglutination of tannic acid treated human erythrocytes despite sequence variation in the mrkD-encoding adhesin gene. Type 3 fimbriae deletion mutants were constructed in 13 representative strains and were used to demonstrate a direct role for type 3 fimbriae in biofilm formation.
The expression of functional type 3 fimbriae is common to many Gram-negative pathogens that cause CAUTI and is strongly associated with biofilm growth. Our data provides additional evidence for the spread of type 3 fimbrial genes by lateral gene transfer. Further work is now required to substantiate the clade structure reported here by examining more strains as well as other bacterial genera that make type 3 fimbriae and cause CAUTI.
Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in the United States and a frequent cause of bacteremia . Nosocomial CAUTI is caused by a range of different bacterial pathogens  and these are often resistant to multiple antibiotics .
Biofilm formation is a trait commonly found among CAUTI isolates and results in the growth of bacteria on the inner surface of the urinary catheter. Biofilm formation promotes encrustation and protects the bacteria from the hydrodynamic forces of urine flow, host defenses and antibiotics . A perquisite to biofilm growth is adherence to the catheter surface. A number of mechanisms by which Gram-negative pathogens mediate adherence to biotic and abiotic surfaces have been described and include fimbriae (e.g. type 1, type 3, type IV, curli and conjugative pili), cell surface adhesins (e.g. autotransporter proteins such as antigen 43, UpaH and UpaG) and flagella [5–16].
The expression of type 3 fimbriae has been described from many Gram-negative pathogens [17–28]. Type 3 fimbriae are 2-4 nm wide and 0.5-2 μm long surface organelles that are characterised by their ability to mediate agglutination of tannic acid-treated human RBC (MR/K agglutination) . Several studies have clearly demonstrated a role for type 3 fimbriae in biofilm formation [17, 28, 30–33]. Type 3 fimbriae also mediate various adherence functions such as binding to epithelial cells (from the respiratory and urinary tracts) and extracellular matrix proteins (e.g. collagen V) [31, 34–36].
Type 3 fimbriae belong to the chaperone-usher class of fimbriae and are encoded by five genes (mrkABCDF) arranged in the same transcriptional orientation [29, 37]. The mrk gene cluster is similar to other fimbrial operons of the chaperone-usher class in that it contains genes encoding major (mrkA) and minor (mrkF) subunit proteins as well as chaperone- (mrkB), usher- (mrkC) and adhesin- (mrkD) encoding genes [37, 38]. A putative regulatory gene (mrkE) located upstream of mrkA has been described previously in Klebsiella pneumoniae . The mrk genes have been shown to reside at multiple genomic locations, including the chromosome , on conjugative plasmids [17, 30] and within a composite transposon . Transfer of an mrk-containing conjugative plasmid to strains of Salmonella enterica serovar Typhimurium, Klebsiella pneumoniae, Enterobacter aerogenes and Kluyvera species has also been demonstrated . Taken together, these data strongly support spread of the mrk genes between Gram-negative pathogens by lateral gene transfer.
Recently, we identified and characterised the role of type 3 fimbriae in biofilm formation from an Escherichia coli strain isolated from a patient with CAUTI . We also demonstrated that the mrkB chaperone-encoding gene and the ability to mediate MR/K agglutination was common in uropathogenic Klebsiella pneumoniae, Klebsiella oxytoca and Citrobacter koseri strains (86.7%, 100% and 100% of strains, respectively) but rare in uropathogenic E. coli and Citrobacter freundii strains (3.2% and 14.3% of strains, respectively) . Despite the occurrence of type 3 fimbriae genes among a range of different Gram-negative bacteria that cause CAUTI, little is known about their molecular relationship. In this study, we have examined the phylogenetic correlation between type 3 fimbrial (mrk) genes from 33 CAUTI strains representing five different uropathogens (E. coli, K. pneumoniae, K. oxytoca, C. koseri and C. freundii). We also demonstrate functional expression of type 3 fimbriae in each of these strains and describe a common role for type 3 fimbriae in biofilm formation.
