M A J O R A R T I C L E
Genomic Heterogeneity in Klebsiella pneumoniae
Strains Is Associated with Primary Pyogenic Liver
Abscess and Metastatic Infection
Li-Chen Ma,1Chi-Tai Fang,2Cha-Ze Lee,2Chia-Tung Shun,3and Jin-Town Wang1,2
1Department of Microbiology, National Taiwan University College of Medicine, and Departments of2Internal Medicine and3Forensic Medicine, National Taiwan University Hospital, Taipei, Taiwan
Background. Primary pyogenic liver abscess (PLA) with septic complication by Klebsiella pneumoniae is an emerging infectious disease.
Methods and results. Using DNA microarray hybridization, we identified a 20-kb chromosomal region that contained 15 open-reading frames (ORFs), including an iron-uptake system (kfu), a phosphoenolpyruvate sugar phosphotransferase system (PTS), and 6 unknown ORFs. The region was more prevalent among tissue-invasive strains (35/46) than among noninvasive strains (19/98) (P!.0001, x2test). To test the role played by this region
in pathogenesis, 3 different deletion mutants (NTUH-K2044 [Dkfu], K2044 [DORF7–9], and K2044 [DPTS]) were constructed. Only the DkfuABC mutants showed decreased virulence in mice, compared with the wild-type strain. An in vitro assay confirmed the involvement of kfu in iron acquisition. There was a high correlation rate (85%) between the kfu/PTS region and 2 tissue invasion–associated chromosomal regions (allS and magA). Moreover, all 3 regions were present in strains that caused PLA plus endophthalmitis or meningitis.
Conclusion. Our results suggest that chromosomal heterogeneity is present in tissue-invasive K. pneumoniae strains. A genotype containing all 3 regions is strongly associated with PLA and metastatic infection. These regions may serve as convenient markers for the rapid diagnosis of emergent tissue-invasive strains.
Klebsiella pneumoniae is an important hospital-acquired
pathogen that is a frequent cause of urinary tract in-fection, septicemia, and pneumonia in immunocom-promised individuals; it is also an important pathogen with respect to community-acquired infectious dis-eases, such as community-acquired pneumonia [1]. In Taiwan, K. pneumoniae–associated primary pyogenic liver abscess (PLA) has recently become an important emerging infectious disease [2–8]. This disease is also a global concern, as is attested by reports from North America [9], Europe [10, 11], and Asia [12–14].
Fulminant tissue-invasive K. pneumoniae infections can attack healthy persons who have no history of
he-Received 3 November 2004; accepted 5 February 2005; electronically published 25 May 2005.
Financial support: Taiwan National Science Council (grant 93-3112-B-002-002). Reprints or correspondence: Dr. Jin-Town Wang, Dept. of Microbiology, National Taiwan University College of Medicine, 1, Sec. 1, Jen-Ai Rd., Taipei, Taiwan (wangjt @ntu.edu.tw).
The Journal of Infectious Diseases 2005; 192:117–28
2005 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2005/19201-0017$15.00
patobiliary disease, and only 50% of patients have a pre-disposing condition, such as diabetes mellitus [2–8]. PLA is manifest with other septic metastatic lesions, including pyogenic meningitis and endophthalmitis, in 10%–12% of patients [3–5, 7]. Despite aggressive antibacterial strat-egies, significant morbidity and mortality still exists, es-pecially in those with diabetes mellitus [2–8].
Using transposon mutagenesis and full genome ex-pression analysis, we recently identified genomic seg-ments of 33 kb (magA) and 22 kb (allS), which were absent in the genome of MGH 78578 and present in most tissue-invasive strains from patients with PLA, meningitis, or endophthalmitis [15, 16]. Therefore, it is probable that these tissue-invasive strains might rep-resent a specific genotype that harbors specific regions involved in pathogenesis and prevalence.
DNA microarray technology provides a useful tool for assessment of the differences and changes in bac-terial genomes [17, 18]. Therefore, we used this tech-nology to compare genomic variations between tissue-invasive strains and nontissue-invasive strains.
