Open Access

Molecular analysis of type 3 fimbrial genes from Escherichia coli, Klebsiella and Citrobacter species

  • Cheryl-lynn Y Ong1,
  • Scott A Beatson1,
  • Makrina Totsika1,
  • Christiane Forestier2,
  • Alastair G McEwan1 and
  • Mark A Schembri1Email author
BMC Microbiology201010:183

DOI: 10.1186/1471-2180-10-183

Received: 5 February 2010

Accepted: 24 June 2010

Published: 24 June 2010

Abstract

Background

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.

Results

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.

Conclusions

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.

Background

Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in the United States and a frequent cause of bacteremia [1]. Nosocomial CAUTI is caused by a range of different bacterial pathogens [2] and these are often resistant to multiple antibiotics [3].

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 [4]. 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 [516].

The expression of type 3 fimbriae has been described from many Gram-negative pathogens [1728]. 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) [29]. Several studies have clearly demonstrated a role for type 3 fimbriae in biofilm formation [17, 28, 3033]. 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, 3436].

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 [37]. The mrk genes have been shown to reside at multiple genomic locations, including the chromosome [39], on conjugative plasmids [17, 30] and within a composite transposon [40]. 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 [17]. 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 [28]. 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) [28]. 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.

Results

Phylogenetic analysis of the mrkABCD genes from uropathogenic bacterial genera

To investigate the phylogenetic relationship of the mrk genes from 33 CAUTI strains (representing E. coli, K. pneumoniae, K. oxytoca, C. koseri and C. freundii) 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 (ML) tree was constructed from the 39 concatenated mrkABCD fragments. The phylogenetic analysis indicated that the sequences clustered into five major clades (referred to as clade A to E) with good bootstrap support (Fig. 1). The five clades range from one member (clade C, represented by C. freundii M46) to 23 members (clade A, represented by K. pneumoniae MGH78578), with an average inter-allelic diversity of 11.2%. Whereas the 10 C. koseri sequences clustered in a single clade (clade E), clade B (3 sequences) and clade A (23 sequences) 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 (data not shown).
https://static-content.springer.com/image/art%3A10.1186%2F1471-2180-10-183/MediaObjects/12866_2010_Article_1139_Fig1_HTML.jpg
Figure 1

Unrooted consensus phylogram of the concatenated mrkABCD nucleotide fragments. Majority-rule consensus tree was based on 500 bootstrap replicates using dnaml, the DNA maximum likelihood algorithm implemented by PHYLIP [54]. Five well-supported clades are labelled A-E; the largest clade, A, is circled. Bootstrap values are shown; small asterisks next to branches denote 100% support. Taxon IDs include species name abbreviations as suffixes (Cf, C. freundii indicated in black; Ck, C. koseri indicated in green; Ec, E. coli indicated in blue; Ko, K. oxytoca indicated in orange; and Kp, K. pneumoniae indicated in red), followed by the strain name. Taxon IDs highlighted in bold and underlined refer to those used in further analyses of the complete sequence of their respective mrk locus. Complete mrk locus sequences available from GenBank are marked with a large asterisk next to the strain name.

The incongruence between the mrk consensus tree and the established phylogeny for enteric bacteria [41] is prima facie evidence for lateral gene transfer (LGT) 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 (e.g. Ec_pOLA52/Kp_M20; EcM202/Kp_MGH78578). Similarly, in clade B, two identical E. coli mrkABCD sequences (M184 and ECOR28) share high nucleotide sequence identity (98%) to the plasmid-borne K. pneumoniae pIA565 mrkABCD. The mrkABCD concatenated nucleotide sequences of K. pneumoniae pIA565 (clade B) and K. pneumoniae MGH78578 (clade A) share only 78.2% 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 (data not shown) with little variation in within-group diversity (Table 1). 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 (Table 1).
Table 1

Diversity of individual mrkA, mrkB, mrkC and mrkD nucleotide sequences

  

