BACKGROUND
catheter-associated urinary tract infection  is the most common nosocomial infection in the united states and a frequent cause of bacteremia  <cit> .
nosocomial cauti is caused by a range of different bacterial pathogens  <cit>  and these are often resistant to multiple antibiotics  <cit> .
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  <cit> .
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 , cell surface adhesins  and flagella  <cit> .
the expression of type  <dig> fimbriae has been described from many gram-negative pathogens  <cit> .
type  <dig> fimbriae are 2- <dig> nm wide and  <dig> - <dig> μm long surface organelles that are characterised by their ability to mediate agglutination of tannic acid-treated human rbc   <cit> .
several studies have clearly demonstrated a role for type  <dig> fimbriae in biofilm formation  <cit> .
type  <dig> fimbriae also mediate various adherence functions such as binding to epithelial cells  and extracellular matrix proteins   <cit> .
type  <dig> fimbriae belong to the chaperone-usher class of fimbriae and are encoded by five genes  arranged in the same transcriptional orientation  <cit> .
the mrk gene cluster is similar to other fimbrial operons of the chaperone-usher class in that it contains genes encoding major  and minor  subunit proteins as well as chaperone- , usher-  and adhesin-  encoding genes  <cit> .
a putative regulatory gene  located upstream of mrka has been described previously in klebsiella pneumoniae  <cit> .
the mrk genes have been shown to reside at multiple genomic locations, including the chromosome  <cit> , on conjugative plasmids  <cit>  and within a composite transposon  <cit> .
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  <cit> .
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  <dig> fimbriae in biofilm formation from an escherichia coli strain isolated from a patient with cauti  <cit> .
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  but rare in uropathogenic e. coli and citrobacter freundii strains   <cit> .
despite the occurrence of type  <dig> 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  <dig> fimbrial  genes from  <dig> cauti strains representing five different uropathogens .
we also demonstrate functional expression of type  <dig> fimbriae in each of these strains and describe a common role for type  <dig> fimbriae in biofilm formation.
RESULTS
phylogenetic analysis of the mrkabcd genes from uropathogenic bacterial genera
to investigate the phylogenetic relationship of the mrk genes from  <dig> cauti strains  we amplified and sequenced an internal segment of the mrka, mrkb, mrkc and mrkd genes from each strain.
we also examined the corresponding sequence from six additional mrk gene clusters available at genbank.
a majority-rule consensus maximum likelihood  tree was constructed from the  <dig> concatenated mrkabcd fragments.
the phylogenetic analysis indicated that the sequences clustered into five major clades  with good bootstrap support .
the five clades range from one member  to  <dig> members , with an average inter-allelic diversity of  <dig> %.
whereas the  <dig> c. koseri sequences clustered in a single clade , clade b  and clade a  consist of sequences from both k. pneumoniae and e. coli.
phylogenetic analysis using parsimony or distance-based methods produced tree topologies very similar to those obtained by using dna maximum likelihood .
the incongruence between the mrk consensus tree and the established phylogeny for enteric bacteria  <cit>  is prima facie evidence for lateral gene transfer  of mrk alleles.
all k. pneumoniae chromosomal alleles cluster in clade a, along with several plasmid-borne or chromosomal alleles from e. coli.
in some cases, the k. pneumoniae and e. coli alleles are identical .
similarly, in clade b, two identical e. coli mrkabcd sequences  share high nucleotide sequence identity  to the plasmid-borne k. pneumoniae pia <dig> mrkabcd.
the mrkabcd concatenated nucleotide sequences of k. pneumoniae pia <dig>  and k. pneumoniae mgh <dig>  share only  <dig> % nucleotide sequence identity.
when analysed individually, the mrka, mrkb, mrkc and mrkd gene fragment alignments produced essentially the same tree topology as the concatenated sequence  with little variation in within-group diversity .
in contrast to other chaperone-usher systems, the mrkd adhesin is more divergent than the mrka major subunit and contributes the most of all mrk alleles to the inter-group diversity .
1mean within group diversity; group c and group d excluded as they contain a single sequence, and two identical sequences, respectively.