Phylogenetic analysis of the mrkABCD genes from uropathogenic bacterial genera
Diversity of individual mrkA, mrkB, mrkC and mrkD nucleotide sequences
Diversity Within Group (%)1
Diversity Between Group (%)2
A and B
A and E
B and E
Sequence comparison of the mrk locus from strains of C. freundii, C. koseri, E. coli and K. oxytoca
Type 3 fimbriae are functionally expressed in C. freundii, C. koseri, E. coli, K. oxytoca and K. pneumoniae
Type 3 fimbriae are strongly associated with biofilm formation
Type 3 fimbriae are adhesive organelles produced by a range of Gram-negative pathogens that cause CAUTI. Here we show that type 3 fimbriae (mrkABCD) genes from 33 CAUTI isolates representing C. freundii, C. koseri, E. coli, K. oxytoca and K. pneumoniae cluster into five well-supported clades on the basis of nucleotide sequence. Type 3 fimbriae were expressed by all of these strains as indicated by their positive MR/K agglutination. Type 3 fimbrial expression was also associated with biofilm growth in the majority of these strains. This is the first report describing the distinct grouping of type 3 fimbrial genes into phylogenetic clades at the species level, with strong evidence supporting inter-species lateral gene transfer. We also demonstrate the functional expression of type 3 fimbriae by strains of C. koseri and C. freundii.
Phylogenetic analysis with individual and concatenated mrkABCD sequences revealed five distinct clades (A-E) which were strongly supported by long internal branches. The majority of the sequences grouped in clade A, which is represented by the chromosomal mrk gene cluster from the genome sequenced K. pneumoniae strain MGH78578. Clades A and B contained mrk gene clusters from K. pneumoniae (both chromosomal and plasmid origin) and E. coli (plasmid origin). Two mrk loci have been fully sequenced from E. coli; in both cases the mrk genes are located on a conjugative plasmid (pMAS2027 and pOLA52, respectively) and flanked by transposon-like sequences [30, 40]. While the genomic location of the mrk genes in the additional seven E. coli strains identified in this study remains to be determined, the data presented here and in previous studies strongly suggests inter-genera lateral gene transfer of the mrk cluster [17, 28]. In contrast, the composition of clade E is entirely C. koseri sequences, while clades C and D are represented by a unique sequence from C freundii and K. oxytoca, respectively. The presence of cko_00966 homologs downstream of representative mrk clusters in all 5 clades strongly suggests that the ancestral mrkABCD locus was also encoded next to a cko_00966 homolog and that the clades are largely related by linear descent. Notably, the relationship determined here is not congruent with the known evolutionary relationship of Klebsiella, Citrobacter, and E. coli , supporting the occurrence of lateral gene transfer. We propose that clade A represents the K. pneumoniae lineage, with mrk regions laterally transferred to E. coli (e.g. pMAS2027 and pOLA52) and clade E represents the C. koseri lineage. Clades B, C and D, which contain mrk sequences from K. pneumoniae, E. coli, C. freundii and K. oxytoca, are clearly under-represented and additional type 3 fimbrial gene sequences are required to confirm the groupings.
Among the four genes used in the phylogenetic analysis, mrkD exhibited the highest inter-group diversity (Table 1). Thus, from the partial sequence comparisons performed in this work, the MrkD adhesin displayed greater sequence variability than the MrkA major subunit. This is inconsistent with other chaperone-usher fimbriae such as type 1 and P fimbriae, where the sequence of the adhesin (e.g. FimH, PapG) is more conserved than the major subunit protein (e.g. FimA, PapA). We note, however, that these findings require substantiation via comparison of the entire sequence of each structural subunit from multiple strains. The MrkD adhesin mediates several phenotypes, including MR/K agglutination, as well as adherence to human endothelial cells, urinary bladder cells, basement membranes and ECM proteins such as collagen IV and V [5, 31, 34, 35]. Interestingly, previous studies have demonstrated that sequence variations in the MrkD adhesin are associated with differential binding properties [42–44]. Our study demonstrates that the degree of sequence variation in MrkD might be even greater than previously predicted .