Diversity Within Group (%)1

Diversity Between Group (%)2

Gene

Length

A

B

E

Mean

A and B

A and E

B and E

mrkA

403 nt

2.3

0.2

2.5

13.8

14.7

15.6

11.8

mrkB

246 nt

1.0

0.8

1.3

9.8

12.2

14.0

8.9

mrkC

655 nt

2.1

0.3

0.6

13.5

18.4

19.6

12.2

mrkD

506 nt

3.3

0.3

0.3

28.1

38.2

26.7

33.3

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: 5 mrk regions were available from GenBank, 3 were sequenced in this study (Fig. 2). As expected, the mrkABCD gene order is conserved in all clades. Predicted insertion sequences were identified flanking both ends of the pMAS2027 and pOLA52 clusters (clade A), and at the 5' end of clusters from ECOR28 (clade B) and C. freundii M46 (clade C), indicative of recent lateral gene transfer. Downstream of mrkF, a conserved 717 bp gene was present in five of the strains, including one from each of the five defined clades. This gene (labelled cko_00966 and kpn_03274 in the genomes of C. koseri ATCC BAA895 and K. pneumoniae MGH78578, respectively) encodes a central EAL domain (Pfam:PF00563, E = 1.7e-29) 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 24 other strains examined (data not shown). Notably, cko_00966 homologs were not encoded downstream of the plasmid-borne mrk clusters in E. coli pMAS2027 and pOLA52, and there is no corresponding sequence information available for this region in pIA565 [37]. The putative mrkE regulatory gene originally identified in pIA565 [37] was not present in any of the strains examined.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2180-10-183/MediaObjects/12866_2010_Article_1139_Fig2_HTML.jpg
Figure 2

Genetic organisation of the type 3 fimbriae ( mrk ) gene cluster. Physical map of the mrk gene cluster and flanking regions from K. pneumoniae MGH78578, plasmid pMAS2027 (E. coli), plasmid pOLA52 (E. coli), plasmid pIA565 (K. pneumoniae), E. coli ECOR28, C. freundii M46, K. oxytoca M126, and C. koseri ATCC BAA895. The mrk genes are indicated in blue and include mrkE (putative regulatory gene), mrkA (major subunit encoding gene), mrkB (chaperone encoding gene), mrkC (usher encoding gene), mrkD (adhesin encoding gene) and mrkF (putative minor subunit encoding gene). ORFs encoding putative transposable elements (yellow) and hypothetical proteins (grey) are indicated. The gene indicated in red (labelled cko_00966 and kpn_03274 in the genomes of C. koseri ATCC BAA895 and K. pneumoniae MGH78578, respectively) encodes a hypothetical protein containing a central EAL domain and was present downstream of mrkF in 29 strains. Sequence information outside the mrk cluster is not known for K. pneumoniae pIA565. Arrows indicate the direction of transcription for each gene.

Type 3 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 (MR/K agglutination), indicating they produced type 3 fimbriae. To specifically demonstrate a direct association between MR/K agglutination and type 3 fimbriae, the mrk locus was deleted from thirteen strains (E. coli MS2027, M184, ECOR15, ECOR28; K. pneumoniae M20, M124, M446, M542, M692; K. oxytoca M126, M239; C. freundii M46; C. koseri M546) 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 3 fimbriae-specific antiserum. A predominant 15 kDa band representing the MrkA major subunit was detected from all wild-type strains except C. freundii M46, which failed to react positively in this assay. In contrast, no reaction was observed for any of the mrk deletion mutants (Fig. 3 and data not shown). 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 (Fig. 4 and data not shown). Finally, the presence of type 3 fimbriae was confirmed by immunogold labelling employing type 3 fimbriae-specific antiserum for E. coli ECOR15 and C. koseri M546, but was absent in their corresponding mrk deletion mutants (Fig. 5). Taken together, the results demonstrate that MR/K agglutination is a conserved phenotype for a range of Gram-negative organisms that express functional type 3 fimbriae.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2180-10-183/MediaObjects/12866_2010_Article_1139_Fig3_HTML.jpg
Figure 3

Western blot analysis employing type 3 fimbriae specific antiserum. The proteins from C. koseri M546 (lane 2), C. koseri M546mrk (lane 3), E. coli ECOR15 (lane 4), E. coli ECOR15mrk (lane 5), K. oxytoca M126 (lane 6), K. oxytoca M126mrk (lane 7), K. pneumoniae M692 (lane 8) and K. pneumoniae M692mrk (lane 9) were acid boiled prior to loading. Molecular size markers are indicated in lane 1. The Type 3 fimbriae major subunit, MrkA, was only observed in the wild-type strains and not in the mrk deletion mutants. The arrow indicates the ~15 kDa band corresponding to MrkA.

https://static-content.springer.com/image/art%3A10.1186%2F1471-2180-10-183/MediaObjects/12866_2010_Article_1139_Fig4_HTML.jpg
Figure 4

Phase contrast microscopy illustrating MR/K agglutination. Parental wild-type strains C. koseri M546, E. coli ECOR15, K. oxytoca M126, K. pneumoniae M692 and C. freundii M46 demonstrated strong agglutination of tannic acid treated human erythrocytes, while their corresponding mrk deletion mutants, M546mrk, ECOR15mrk, M126mrk, M692mrk and M46mrk were negative for agglutination.

https://static-content.springer.com/image/art%3A10.1186%2F1471-2180-10-183/MediaObjects/12866_2010_Article_1139_Fig5_HTML.jpg
Figure 5

Immunogold electron microscopy demonstrating expression of type 3 fimbriae in E. coli ECOR15 and C. koseri M546. Expression of type 3 fimbriae at the cell surface was demonstrated by abundant labelling with anti-type 3 fimbriae-gold particles. In contrast, the deletion mutants, E. coli ECOR15mrk and C. koseri M546mrk were virtually devoid of gold labelling. Scale bar represents 1 μm.