Table 1. Prevalences of 3 chromosomal fragments among Klebsiella pneumoniae strains used in the present study. Strain type Clinical diagnosis of patient Origin No. of isolates
No. of isolates positive for region 20-kb region (kfu/PTS) Large plasmid 22-kb region (allS) 33-kb region (magA)
Tissue-invasive strains (n p 46) PLA NTUH (Taiwan) 33 24 30 24 26
PLA plus endopthalmitis NTUH (Taiwan) 8 8 7 8 8
Menignitis NTUH (Taiwan) 4 2 4 0 2
PLA plus meningitis NTUH (Taiwan) 1 1 1 1 1
Noninvasive strains (n p 98) Disease other than PLA NTUH (Taiwan) 33 9 13 2 2
Disease other than PLA FEMH (Taiwan) 32 0 30 0 0
Disease other than PLA ECKH (Taiwan) 11 3 11 1 1
Disease other than PLA ATCC (USA) 22 7 22 0 3
NOTE. ECKH, En Chu Kong Hospital; FEMH, Far Eastern Memorial Hospital; NTUH, National Taiwan University Hospital; PLA, primary pyogenic liver abscess; PTS, phosphoenolpyruvate sugar phosphotransferase system.
PATIENTS, MATERIALS, AND METHODS
K. pneumoniae strains and culture conditions. K.
pneumoni-ae isolates were obtained consecutively from cultures of blood
samples obtained from patients between 1997 and 2003. A total of 46 tissue-invasive K. pneumoniae strains were obtained from patients with PLA or meningitis at National Taiwan University Hospital (NTUH). Of these, 22 were isolated from patients with diabetes mellitus, and 24 were isolated from patients who had previously been healthy.
For comparison, 98 noninvasive strains were obtained from patients with sepsis but without any tissue-invasive disease (such as PLA, meningitis, and endophthalmitis). Of these strains, 33 were obtained from patients at NTUH, 32 were obtained from patients at Far Eastern Memorial Hospital (FEMH; Banciao, Taiwan), and 11 were obtained from pa-tients at En Chu Kong Hospital (ECKH; Sansia, Taiwan). Al-so, 22 strains from the United States, including MGH 78578, were purchased from the American Type Culture Collection. MGH 78578 was chosen for full genome sequencing at Wash-ington University (St. Louis, MO; available at: http://www .genome.wustl.edu/projects/bacterial/). Information pertain-ing to the 144 strains is listed in table 1. All strains were identified and cultured according to standard clinical micro-biology methods [19].
Microarray construction and hybridization. The genomic library was constructed from a clinical isolate, NTUH-K2044, obtained from a patient with PLA plus meningitis in a phag-emid [20]. DNA fragments in phagphag-emids of K. pneumoniae were amplified by polymerase chain reaction (PCR) with prim-ers in vectors and were spotted onto a nylon membrane (Roche) by a computer-controlled XYZ translation system (PM500; Newport) [21].
Probe preparation and hybridization. Genomic DNA from 4 PLA strains (NTUH-K2044, A1208, A3021, and A5011) and 3 noninvasive strains (N3423, N3529, and N5322)
were extracted and were labeled with biotin-16-dUTP (Perkin Elmer) by a randomly primed polymerization reaction. The microarray membrane was prehybridized in 2 mL of hybrid-ization buffer for 4 h at 65C and was hybridized for 16 h at 68C. The membrane was washed twice with 2⫻ standard saline citrate (SSC) containing 0.1% SDS for 5 min at room temperature and was then washed 3 times with 0.1⫻ SSC containing 0.1% SDS for 15 min at 65C each time. Color-imetry detection and image analysis were then performed as described elsewhere [22].
Recombinant DNA techniques and plasmids. K. pneu-moniae deletion mutants were constructed by replacing the
deletion region with a kanamycin (Km) cassette in a double-crossover integration of chromosomal DNA. All primers used in the present study are listed in table 2. To generate the NTUH-K2044 (Dkfu) mutant, a PCR fragment amplified by primers PVAR KO-1 and PVAR KO-2 was cloned into a pGEM-T Easy vector (Promega). PVAR KO-1(iPCR) and PVAR KO-2(iPCR) primers were used for inverse PCR with plaque-forming-unit polymerase (MBI Fermentas). A blunt-end Km gene was phosphorylated by use of a polynucleo-tide kinase (New England Biolabs) and was ligated to the inverse PCR product to generate the kfu disruption fragment. We cloned the fragment into a pUT suicide vector [23] con-taining an EcoRI site and added a second marker (spection-mycin; Spe) to the ApaLI site. pUT-(Dkfu) was transformed into wild-type NTUH-K2044 to generate a kfu deletion mu-tant by conjugation. Deletion clones were selected by Kmr
and Spes. The same procedures were used for open-reading
frames (ORFs) 7–9 and phosphoenolpyruvate sugar phospho-transferase system (PTS) deletion constructs. All of the deletion mutants were confirmed by PCR with multiple primer pairs and sequence determination.