2mean between group diversity calculated with all five groups.
sequence comparison of the mrk locus from strains of c. freundii, c. koseri, e. coli and k. oxytoca
we compared the mrk gene clusters from representatives of each of the five clades:  <dig> mrk regions were available from genbank,  <dig> were sequenced in this study .
as expected, the mrkabcd gene order is conserved in all clades.
predicted insertion sequences were identified flanking both ends of the pmas <dig> and pola <dig> clusters , and at the 5' end of clusters from ecor <dig>  and c. freundii m <dig> , indicative of recent lateral gene transfer.
downstream of mrkf, a conserved  <dig> bp gene was present in five of the strains, including one from each of the five defined clades.
this gene  encodes a central eal domain  suggesting that it may have a role in signalling, however, no close homologs have been functionally characterised.
pcr primers designed from these sequences demonstrated that this region was also conserved in  <dig> other strains examined .
notably, cko_ <dig> homologs were not encoded downstream of the plasmid-borne mrk clusters in e. coli pmas <dig> and pola <dig>  and there is no corresponding sequence information available for this region in pia <dig>  <cit> .
the putative mrke regulatory gene originally identified in pia <dig>  <cit>  was not present in any of the strains examined.
type  <dig> fimbriae are functionally expressed in c. freundii, c. koseri, e. coli, k. oxytoca and k. pneumoniae
all of the mrk-positive strains examined in this study mediated mannose-resistant hemagglutination of tannic acid treated human rbc , indicating they produced type  <dig> fimbriae.
to specifically demonstrate a direct association between mr/k agglutination and type  <dig> fimbriae, the mrk locus was deleted from thirteen strains  employing λ-red mediated homologous recombination.
the strains were selected on the basis of their transformation efficiency and included at least one representative from each of the mrk phylogenetic clades.
several different assays were employed to compare the thirteen sets of wild-type and mrk deletion strains.
first, sds-page analysis of crude cell lysates and subsequent western blotting was performed using a type  <dig> fimbriae-specific antiserum.
a predominant  <dig> kda band representing the mrka major subunit was detected from all wild-type strains except c. freundii m <dig>  which failed to react positively in this assay.
in contrast, no reaction was observed for any of the mrk deletion mutants .
next, the wild-type and mrk mutant strains were compared for their ability to mediate mr/k agglutination.
only the wild-type strains produced a positive phenotype .
finally, the presence of type  <dig> fimbriae was confirmed by immunogold labelling employing type  <dig> fimbriae-specific antiserum for e. coli ecor <dig> and c. koseri m <dig>  but was absent in their corresponding mrk deletion mutants .
taken together, the results demonstrate that mr/k agglutination is a conserved phenotype for a range of gram-negative organisms that express functional type  <dig> fimbriae.
type  <dig> fimbriae are strongly associated with biofilm formation
the thirteen sets of isogenic wild-type and mrk deletion strains generated above were examined for their ability to produce a biofilm following growth in m <dig> minimal medium  under dynamic culture conditions.
strong biofilm growth was observed from all wild-type strains except c. freundii m <dig>  in contrast, deletion of the mrk gene cluster caused a significant reduction in biofilm growth  in all strains except e. coli m <dig> .
similar results were also observed following growth in synthetic urine .
thus, type  <dig> fimbriae contribute significantly to biofilm formation when expressed in e. coli, k. pneumoniae, k. oxytoca and c. koseri.
discussion
type  <dig> fimbriae are adhesive organelles produced by a range of gram-negative pathogens that cause cauti.
here we show that type  <dig> fimbriae  genes from  <dig> 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  <dig> fimbriae were expressed by all of these strains as indicated by their positive mr/k agglutination.
type  <dig> 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  <dig> 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  <dig> fimbriae by strains of c. koseri and c. freundii.
phylogenetic analysis with individual and concatenated mrkabcd sequences revealed five distinct clades  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 mgh <dig>  clades a and b contained mrk gene clusters from k. pneumoniae  and e. coli .
two mrk loci have been fully sequenced from e. coli; in both cases the mrk genes are located on a conjugative plasmid  and flanked by transposon-like sequences  <cit> .
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  <cit> .
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_ <dig> homologs downstream of representative mrk clusters in all  <dig> clades strongly suggests that the ancestral mrkabcd locus was also encoded next to a cko_ <dig> 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  <cit> , 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  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  <dig> 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 .
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  <dig> and p fimbriae, where the sequence of the adhesin  is more conserved than the major subunit protein .
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  <cit> .
interestingly, previous studies have demonstrated that sequence variations in the mrkd adhesin are associated with differential binding properties  <cit> .
our study demonstrates that the degree of sequence variation in mrkd might be even greater than previously predicted  <cit> .
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  <dig> 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  <dig> fimbriae.
in biofilm assays, 11/ <dig> mrk mutants displayed a significant reduction in biofilm growth compared to their respective parent strain, demonstrating that type  <dig> fimbriae contribute to this phenotype across a range of different genera and species.
the exceptions were c. freundii m <dig> and e. coli m <dig>  c. freundii m <dig> 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 m <dig> showed no reduction in biofilm growth upon deletion of the mrk genes.
it is likely that e. coli m <dig> contains additional mechanisms that promote biofilm growth and therefore deletion of the mrk genes did not result in loss of this phenotype.