Type 3 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 M9 minimal medium (containing 0.2% glucose) under dynamic culture conditions. Strong biofilm growth was observed from all wild-type strains except C. freundii M46. In contrast, deletion of the mrk gene cluster caused a significant reduction in biofilm growth (p < 0.0001) in all strains except E. coli M184 (Fig. 6). Similar results were also observed following growth in synthetic urine (data not shown). Thus, type 3 fimbriae contribute significantly to biofilm formation when expressed in E. coli, K. pneumoniae, K. oxytoca and C. koseri.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2180-10-183/MediaObjects/12866_2010_Article_1139_Fig6_HTML.jpg
Figure 6

Biofilm formation by wild-type and isogenic mrk deletion strains. Strains were grown at 37°C under shaking conditions for 16 h in PVC microtitre plates containing M9 minimal medium, washed to remove unbound cells and stained with 0.1% crystal violet. Biofilm formation was quantified by resuspending adherent cells in ethanol-acetate (80:20) and measuring the absorbance at 595 nm. Shown are the results for E. coli MS2027, M184, ECOR15 and, ECOR28, K. pneumoniae M20, M124, M446, M542 and, M692, K. oxytoca M126 and, M239, C. koseri M546 and C. freundii M46 and their respective mrk deletion mutants.

Discussion

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 [41], 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 [4244]. Our study demonstrates that the degree of sequence variation in MrkD might be even greater than previously predicted [44].

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.

Conclusions

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.

Methods

Bacterial strains, plasmids & growth conditions

The strains and plasmids used in this study are described in Table 2. Clinical UTI isolates were obtained from urine samples of patients at the Princess Alexandra Hospital (Brisbane, Australia) and have been described previously [45]. E. coli ECOR15, ECOR23 and ECOR28 were from the E. coli reference (ECOR) collection [46]. Cells were routinely grown at 37 °C on solid or in liquid Luria-Bertani (LB) medium supplemented with appropriate antibiotics unless otherwise stated. M9 minimal medium and synthetic urine were formulated as previously described [47, 48].
Table 2

Bacterial strains and plasmids used in this study

Strains/Plasmids

Description

Reference

Strains

  

MS2027

E. coli CAUTI isolate

[28]

M20

K. pneumoniae CAUTI isolate

[28]

M46

C. freundii ABU isolate

[28]

M124

K. pneumoniae CAUTI isolate

[28]

M126

K. oxytoca CAUTI isolate

[28]

M184

E. coli pyelonephritis isolate

[28]

M239

K. oxytoca CAUTI isolate

[28]

M446

K. pneumoniae CAUTI isolate

[28]

M542

K. pneumoniae CAUTI isolate

[28]

M546

C. koseri CAUTI isolate

[28]

M692

K. pneumoniae CAUTI isolate

[28]

MS2181

CAUTI E. coli MS2027mrk::cam

This study

MS2266

Pyelonephritis E. coli M184mrk::cam

This study

MS2267

E. coli ECOR15mrk::cam

This study

MS2332

CAUTI K. pneumoniae M124mrk::kan

This study

MS2334

CAUTI K. pneumoniae M446mrk::kan

This study

MS2335

CAUTI K. pneumoniae M542mrk::kan

This study

MS2374

CAUTI K. pneumoniae M20Δrk::kan

This study

MS2377

CAUTI K. oxytoca M126mrk::kan

This study

MS2379

CAUTI K. oxytoca M239mrk:: kan

This study

MS2454

CAUTI C. koseri M546mrk::kan

This study

MS2456

ABU C. freundii M46mrk::kan

This study

MS2458

E. coli ECOR28mrk::kan

This study

MS2515

CAUTI K. pneumoniae M692mrk::kan

This study

Plasmids

  

pKD3

Deletion mutant template plasmid (cam)

[49]

pKD4

Deletion mutant template plasmid (kan)

[49]

pKD46

Temperature-sensitive plasmid containing λ-Red recombinase system

[49]

pKOBEG199

Plasmid with λ-Red genes under the control of the arabinose-inducible promoter

[50]