CAUTI is associated with biofilm formation on the inner surface of indwelling catheters. Thirteen independent mrk deletion mutants were generated and used to examine type 3 fimbriae associated phenotypes including MR/K agglutination and biofilm formation. All of the mrk mutants were unable to cause MR/K agglutination, confirming that this property is highly specific for type 3 fimbriae. In biofilm assays, 11/13 mrk mutants displayed a significant reduction in biofilm growth compared to their respective parent strain, demonstrating that type 3 fimbriae contribute to this phenotype across a range of different genera and species. The exceptions were C. freundii M46 and E. coli M184. C. freundii M46 failed to produce a significant biofilm in the assay conditions employed irrespective of its mrk genotype. Although this strain caused MR/K agglutination, we were also unable to detect the MrkA major subunit protein by western blot analysis. E. coli M184 showed no reduction in biofilm growth upon deletion of the mrk genes. It is likely that E. coli M184 contains additional mechanisms that promote biofilm growth and therefore deletion of the mrk genes did not result in loss of this phenotype.
This study demonstrated that the expression of functional type 3 fimbriae is common to many Gram-negative pathogens that cause CAUTI. Biofilm growth mediated by type 3 fimbriae may be important for the survival of these organisms on the surface of urinary catheters and within the hospital environment. Although our analysis provides additional evidence for the spread of type 3 fimbrial genes by lateral gene transfer, further work is required to substantiate the clade structure reported here by examining more strains as well as other genera that make type 3 fimbriae and cause CAUTI such as Proteus and Providentia.
Bacterial strains, plasmids & growth conditions
Bacterial strains and plasmids used in this study
E. coli CAUTI isolate
K. pneumoniae CAUTI isolate
C. freundii ABU isolate
K. pneumoniae CAUTI isolate
K. oxytoca CAUTI isolate
E. coli pyelonephritis isolate
K. oxytoca CAUTI isolate
K. pneumoniae CAUTI isolate
K. pneumoniae CAUTI isolate
C. koseri CAUTI isolate
K. pneumoniae CAUTI isolate
CAUTI E. coli MS2027mrk::cam
Pyelonephritis E. coli M184mrk::cam
E. coli ECOR15mrk::cam
CAUTI K. pneumoniae M124mrk::kan
CAUTI K. pneumoniae M446mrk::kan
CAUTI K. pneumoniae M542mrk::kan
CAUTI K. pneumoniae M20Δrk::kan
CAUTI K. oxytoca M126mrk::kan
CAUTI K. oxytoca M239mrk:: kan
CAUTI C. koseri M546mrk::kan
ABU C. freundii M46mrk::kan
E. coli ECOR28mrk::kan
CAUTI K. pneumoniae M692mrk::kan
Deletion mutant template plasmid (cam)
Deletion mutant template plasmid (kan)
Temperature-sensitive plasmid containing λ-Red recombinase system
Plasmid with λ-Red genes under the control of the arabinose-inducible promoter
DNA manipulations and genetic techniques
Primers used in this study
Type 3 fimbriae deletion primers
50 bp overhang mrk knockout F-primer 1
50 bp overhang mrk knockout R-primer 1
Knockout screening F 1
Knockout screening R 1
50 bp overhang mrk knockout F-primer 2
50 bp overhang mrk knockout R-primer 2
Knockout screening R 2
50 bp overhang mrk knockout F-primer 3
50 bp overhang mrk knockout R-primer 3
Knockout screening F 3
Knockout screening R 3
50 bp overhang mrk knockout F-primer 4
50 bp overhang mrk knockout R-primer 4
Knockout screening F 4
Knockout screening R 4
50 bp overhang mrk knockout F-primer 5
Knockout screening F 5
Inverse PCR primers
mrkA R-primer A
mrkA R-primer C
mrkD F-primer A
mrkD F-primer B
mrkD F-primer C
mrkD F-primer D
Outside mrk cluster screening primers
F-primer (outside mrkA of K. pneumoniae MGH78578)
R-primer (outside mrkF of K. pneumoniae MGH78578)
F-primer (mrkE of K. pneumoniae pIA565)
F-primer (outside mrkA of C. koseri ATCC BAA-895)
R-primer (outside mrkF of C. koseri ATCC BAA-895)
F-primer (outside mrkA of E. coli MS2027)
R-primer (outside mrkF of E. coli MS2027)
pMAS2027 screening primers
pMAS2027 screening primer F1
pMAS2027 screening primer R1
pMAS2027 screening primer F2
pMAS2027 screening primer R2
pMAS2027 screening primer F3
pMAS2027 screening primer R3
pMAS2027 screening primer F4
pMAS2027 screening primer R4
pMAS2027 screening primer F5
pMAS2027 screening primer R5
Bacterial agglutination of tannic acid treated human erythrocytes (MR/K agglutination) was performed as previously described to detect the expression of Type 3 fimbriae . Bacterial strains were grown overnight as shaking cultures in M9 minimal medium. Strains which produced a negative result in this assay were enriched for type 3 fimbriae production by three successive rounds of 48 h static growth in M9 minimal medium and then re-tested.