Transcomplementation of Escherichia coli H1443. An
Table 2. Polymerase chain reaction primers used in the present study.
Primer Sequence Description Reference
PVAR KO-1 5-GCAGCAGATGAATATTCTGG-3 kfu deletion construct Present study
PVAR KO-1(iPCR) 5-TCTGGGTGCAGAACCAAATG-3 kfu deletion construct Present study
PVAR KO-2 5-TTCCGACGCCAATGCTGATC-3 kfu deletion construct Present study
PVAR KO-2(iPCR) 5-ATGGAGTGGTAGTACGTTGG-3 kfu deletion construct Present study
PVAR KO-3 5-GGTCCTCGATAATCTCAATC-3 ORF7–9 deletion construct Present study
PVAR KO-3(iPCR) 5-TGACCGTCAGTCTCACCATC-3 ORF7–9 deletion construct Present study
PVAR KO-4 5-GACGTTGCCGAATGTCCATC-3 ORF7–9 deletion construct Present study
PVAR KO-4(iPCR) 5-GCAGCAGGAAGCTCTTGATG-3 ORF7–9 deletion construct Present study
PVAR g-F 5-ATTTGCGGCCAATCAGCGTC-3 PTS deletion construct Present study
PVAR g-R 5-CGACGCCGATATCAAAGAGT-3 PTS deletion construct Present study
PVAR h-F 5-AACCCGACGCTGTTTCATGC-3 PTS deletion construct Present study
PVAR h-R 5-GGAACTGGATAAAACGCATC-3 PTS deletion construct Present study
PVAR d-F 5-GCGTGCTAAGGTAATTAGGC-3 kfu operon analysis Present study
PVAR d-R 5-CGAAAATCTGGTGAAGTCGT-3 kfu operon analysis Present study
6C12E1-Sp63 5-ACTTCAAACACCAGGATCCG-3 kfu operon analysis Present study
6C12E1-T73 5-AGGCGTAGCTCTGGACAAAG-3 kfu operon analysis Present study
kfuB-F1179 5-GAAGTGACGCTGTTTCTGGC-3 kfu operon analysis Present study
kfuC-R649 5-TTTCGTGTGGCCAGTGACTC-3 kfu operon analysis Present study
PVAR-F 5-GCTTTTACCTGTAGCCGTCC-3 20-kb region (PVAR) prevalence analysisa Present study PVAR-R 5-GCAGGATATGACCATTTTGC-3 20-kb region (PVAR) prevalence analysisa Present study 7C4-T71 5-ATCCTGGAAGCTCACCGTTC-3 20-kb region (PVAR) prevalence analysisb Present study 26D6-T32 5-ATTTCCACGCGGATACCGTC-3 20-kb region (PVAR) prevalence analysisb Present study
26A1-T34 5-ACGTTCGCAGGTTAAAGCTG-3 Large plasmid prevalence analysis Present study
26A1-T73 5-CCATGTAGCTGCAGATACTG-3 Large plasmid prevalence analysis Present study
28G9-T33 5-AAGCGTCTGGTTTCCTGGTG-3 Large plasmid prevalence analysis Present study
28G9-T72 5-TATTTGTCGGCAATTACGGC-3 Large plasmid prevalence analysis Present study
2801769-F 5-GTACACCAGCGGCCTGTTCTGGGCGCAATC-3 22-kb region (all) prevalence analysisa [15] 2803831-R 5-CAGAACAGCATTTACTATGATGTGGTG-3 22-kb region (all) prevalence analysisa [15]
10E4-2-5F 5-AGTCGGCCTGGGGTTTAAGG-3 22-kb region (all) prevalence analysisb [15]
10E4-2-475R 5-CAGTCAACGTGGCGATTCGC-3 22-kb region (all) prevalence analysisb [15]
TF-F 5-GCGATACTGGTAACTGGGTT-3 33-kb region (magA) prevalence analysisc [16]
M5YM-R 5-GTCACTTTCTTGGCTTCCTG-3 33-kb region (magA) prevalence analysisc [16]
M3YM-F 5-AACCCATCGCATTGGACAGA-3 33-kb region (magA) prevalence analysisd [16]
M3M-R 5-ACCTTTTACTCGCGCACCAA-3 33-kb region (magA) prevalence analysisd [16]
1040-F 5-CGTTACGAACTTGAACGAGC-3 33-kb region (magA) prevalence analysisb [16]
936-R 5-GCAATGGCCATTTGCGTTAG-3 33-kb region (magA) prevalence analysisb [16]
NOTE. ORF, open-reading frame; PTS, phosphoenolpyruvate sugar phosphotransferase system.