DNA manipulations and genetic techniques

Plasmid DNA was isolated using the QIAprep Spin Miniprep Kit (Qiagen, Australia). Restriction endonucleases were used according to the manufacturer's specifications (New England Biolabs, USA). Chromosomal DNA was purified as previously described [48]. PCR was performed using Taq polymerase according to the manufacturer's instructions (New England Biolabs, USA). DNA sequencing was performed by the Australian Equine Genome Research Centre. Deletion mutants were constructed essentially as previously described using either pKD46 [49] or pKOBEG199 [50, 51], with the exception that C. freundii and C. koseri strains were heated at 42°C for 2 min prior to electroporation. Primers used to generate deletion mutants were as follows: 1293 and 1294 (E. coli MS2027), 1456 and 1457 (E. coli ECOR15 and K. pneumoniae strains), 1458 and 1459 (E. coli ECOR28), 1456 and 1459 (E. coli M184), 1460 and 1459 (K. oxytoca strains), 1456 and 1461 (C. koseri M546), 1462 and 1459 (C. freundii M46) (Table 3). All deletion mutants were checked by PCR using specific primers (Table 3) in conjunction with primers targeting the kanamycin or chloramphenicol resistance gene [49] and further confirmed by sequencing. Sequence information outside the mrk cluster was obtained by inverse PCR (using primer combinations 1450/1452, 1450/1454, 1450/1453, 1451/1455, or 1451/1453) or standard PCR employing primers designed from the genome sequenced K. pneumoniae MGH78578 or C. koseri ATCC BAA895 (Table 3).
Table 3

Primers used in this study

Primer

Description

Sequence (5'-3')

Type 3 fimbriae deletion primers

 

1293

50 bp overhang mrk knockout F-primer 1

TCTTCTCTCTGCAGCAATGGCAACCGCGTTTTTTGGCATGACTGCTGCCCGTGTAGGCTGGAGCTGCTTCG

1294

50 bp overhang mrk knockout R-primer 1

GGTGTGAGCGGGATAGTTGTCTGAGTCACAGGCAGTTTCCTCTTCACCAGCATATGAATATCCTCCTTAG

1295

Knockout screening F 1

GGCAGCATAACCGAACAAAT

1296

Knockout screening R 1

TAAATTTTCTGCGGCAAACC

1456

50 bp overhang mrk knockout F-primer 2

GTTAACGGTACCCGCTTTATTTATCCAGGAAATGAAAAAGAAATAACGGTGTGTAGGCTGGAGCTGCTTCG

1457

50 bp overhang mrk knockout R-primer 2

CCTTTGTCCCAGAACTCCGGGCTGACATAGTTTTTCAGGCGTTGATCTTCCATATGAATATCCTCCTTAG

1463

Knockout screening R 2

GGTCTGGTTGCTGTTCCAGT

1458

50 bp overhang mrk knockout F-primer 3

GAGAGCTGCACCGTTATTTCTTTTTCATTTCCTGGATAAATAAAGCGGGTGTGTAGGCTGGAGCTGCTTCG

1459

50 bp overhang mrk knockout R-primer 3

CTTTACCGGCGGTGATTTTCAGGTCGTAGCGCCCTTTCCATTCGCCGTTGCATATGAATATCCTCCTTAG

1464

Knockout screening F 3

CTGCCGAGCTTAATACGCA

1465

Knockout screening R 3

GCAACGCCTTTATCCCAGA

1460

50 bp overhang mrk knockout F-primer 4

CTGCCGTTCAGGACGGCGCGCGCTTCACTTACTACGTTGGCTACGCTACCGTGTAGGCTGGAGCTGCTTCG

1461

50 bp overhang mrk knockout R-primer 4

GTGTAGCAGACGCCCATTTTGCCGTCTTTTCCACGGGTGATATTCAGGTCCATATGAATATCCTCCTTAG

1466

Knockout screening F 4

ATCCAGCTGGTTCTGTCCAC

1467

Knockout screening R 4

CCGGGCTGACAAAATTCTTA

1462

50 bp overhang mrk knockout F-primer 5

GGCTGGATAACGGTAATGCTGACGCAACGCCGGATACTATTACGACTCCAGTGTAGGCTGGAGCTGCTTCG

1468

Knockout screening F 5

AAAAGAAATAACGGTGCAGCTC

Inverse PCR primers

 

1450

mrkA R-primer A

CTTTACCGAAGAAATTAACC

1451

mrkA R-primer C

GAAGAAATTAACCTGACCGCC

1452

mrkD F-primer A

AAACCTATCTGAGCGCCAAC

1453

mrkD F-primer B

CCTCTTATGACTGGGAGAACG

1454

mrkD F-primer C

TGCGCGCTTCTATCAATATG

1455

mrkD F-primer D

GGCGTCCAGGTACTGAAAGA

Outside mrk cluster screening primers

 

1469

F-primer (outside mrkA of K. pneumoniae MGH78578)