Biofilm formation on polyvinyl chloride (PVC) surfaces was monitored by using 96-well microtitre plates (Falcon) essentially as previously described . Briefly, cells were grown for 24 h in M9 minimal medium (containing 0.2% glucose) or 48 h in synthetic urine at 37°C under shaking conditions, washed to remove unbound cells and stained with crystal violet. Quantification of biofilm mass was performed by addition of acetone-ethanol (20:80) and measurement of the dissolved crystal violet at an optical density of 595 nm. All experiments were performed in a minimum of eight replicates.
Immunoblotting and immunogold-labelled electron microscopy
Crude cell lysates were prepared from overnight cultures and boiled in acid as previously described . Protein samples were analysed by SDS-PAGE and western blotting as previously described  employing a type 3 fimbriae specific antiserum. Immunogold labelling was performed using the same Type 3 fimbriae specific antiserum as previously described . Cells were examined under a JEOL JEM1010 TEM operated at 80 kV. Images were captured using an analysis Megaview digital camera.
Phylogenetic and sequence analysis
PCR products were generated from an internal region of mrkA (416 bp), mrkB (243 bp), mrkC (657 bp) and mrkD (778), respectively, from each of the 33 CAUTI strains and sequenced on both strands. These sequences correspond to nucleotides 112 to 530 of mrkA, 66 to 308 of mrkB, 173 to 829 of mrkC and 157 to 934 of mrkD in the reference strain K. pneumoniae MGH78578 (CP000647). Individual and concatenated gene fragments from the 33 CAUTI strains (and six additional mrk sequences available at GenBank from strains causing other infections; accession numbers: CP000647, EU682505, CP000964, M55912, CP000822, EU370913) were aligned using ClustalX , and subjected to phylogenetic analysis using PHYLIP . Maximum likelihood (ML) trees were built from a concatenated alignment of 2104 nucleotides (comprising 1269 conserved sites and 775 informative sites) using the dnaml algorithm in PHYLIP . A consensus tree of 500 ML bootstrap replicates was prepared using the majority rule method as implemented by Splitstree version 4 [55, 56]. We were unable to amplify mrkD from E. coli M202 and only used the mrkABC concatenated fragments in the analysis. For comparative analysis, the complete mrk cluster (and adjacent regions) from E. coli ECOR28, C. freundii M46 and K. oxytoca M126 were amplified using an inverse PCR strategy and sequenced.
Differences in biofilm formation between wild-type and mrk mutant strains were analysed using the ANOVA single factor test (Minitab 15 Statistical Software).
Nucleotide sequence accession numbers
Gene fragments were deposited in GenBank under the accession numbers: FJ96754, FJ96756-FJ96774, and FJ96777-FJ96789 (for mrkA), FJ96793, FJ96795-FJ96811, FJ96813-FJ96814, and FJ96817-FJ96829 (for mrkC) and FJ96832, FJ96834-FJ96849, FJ96851-FJ96852, and FJ96855-FJ96867 (for mrkD). The mrkB sequences were described previously . The complete mrk cluster (and adjacent regions) from E. coli ECOR28, C. freundii M46 and K. oxytoca M126 were deposited in GenBank under accession numbers FJ96870, FJ96871 and FJ96872, respectively.
Approval for this study was obtained from the Princess Alexandra Hospital Human Research Ethics Committee (2005/098). Since the study used E. coli isolates collected as part of routine methods for the diagnosis of UTI and no additional procedures on patients were involved, individual informed consent was not obtained.
This work was supported by grants from the National Health and Medical Research Council (455914 and 631654) and the Australian Research Council (DP0666852). SAB is supported by an ARC Australian Research Fellowship (DP0881247). We thank Prof Timo Korhonen for providing Type 3 fimbriae antiserum.
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