a
Outside primers of the region.
b
Inside primer of the region.
c
Left junction primer of the 33-kb (magA) region.
d
Right junction primer of the 33-kb (magA) region.
when cultured in medium containing iron chelator 2,2 -dipyr-idyl (Sigma) [24, 25]. Transformed E. coli H1443 with plasmid pBR322::kfuABC, plasmid pSZ1 [25], or pBR322 were grown overnight at 37C in Luria-Bertani (LB) broth. Each culture was diluted in fresh LB broth supplemented with 0.1, 0.2, or 0.5 mmol/L 2,2-dipyridyl. The growth rate was monitored spectrophotometrically at 620 nm.
RNA isolation and reverse-transcription (RT) PCR. Total RNA was isolated from K. pneumoniae cultured at the expo-nential phase of growth, as described elsewhere [15]. For each RT, 5 mg of RNA was used with 2 pmol of RT-PCR primer and 200 U of M-MLV reverse transcriptase (Invitrogen). Reaction mixtures without reverse transcriptase were included as
nega-tive controls. PCR was performed with 10% of each RT reac-tion volume under 30 cycles of amplificareac-tion.
Murine experiments. Initially, we infected BALB/cByl mice intraperitoneally (ip). However, later experiments showed that intragastric (ig) inoculation had a higher sen-sitivity to differential virulence [15, 16]. Mice were admin-istered ig [26] either wild-type NTUH-K2044, MGH 78578, NTUH-K2044 (Dkfu), K2044 (DORF7–9), or K2044 (DPTS) (103–106 cfu; 4 mice for each dose). For ig inoculation, we
carefully slipped a 1-mm polyethylene flexible tube (Becton Dickinson) past the pharynx into the stomach (∼5 cm of intubation) of each mouse and delivered 0.2 mL (103–106cfu)
pro-Figure 1. DNA hybridization and colorimetric detection of 4 tissue-invasive and 3 noninvasive Klebsiella pneumoniae strains on a microarray. Circles show decreased hybridization signals in noninvasive strains.
Table 3. Sequences of 12 clones with decreased hybridization signals in noninvasive strains.
Clone no. Predicted protein (GenBank accession no.)
Length of comparable amino acid sequence
(% identity)
Comparison with MGH 78578
1 Ferric enterobactin (enterochelin) transport (NP 286317.1) 333 (86) MGH contig 123
Hypothetical protein …
Hypothetical membrane protein P43 (NP752609.1) 406 (85)
Ferrienterobactin-binding periplasmic protein precursor (NP 752610.1) 307 (84)
2 Class II aldolase/adducin domain protein (NP745015.1) 196 (58) MGH contig 112
Putative 2-hydroxyacid dehydrogenase (NP879163.1) 269 (47)
Probable short-chain dehydrogenase (NP250035.1) 263 (60)
Conserved hypothetical protein (NP929872.1) 191 (44)
Probable extracellular solute-binding protein (NP406806.1) 250 (63)
3∼10a Iron-uptake system … No similarity
PTS …
Six uncharacterized hypothetical proteins …
11b CobW (AAR07790) 406 (100) No similarity
Conserved hypothetical protein (AAR07791) 68 (100)
Ferrous ion transporter protein (AAR07792) 549 (100)
12b Putative partitioning (parA) (AAR07854) 296 (100) No similarity
Putative partitioning (parB) (AAR07855) 343 (100)
Conserved hypothetical protein (AAR07856) 147 (99)
NOTE. PTS, phosphoenolpyruvate sugar phosphotransferase system.