TGGCGGTACATACTTCACTCA

1470

R-primer (outside mrkF of K. pneumoniae MGH78578)

ATCCTCTGCCTATTGTCTGACT

1471

F-primer (mrkE of K. pneumoniae pIA565)

TTTGCATATCCGCAATCTGA

1472

F-primer (outside mrkA of C. koseri ATCC BAA-895)

AATTGCAGGAACAGGGTCTG

1473

R-primer (outside mrkF of C. koseri ATCC BAA-895)

TTCCTTTCCCGTAACCA

1474

F-primer (outside mrkA of E. coli MS2027)

AGCGTTCTGCATGGCTTTAT

1475

R-primer (outside mrkF of E. coli MS2027)

AACGACGTTGGGTCGTTAGT

pMAS2027 screening primers

 

1476

pMAS2027 screening primer F1

GCGCAGAACTGGTTATGAAT

1477

pMAS2027 screening primer R1

TCATGGATTTTCTTCCTTAACAA

1478

pMAS2027 screening primer F2

ACAACTATCCCGCTCACACC

1479

pMAS2027 screening primer R2

ACCGTTAACGCGTAGTCACC

1480

pMAS2027 screening primer F3

TGCTTCAGCAGCATATCAGG

1481

pMAS2027 screening primer R3

GGAAAGCGTTAAAGCAGGTG

1482

pMAS2027 screening primer F4

CCGTATGCGCTTTTTCAAGT

1483

pMAS2027 screening primer R4

AAAGTTGAAGCCCGCTTTCT

1484

pMAS2027 screening primer F5

ACGGGTAAGACCGCTAACCT

1485

pMAS2027 screening primer R5

TCGATAAGGTAGGCATCAACAA

MR/K agglutination

Bacterial agglutination of tannic acid treated human erythrocytes (MR/K agglutination) was performed as previously described to detect the expression of Type 3 fimbriae [29]. 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 study

Biofilm formation on polyvinyl chloride (PVC) surfaces was monitored by using 96-well microtitre plates (Falcon) essentially as previously described [16]. 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 [14]. Protein samples were analysed by SDS-PAGE and western blotting as previously described [52] employing a type 3 fimbriae specific antiserum. Immunogold labelling was performed using the same Type 3 fimbriae specific antiserum as previously described [14]. 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 [53], and subjected to phylogenetic analysis using PHYLIP [54]. 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 [54]. 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.

Statistical analysis

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 [28]. 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.

Ethical approval

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.

Declarations

Acknowledgements

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.

Authors’ Affiliations

(1)
Centre for Infectious Disease Research, School of Chemistry and Molecular Biosciences, University of Queensland
(2)
UFR Pharmacie, Laboratoire de Bactériologie, Université de Clermont 1