CONCLUSIONS
this study demonstrated that the expression of functional type  <dig> fimbriae is common to many gram-negative pathogens that cause cauti.
biofilm growth mediated by type  <dig> 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  <dig> 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  <dig> fimbriae and cause cauti such as proteus and providentia.

 catheter-associated urinary tract infection 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, cell surface adhesins and flagella. the expression of type fimbriae has been described from many gram-negative pathogens. type fimbriae are 2- nm wide and - μm long surface organelles that are characterised by their ability to mediate agglutination of tannic acid-treated human rbc. several studies have clearly demonstrated a role for type fimbriae in biofilm formation. type fimbriae also mediate various adherence functions such as binding to epithelial cells and extracellular matrix proteins. type fimbriae belong to the chaperone-usher class of fimbriae and are encoded by five genes arranged in the same transcriptional orientation. the mrk gene cluster is similar to other fimbrial operons of the chaperone-usher class in that it contains genes encoding major and minor subunit proteins as well as chaperone-, usher- and adhesin- encoding genes. a putative regulatory gene 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 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 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 but rare in uropathogenic e. coli and citrobacter freundii strains. despite the occurrence of type 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 fimbrial genes from cauti strains representing five different uropathogens. we also demonstrate functional expression of type fimbriae in each of these strains and describe a common role for type fimbriae in biofilm formation.  phylogenetic analysis of the mrkabcd genes from uropathogenic bacterial genera to investigate the phylogenetic relationship of the mrk genes from cauti strains we amplified and sequenced an internal segment of the mrka, mrkb, mrkc and mrkd genes from each strain. we also examined the corresponding sequence from six additional mrk gene clusters available at genbank. a majority-rule consensus maximum likelihood tree was constructed from the concatenated mrkabcd fragments. the phylogenetic analysis indicated that the sequences clustered into five major clades with good bootstrap support. the five clades range from one member to members, with an average inter-allelic diversity of %. whereas the c. koseri sequences clustered in a single clade, clade b and clade a consist of sequences from both k. pneumoniae and e. coli. phylogenetic analysis using parsimony or distance-based methods produced tree topologies very similar to those obtained by using dna maximum likelihood. the incongruence between the mrk consensus tree and the established phylogeny for enteric bacteria is prima facie evidence for lateral gene transfer of mrk alleles. all k. pneumoniae chromosomal alleles cluster in clade a, along with several plasmid-borne or chromosomal alleles from e. coli. in some cases, the k. pneumoniae and e. coli alleles are identical. similarly, in clade b, two identical e. coli mrkabcd sequences share high nucleotide sequence identity to the plasmid-borne k. pneumoniae pia mrkabcd. the mrkabcd concatenated nucleotide sequences of k. pneumoniae pia and k. pneumoniae mgh share only % nucleotide sequence identity. when analysed individually, the mrka, mrkb, mrkc and mrkd gene fragment alignments produced essentially the same tree topology as the concatenated sequence with little variation in within-group diversity. in contrast to other chaperone-usher systems, the mrkd adhesin is more divergent than the mrka major subunit and contributes the most of all mrk alleles to the inter-group diversity. 1mean within group diversity; group c and group d excluded as they contain a single sequence, and two identical sequences, respectively. 2mean between group diversity calculated with all five groups. sequence comparison of the mrk locus from strains of c. freundii, c. koseri, e. coli and k. oxytoca we compared the mrk gene clusters from representatives of each of the five clades: mrk regions were available from genbank, were sequenced in this study. as expected, the mrkabcd gene order is conserved in all clades. predicted insertion sequences were identified flanking both ends of the pmas and pola clusters, and at the 5' end of clusters from ecor and c. freundii m, indicative of recent lateral gene transfer. downstream of mrkf, a conserved bp gene was present in five of the strains, including one from each of the five defined clades. this gene encodes a central eal domain suggesting that it may have a role in signalling, however, no close homologs have been functionally characterised. pcr primers designed from these sequences demonstrated that this region was also conserved in other strains examined. notably, cko_ homologs were not encoded downstream of the plasmid-borne mrk clusters in e. coli pmas and pola and there is no corresponding sequence information available for this region in pia. the putative mrke regulatory gene originally identified in pia was not present in any of the strains examined. type fimbriae are functionally expressed in c. freundii, c. koseri, e. coli, k. oxytoca and k. pneumoniae all of the mrk-positive strains examined in this study mediated mannose-resistant hemagglutination of tannic acid treated human rbc, indicating they produced type fimbriae. to specifically demonstrate a direct association between mr/k agglutination and type fimbriae, the mrk locus was deleted from thirteen strains employing λ-red mediated homologous recombination. the strains were selected on the basis of their transformation efficiency and included at least one representative from each of the mrk phylogenetic clades. several different assays were employed to compare the thirteen sets of wild-type and mrk deletion strains. first, sds-page analysis of crude cell lysates and subsequent western blotting was performed using a type fimbriae-specific antiserum. a predominant kda band representing the mrka major subunit was detected from