a
Sequence could be connected to∼9 kb; see table 4 for further analysis.
b
Proved, by BLAST analysis, to be localized in the large plasmid of Klebsiella pneumoniae CG43; the position of clone 11 was between 128577 and 131336, and the position of clone 12 was between 188902 and 191960.
cedure. Mice were monitored for 4 weeks; upon death, the liver and brain were removed, and histopathological exami-nation was conducted. Surviving mice were killed at the end of the 4 weeks. The LD50was calculated as described elsewhere
[27]. Survival was analyzed by Kaplan-Meier analysis with a
log-rank test; P!.05 was considered to be statistically significant.
Slot-blot hybridization. Ten micrograms of genomic DNA from K. pneumoniae strains were vacuum blotted onto nylon membranes. Hybridization was performed for 16 h at 68C
Figure 2. Genetic organization of the 20-kb kfu/phosphoenolpyruvate sugar phosphotransferase system (PTS) chromosomal region. A 5292-bp fragment in MGH 78578 was replaced by a 19,640-bp fragment in NTUH-K2044. The locations and orientation of the open-reading frames (ORFs) described in the present study are indicated by arrows.
Table 4. Characteristics of the open-reading frames (ORFs) and deduced amino acid sequences present in the 20-kb DNA fragment.
ORF no., name
Product
size, aa ORF product exhibits homology to: Source
Identity, %
GenBank accession no.
01, ORF1 808 Glycogen phosphorylase Thermosynechococcus elongatus BP-1 50 NP681571.1
02, ORF2 115 Anti–anti-j factor Rhodobacter sphaeroides 40 ZP00006519.1
03, ORF3 511 Putative protease Shigella flexneri 2a strain 301 50 NP707066.1
04, kfuA 338 Iron(III)-binding periplasmic protein Yersinia pestis 78 NP406453.1
05, kfuB 524 Iron(III)-transport system permease Y. pestis 68 NP406455.1
06, kfuC 342 Iron(III)-transport ATP-binding protein Y. pestis 58 NP103033.1
07, ORF7 306 … … … …
08, yijO 277 Putative ARAC-type regulatory protein Escherichia coli O157:H7 57 NP290591.1 09, mmcH 297 Biosynthesis of mitomycin antibiotics Streptomyces lavendulae 29 AF127374.1
10, frwD 113 PTS fructose-like IIB component 2 Salmonella typhimurium LT2 59 NP462996.1
11, pflC 328 Pyruvate formate-lyase activating enzyme S. typhimurium LT2 63 NP462995.1
12, pflD 769 Formate acetyltransferase 2 S. flexneri 2a strain 301 85 NP838933.1
13, frwB 106 PTS fructose-like IIB component 1 S. typhimurium LT2 92 NP462993.1
14, frwC 389 PTS fructose-like IIC component S. flexneri 2a strain 301 85 NP709749.1
15, ptsA 833 Phosphoenolpyruvate-protein phosphotransferase E. coli CFT073 78 833 79 NP756760.1
NOTE. PTS, phosphoenolpyruvate sugar phosphotransferase system.
with each biotin-labeled probe generated by PCR. The gene encoding 23S rRNA was used as a positive control. Detection was performed by use of the Southern-Light Chemiluminescent detection system (Tropix), in accordance with the manufac-turer’s instructions.
RESULTS
DNA microarray hybridization. A total of 3146 PCR clones were randomly selected for the microarray. The coverage rate was∼88%, according to the formula N p ln(1 ⫺ P)/ln(1 ⫺ f )
Figure 3. Polymerase chain reaction primer alignments used in the present study to analyze the prevalences of the kfu/phosphoenolpyruvate sugar phosphotransferase system (PTS) chromosomal region.
Figure 4. A, Schematic diagram of the deletion constructs. Deletion regions (shown by dashed lines) were replaced by a kanamycin (Km) cassette.
B and C, Survival of BALB/cByl mice inoculated with various deletion mutants at a dose of 104cfu (B) or 105cfu (C)., wild-type strain; ,
NTUH-K2044 (Dkfu);, NTUH-K2044 (DORF7–9); 䉭, NTUH-K2044 (DPTS); ORF, open-reading frame. [28]. To test the redundancy of the library, 798 of the 3146
clones were randomly selected for sequencing. These clones contained 678 distinct sequences, representing a redundancy rate of 15%.