References

  1. Stamm WE: Catheter-associated urinary tract infections: epidemiology, pathogenesis, and prevention. Am J Med. 1991, 91 (3B): 65S-71S. 10.1016/0002-9343(91)90345-X.View ArticlePubMed
  2. Warren JW, Tenney JH, Hoopes JM, Muncie HL, Anthony WC: A prospective microbiologic study of bacteriuria in patients with chronic indwelling urethral catheters. J Infect Dis. 1982, 146 (6): 719-723.View ArticlePubMed
  3. Paterson DL, Lipman J: Returning to the pre-antibiotic era in the critically ill: the XDR problem. Crit Care Med. 2007, 35 (7): 1789-1791. 10.1097/01.CCM.0000269352.39174.A4.View ArticlePubMed
  4. Warren JW: Catheter-associated urinary tract infections. Int J Antimicrob Agents. 2001, 17 (4): 299-303. 10.1016/S0924-8579(00)00359-9.View ArticlePubMed
  5. Sebghati TA, Korhonen TK, Hornick DB, Clegg S: Characterization of the type 3 fimbrial adhesins of Klebsiella strains. Infect Immun. 1998, 66 (6): 2887-2894.PubMed CentralPubMed
  6. Giltner CL, van Schaik EJ, Audette GF, Kao D, Hodges RS, Hassett DJ, Irvin RT: The Pseudomonas aeruginosa type IV pilin receptor binding domain functions as an adhesin for both biotic and abiotic surfaces. Mol Microbiol. 2006, 59 (4): 1083-1096. 10.1111/j.1365-2958.2005.05002.x.View ArticlePubMed
  7. Zogaj X, Bokranz W, Nimtz M, Romling U: Production of cellulose and curli fimbriae by members of the family Enterobacteriaceae isolated from the human gastrointestinal tract. Infect Immun. 2003, 71 (7): 4151-4158. 10.1128/IAI.71.7.4151-4158.2003.PubMed CentralView ArticlePubMed
  8. Ghigo JM: Natural conjugative plasmids induce bacterial biofilm development. Nature. 2001, 412 (6845): 442-445. 10.1038/35086581.View ArticlePubMed
  9. Reisner A, Haagensen JA, Schembri MA, Zechner EL, Molin S: Development and maturation of Escherichia coli K-12 biofilms. Mol Microbiol. 2003, 48 (4): 933-946. 10.1046/j.1365-2958.2003.03490.x.View ArticlePubMed
  10. Valle J, Mabbett AN, Ulett GC, Toledo-Arana A, Wecker K, Totsika M, Schembri MA, Ghigo JM, Beloin C: UpaG, a new member of the trimeric autotransporter family of adhesins in uropathogenic Escherichia coli. J Bacteriol. 2008, 190 (12): 4147-4161. 10.1128/JB.00122-08.PubMed CentralView ArticlePubMed
  11. Kjaergaard K, Schembri MA, Ramos C, Molin S, Klemm P: Antigen 43 facilitates formation of multispecies biofilms. Environ Microbiol. 2000, 2 (6): 695-702. 10.1046/j.1462-2920.2000.00152.x.View ArticlePubMed
  12. Lane MC, Lockatell V, Monterosso G, Lamphier D, Weinert J, Hebel JR, Johnson DE, Mobley HL: Role of motility in the colonization of uropathogenic Escherichia coli in the urinary tract. Infect Immun. 2005, 73 (11): 7644-7656. 10.1128/IAI.73.11.7644-7656.2005.PubMed CentralView ArticlePubMed
  13. Allsopp LP, Totsika M, Tree JJ, Ulett GC, Mabbett AN, Wells TJ, Kobe B, Beatson SA, Schembri MA: UpaH is a newly identified autotransporter protein that contributes to biofilm formation and bladder colonization by uropathogenic Escherichia coli CFT073. Infect Immun. 2010, 78 (4): 1659-1669. 10.1128/IAI.01010-09.PubMed CentralView ArticlePubMed
  14. Ulett GC, Mabbett AN, Fung KC, Webb RI, Schembri MA: The role of F9 fimbriae of uropathogenic Escherichia coli in biofilm formation. Microbiology. 2007, 153 (Pt 7): 2321-2331. 10.1099/mic.0.2006/004648-0.View ArticlePubMed
  15. Connell I, Agace W, Klemm P, Schembri M, Marild S, Svanborg C: Type 1 fimbrial expression enhances Escherichia coli virulence for the urinary tract. Proc Natl Acad Sci USA. 1996, 93 (18): 9827-9832. 10.1073/pnas.93.18.9827.PubMed CentralView ArticlePubMed
  16. Schembri MA, Klemm P: Biofilm formation in a hydrodynamic environment by novel fimH variants and ramifications for virulence. Infect Immun. 2001, 69 (3): 1322-1328. 10.1128/IAI.69.3.1322-1328.2001.PubMed CentralView ArticlePubMed
  17. Burmolle M, Bahl MI, Jensen LB, Sorensen SJ, Hansen LH: Type 3 fimbriae, encoded by the conjugative plasmid pOLA52, enhance biofilm formation and transfer frequencies in Enterobacteriaceae strains. Microbiology. 2008, 154 (Pt 1): 187-195. 10.1099/mic.0.2007/010454-0.View ArticlePubMed