all wild-type strains except c. freundii m which failed to react positively in this assay. in contrast, no reaction was observed for any of the mrk deletion mutants. next, the wild-type and mrk mutant strains were compared for their ability to mediate mr/k agglutination. only the wild-type strains produced a positive phenotype. finally, the presence of type fimbriae was confirmed by immunogold labelling employing type fimbriae-specific antiserum for e. coli ecor and c. koseri m but was absent in their corresponding mrk deletion mutants. taken together, the results demonstrate that mr/k agglutination is a conserved phenotype for a range of gram-negative organisms that express functional type fimbriae. type fimbriae are strongly associated with biofilm formation the thirteen sets of isogenic wild-type and mrk deletion strains generated above were examined for their ability to produce a biofilm following growth in m minimal medium under dynamic culture conditions. strong biofilm growth was observed from all wild-type strains except c. freundii m in contrast, deletion of the mrk gene cluster caused a significant reduction in biofilm growth in all strains except e. coli m. similar results were also observed following growth in synthetic urine. thus, type fimbriae contribute significantly to biofilm formation when expressed in e. coli, k. pneumoniae, k. oxytoca and c. koseri. discussion type fimbriae are adhesive organelles produced by a range of gram-negative pathogens that cause cauti. here we show that type fimbriae genes from 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 fimbriae were expressed by all of these strains as indicated by their positive mr/k agglutination. type 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 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 fimbriae by strains of c. koseri and c. freundii. phylogenetic analysis with individual and concatenated mrkabcd sequences revealed five distinct clades 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 mgh clades a and b contained mrk gene clusters from k. pneumoniae and e. coli. two mrk loci have been fully sequenced from e. coli; in both cases the mrk genes are located on a conjugative plasmid and flanked by transposon-like sequences. 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. 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_ homologs downstream of representative mrk clusters in all clades strongly suggests that the ancestral mrkabcd locus was also encoded next to a cko_ 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 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 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. 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 and p fimbriae, where the sequence of the adhesin is more conserved than the major subunit protein. 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. interestingly, previous studies have demonstrated that sequence variations in the mrkd adhesin are associated with differential binding properties. 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 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 fimbriae. in biofilm assays, 11/ mrk mutants displayed a significant reduction in biofilm growth compared to their respective parent strain, demonstrating that type fimbriae contribute to this phenotype across a range of different genera and species. the exceptions were c. freundii m and e. coli m c. freundii m 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 m showed no reduction in biofilm growth upon deletion of the mrk genes. it is likely that e. coli m 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 fimbriae is common to many gram-negative pathogens that cause cauti. biofilm growth mediated by type 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 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 fimbriae and cause cauti such as proteus and providentia. 
biofilm formation by uropathogens that cause cauti is often mediated by cell surface structures such as fimbriae. type fimbriae deletion mutants were constructed in representative strains and were used to demonstrate a direct role for type fimbriae in biofilm formation. in this study, we characterised the genes encoding type fimbriae from cauti strains of escherichia coli, klebsiella pneumoniae, klebsiella oxytoca, citrobacter koseri and citrobacter freundii. the majority of sequences grouped in clade a, which was represented by the mrk gene cluster from the genome sequenced k. pneumoniae mgh 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. our data provides additional evidence for the spread of type 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 fimbriae and cause cauti. all of the strains examined caused type fimbriae mediated agglutination of tannic acid treated human erythrocytes despite sequence variation in the mrkd-encoding adhesin gene. catheter-associated urinary tract infection is the most common nosocomial infection in the united states and is caused by a range of uropathogens. phylogenetic analysis of the type fimbrial genes from strains revealed they clustered into five distinct clades ranging from one to twenty-three members. the expression of functional type fimbriae is common to many gram-negative pathogens that cause cauti and is strongly associated with biofilm growth.
 catheter-associated urinary tract infection 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 fimbriae from cauti strains of escherichia coli, klebsiella pneumoniae, klebsiella oxytoca, citrobacter koseri and citrobacter freundii.  phylogenetic analysis of the type fimbrial genes from strains revealed they clustered into five distinct clades 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 mgh 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 fimbriae mediated agglutination of tannic acid treated human erythrocytes despite sequence variation in the mrkd-encoding adhesin gene. type fimbriae deletion mutants were constructed in representative strains and were used to demonstrate a direct role for type fimbriae in biofilm formation.  the expression of functional type 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 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 fimbriae and cause cauti. 