Comparison of 4 tissue-invasive strains (A1208, A3021, A5011, and NTUH-K2044) and 3 noninvasive strains (N3423, N3529, and N5322) revealed 12 clones with significantly de-creased hybridization signals (defined as⭓3 SDs of the mean ratio) in the noninvasive strains (figure 1 and table 3). These 12 clones were sequenced and then compared with the 10⫻ shotgun sequences of K. pneumoniae MGH 78578 (available at: http://www.genome.wustl.edu/projects/bacterial/kpneumoniae/). Of them, there were 10 clones (clones 3–12) that bore no sim-ilarity in sequence to MGH 78578. Of these 10 clones, 8 over-lapped and extended to encompass an∼9-kb fragment, whereas
the other 2 matched to a 200-kb plasmid (pLVPK) of K.
pneu-moniae CG43 [29] (hereafter, “the large plasmid”).
Sequencing of the flanking regions of the 8 clones in NTUH-K2044. The flanking regions of the∼9-kb fragment were se-quenced from the genomic library until both ends matched in MGH 78578. A 19,640-bp fragment (GenBank accession num-ber AB115591) that was obtained replaced a 5292-bp fragment in the genome of MGH 78578 (figure 2). BLAST searches re-vealed that this fragment contained 15 ORFs (figure 2 and table 4). The common flanking regions of NTUH-K2044 and MGH 78578 respectively contained oppA and an ORF encoding a pu-tative diogenase b subunit. The overall GC content of this region was 56.9%, similar to the 57.7% GC content of the remainder of the genome.
Figure 5. Histological examination of the livers and brains from infected mice. The wild-type strain and deletion mutants were administered to mice via intragastric inoculation at a dose of 104cfu. Half of the mice inoculated with 104
cfu of the wild-type strain, NTUH-K2044 (DORF7–9), or K2044 (DPTS) were dead within 2 weeks; however, all of the mice inoculated with 104
cfu of K2044 (Dkfu) or MGH 78578 remained healthy until they were killed at the end of 4 weeks. The livers from the mice inoculated with the wild-type strain (a), K2044 (DORF7–9) (b), K2044 (DPTS) (c), or K2044 (Dkfu) (d) were examined. The first 3 had severe liver abscesses, whereas the livers from the mice inoculated with K2044 (Dkfu) had normal histological structure. Brain abscesses could also be found in the mice inoculated with the wild-type strain (e), whereas the brains from the mice inoculated with K2044 (Dkfu) had normal histological structure (f). The stain used was hematoxylin-eosin. Original magnification,⫻100.
with 4 regions (figure 2 and table 4). Region 1 (293–4704) carried ORF1–3. These ORFs showed homology to proteins involved in glycogen phosphorylase (ORF1), an anti–anti-j fac-tor (ORF2), and a putative protease (ORF3). Region 2 (5104– 8743) harbored ORF4–6. These ORFs exhibited high homology to the bacterial ferric iron–uptake system, which is a bacterial ABC iron transport system [30]. They include Sfu of Serratia
marcescens [25, 31], Hit of Haemophilus influenzae [32], Yfu
of Yersinia pestis [33], Afu of Actinobacillus pleuropneumoniae [34], and Fbp of Neisseria gonorrhoeae [35]. Therefore, these ORFs were putatively designated kfuA, kfuB, and kfuC, respec-tively (“kfu” stands for Klebsiella ferric iron uptake). Region 3 (8990–11,786) contained ORF7–9. ORF7 displayed no signif-icant homology to any sequences in the database. However, ORF8 revealed similarity to the gene yijO, which encodes a putative ARAC-type regulatory protein in E. coli. ORF9 showed homology to a protein involved in the biosynthesis of mito-mycin [36]. Region 4 (11,786–19,553) extended from ORF10 to ORF15. These 6 ORFs showed high homology to the PTS.
PTS catalyzes translocation with concomitant phosphorylation of sugars and hexitols and regulates metabolism in response to the availability of carbohydrates [37]. Some PTS proteins have been tentatively linked with bacterial virulence [38–40]. This 20-kb region was designated “the kfu/PTS region.”