  18. Hornick DB, Allen BL, Horn MA, Clegg S: Fimbrial types among respiratory isolates belonging to the family Enterobacteriaceae. J Clin Microbiol. 1991, 29 (9): 1795-1800.PubMed CentralPubMed
  19. Yakubu DE, Old DC, Senior BW: The haemagglutinins and fimbriae of Proteus penneri. J Med Microbiol. 1989, 30 (4): 279-284. 10.1099/00222615-30-4-279.View ArticlePubMed
  20. Old DC, Adegbola RA: Antigenic relationships among type-3 fimbriae of Enterobacteriaceae revealed by immunoelectronmicroscopy. J Med Microbiol. 1985, 20 (1): 113-121. 10.1099/00222615-20-1-113.View ArticlePubMed
  21. Adegbola RA, Old DC: Fimbrial and non-fimbrial haemagglutinins in Enterobacter aerogenes. J Med Microbiol. 1985, 19 (1): 35-43. 10.1099/00222615-19-1-35.View ArticlePubMed
  22. Old DC, Adegbola RA: Relationships among broad-spectrum and narrow-spectrum mannose-resistant fimbrial hemagglutinins in different Yersinia species. Microbiol Immunol. 1984, 28 (12): 1303-1311.View ArticlePubMed
  23. Adegbola RA, Old DC, Senior BW: The adhesins and fimbriae of Proteus mirabilis strains associated with high and low affinity for the urinary tract. J Med Microbiol. 1983, 16 (4): 427-431. 10.1099/00222615-16-4-427.View ArticlePubMed
  24. Adegbola RA, Old DC, Aleksic S: Rare MR/K-like hemagglutinins (and type-3-like fimbriae) of Salmonella strains. FEMS Microbiol Lett. 1983, 19 (2-3): 233-238. 10.1111/j.1574-6968.1983.tb00548.x.View Article
  25. Adegbola RA, Old DC: Fimbrial haemagglutinins in Enterobacter species. J Gen Microbiol. 1983, 129 (7): 2175-2180.PubMed
  26. Old DC, Adegbola R, Scott SS: Multiple fimbrial haemagglutinins in Serratia species. Med Microbiol Immunol. 1983, 172 (2): 107-115. 10.1007/BF02124511.View ArticlePubMed
  27. Old DC, Adegbola RA: Haemagglutinins and fimbriae of Morganella, Proteus and Providencia. J Med Microbiol. 1982, 15 (4): 551-564. 10.1099/00222615-15-4-551.View ArticlePubMed
  28. Ong CL, Ulett GC, Mabbett AN, Beatson SA, Webb RI, Monaghan W, Nimmo GR, Looke DF, McEwan AG, Schembri MA: Identification of type 3 fimbriae in uropathogenic Escherichia coli reveals a role in biofilm formation. J Bacteriol. 2008, 190 (3): 1054-1063. 10.1128/JB.01523-07.PubMed CentralView ArticlePubMed
  29. Duguid JP: Fimbriae and adhesive properties in Klebsiella strains. J Gen Microbiol. 1959, 21: 271-286.View ArticlePubMed
  30. Ong CL, Beatson SA, McEwan AG, Schembri MA: Conjugative plasmid transfer and adhesion dynamics in an Escherichia coli biofilm. Appl Environ Microbiol. 2009, 75 (21): 6783-6791. 10.1128/AEM.00974-09.PubMed CentralView ArticlePubMed
  31. Jagnow J, Clegg S: Klebsiella pneumoniae MrkD-mediated biofilm formation on extracellular matrix- and collagen-coated surfaces. Microbiology. 2003, 149 (Pt 9): 2397-2405. 10.1099/mic.0.26434-0.View ArticlePubMed
  32. Boddicker JD, Anderson RA, Jagnow J, Clegg S: Signature-tagged mutagenesis of Klebsiella pneumoniae to identify genes that influence biofilm formation on extracellular matrix material. Infect Immun. 2006, 74 (8): 4590-4597. 10.1128/IAI.00129-06.PubMed CentralView ArticlePubMed
  33. Langstraat J, Bohse M, Clegg S: Type 3 fimbrial shaft (MrkA) of Klebsiella pneumoniae, but not the fimbrial adhesin (MrkD), facilitates biofilm formation. Infect Immun. 2001, 69 (9): 5805-5812. 10.1128/IAI.69.9.5805-5812.2001.PubMed CentralView ArticlePubMed
  34. Sebghati TA, Clegg S: Construction and characterization of mutations within the Klebsiella mrkD1P gene that affect binding to collagen type V. Infect Immun. 1999, 67 (4): 1672-1676.PubMed CentralPubMed
  35. Tarkkanen AM, Virkola R, Clegg S, Korhonen TK: Binding of the type 3 fimbriae of Klebsiella pneumoniae to human endothelial and urinary bladder cells. Infect Immun. 1997, 65 (4): 1546-1549.PubMed CentralPubMed
  36. Tarkkanen AM, Allen BL, Westerlund B, Holthofer H, Kuusela P, Risteli L, Clegg S, Korhonen TK: Type V collagen as the target for type-3 fimbriae, enterobacterial adherence organelles. Mol Microbiol. 1990, 4 (8): 1353-1361. 10.1111/j.1365-2958.1990.tb00714.x.View ArticlePubMed
  37. Allen BL, Gerlach GF, Clegg S: Nucleotide sequence and functions of mrk determinants necessary for expression of type 3 fimbriae in Klebsiella pneumoniae. J Bacteriol. 1991, 173 (2): 916-920.PubMed CentralPubMed
  38. Huang YJ, Liao HW, Wu CC, Peng HL: MrkF is a component of type 3 fimbriae in Klebsiella pneumoniae. Res Microbiol. 2009, 160 (1): 71-79. 10.1016/j.resmic.2008.10.009.View ArticlePubMed
  39. Struve C, Bojer M, Krogfelt KA: Identification of a conserved chromosomal region encoding Klebsiella pneumoniae type 1 and type 3 fimbriae and assessment of the role of fimbriae in pathogenicity. Infect Immun. 2009, 77 (11): 5016-5024. 10.1128/IAI.00585-09.PubMed CentralView ArticlePubMed
  40. Norman A, Hansen LH, She Q, Sorensen SJ: Nucleotide sequence of pOLA52: a conjugative IncX1 plasmid from Escherichia coli which enables biofilm formation and multidrug efflux. Plasmid. 2008, 60 (1): 59-74. 10.1016/j.plasmid.2008.03.003.View ArticlePubMed
  41. Wertz JE, Goldstone C, Gordon DM, Riley MA: A molecular phylogeny of enteric bacteria and implications for a bacterial species concept. J Evol Biol. 2003, 16 (6): 1236-1248. 10.1046/j.1420-9101.2003.00612.x.View ArticlePubMed
  42. Hornick DB, Thommandru J, Smits W, Clegg S: Adherence properties of an mrkD-negative mutant of Klebsiella pneumoniae. Infect Immun. 1995, 63 (5): 2026-2032.PubMed CentralPubMed
  43. Schurtz TA, Hornick DB, Korhonen TK, Clegg S: The type 3 fimbrial adhesin gene (mrkD) of Klebsiella species is not conserved among all fimbriate strains. Infect Immun. 1994, 62 (10): 4186-4191.PubMed CentralPubMed
  44. Huang YJ, Wu CC, Chen MC, Fung CP, Peng HL: Characterization of the type 3 fimbriae with different MrkD adhesins: possible role of the MrkD containing an RGD motif. Biochem Biophys Res Commun. 2006, 350 (3): 537-542. 10.1016/j.bbrc.2006.09.070.View ArticlePubMed
  45. Mabbett AN, Ulett GC, Watts RE, Tree JJ, Totsika M, Ong CL, Wood JM, Monaghan W, Looke DF, Nimmo GR: Virulence properties of asymptomatic bacteriuria Escherichia coli. Int J Med Microbiol. 2009, 299 (1): 53-63. 10.1016/j.ijmm.2008.06.003.View ArticlePubMed
  46. Ochman H, Selander RK: Standard reference strains of Escherichia coli from natural populations. J Bacteriol. 1984, 157 (2): 690-693.PubMed CentralPubMed
  47. Martino PD, Fursy R, Bret L, Sundararaju B, Phillips RS: Indole can act as an extracellular signal to regulate biofilm formation of Escherichia coli and other indole producing bacteria. Can J Microbiol. 2003, 49 (7): 443-449. 10.1139/w03-056.View ArticlePubMed
  48. Sambrook J, Russell DW: Molecular Cloning: A Laboratory Manual. 2001, New York: Cold Spring Harbor Laboratory Press, 1: Third
  49. Datsenko KA, Wanner BL: One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA. 2000, 97 (12): 6640-6645. 10.1073/pnas.120163297.PubMed CentralView ArticlePubMed
  50. Balestrino D, Haagensen JA, Rich C, Forestier C: Characterization of type 2 quorum sensing in Klebsiella pneumoniae and relationship with biofilm formation. J Bacteriol. 2005, 187 (8): 2870-2880. 10.1128/JB.187.8.2870-2880.2005.PubMed CentralView ArticlePubMed
  51. Coudeyras S, Nakusi L, Charbonnel N, Forestier C: A tripartite efflux pump involved in gastrointestinal colonization by Klebsiella pneumoniae confers a tolerance response to inorganic acid. Infect Immun. 2008, 76 (10): 4633-4641. 10.1128/IAI.00356-08.PubMed CentralView ArticlePubMed
  52. Ulett GC, Webb RI, Schembri MA: Antigen-43-mediated autoaggregation impairs motility in Escherichia coli. Microbiology. 2006, 152 (Pt 7): 2101-2110. 10.1099/mic.0.28607-0.View ArticlePubMed
  53. Jeanmougin F, Thompson JD, Gouy M, Higgins DG, Gibson TJ: Multiple sequence alignment with Clustal X. Trends Biochem Sci. 1998, 23 (10): 403-405. 10.1016/S0968-0004(98)01285-7.View ArticlePubMed
  54. Felsenstein J: PHYLIP (Phylogeny Inference Package) version 3.6. 2004, Seattle: Department of Genome Sciences
  55. Kloepper TH, Huson DH: Drawing explicit phylogenetic networks and their integration into SplitsTree. BMC Evol Biol. 2008, 8: 22-10.1186/1471-2148-8-22.PubMed CentralView ArticlePubMed
  56. Huson DH: SplitsTree: analyzing and visualizing evolutionary data. Bioinformatics. 1998, 14 (1): 68-73. 10.1093/bioinformatics/14.1.68.View ArticlePubMed

Copyright

© Ong et al; licensee BioMed Central Ltd. 2010

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Advertisement