Prevalences of the kfu/PTS region and the large plasmid among K. pneumoniae strains. Because sequences of clones 1 and 2 were present in the noninvasive strain MGH 78578 (table 3), we studied the prevalences of the kfu/PTS region and the large plasmid among the clinical isolates to find regions that are specific to tissue-invasive strains. Genomic DNA ex-tracted from 46 tissue-invasive and 98 noninvasive strains was used for PCR analysis. We used the inside and outside primers to detect the presence of the kfu/PTS region (figure 3), on the following basis: If a clinical strain contained the region being tested, then the primer pairs for the flanking regions (outside primers) should fail to amplify the fragments; however, the inside primers should amplify products with a predicted length. Conversely, if the clinical strain did not contain the region,
Figure 6. A, Promoter sequence of the Klebsiella pneumoniae kfu operon. The potential ribosome binding site (RBS) is underscored. The box indicates the conserved Fur-binding sequences (Fur box) [41]. The transcription initiation start site (predicted by the Berkeley Drosophila Genome Project with a score of 1.0 [available at: http://www.fruitfly.org/seq_tools/promoter.html/]) and translation initiation condon are indicated by boldface. B, Genetic organization of the kfu locus. The thick black line represents chromosomal DNA, and the open arrows represent kfu open-reading frames. Ethidium bromine–stained agarose gels show transcriptional analysis of the kfu locus. Each panel contains products with the same primer pairs for polymerase chain reaction (PCR) with K. pneumoniae chromosomal DNA as the template (lane 1), for reverse transcription PCR with K. pneumoniae RNA as the template (lane 2), and for the same PCRs containing no reverse transcriptase, which generate no product (lane 3). Bars represent the corresponding target mRNA segments for each primer pairs used; arrows indicate the expected size of the RT-PCR products. C, Growth experiments with the wild-type strain, the NTUH-K2044 (Dkfu) deletion mutant, MGH 78578, and a noninvasive strain, N5322, in Luria-Bertani (LB) broth containing 0.2 mmol/ L 2,2-dipyridyl (DIP). D, Functional assay of the kfuABC operon of K. pneumoniae in LB broth containing 0.2 mmol/L DIP. Growth was monitored spectrophotometrically at 620 nm. The cultures were inoculated with fresh precultures to an OD620 nmof 0.01. The optical-density values are the means
of 3 experiments; error bars indicate SDs. H1443 pBR322 is the Escherichia coli aroB mutant strain transformed with pBR322 vector only; H1443 pSZ1 is the E. coli aroB mutant strain transformed with pBR322 containing an intact sfuABC as a positive control; and H1443 pBR322::kfu is the E. coli
aroB mutant strain transformed with pBR322 containing an intact kfuABC.
then PCR with the outside primer pairs would be positive, whereas PCR with the inside primers would be negative. We also used the inside sequences of clones 11 and 12 to detect the existence of the large plasmid.
The prevalence of the kfu/PTS region was significantly higher in tissue-invasive strains than in noninvasive strains (35/46 vs.
19/98; P p .0001, x2 test) (table 1). However, no significant
correlation was observed between the large plasmid and clin-ically invasive disease (42/46 vs. 76/98;P p .077, x2test).
Analysis of deletion mutants. Three deletion mutants (NTUH-K2044 [Dkfu], K2044 [DORF7–9], and K2044 [DPTS]) were constructed by use of a suicide vector (figure 4A). The
Figure 7. Polymerase chain reaction (PCR) analysis and slot-blot hybridization for 3 chromosomal regions. A, PCR primer alignments used in the analysis of the prevalences of 2 chromosomal regions among tissue-invasive and noninvasive Klebsiella pneumoniae strains. B, Slot-blot analyses performed to confirm the PCR results. Hybridization was performed by use of probes located inside the 3 regions. Lanes 1–6 represent 6 tissue-invasive strains; lanes 7–10 represent 4 nontissue-invasive strains. The 23S rRNA gene (upper panel), NTUH-K2044 (lane 11), and the PCR product of each probe (lane 12) served as positive controls. PTS, phosphoenolpyruvate sugar phosphotransferase system.
growth of the mutants was compared with that of the wild-type strain in nutrient-rich, undefined LB medium. However, no significant differences were found.
The effect on virulence of each mutation was investigated in a mouse model (figure 4B and 4C). All of the mice inoculated with 103
cfu survived. Half of the mice died when inoculated with 104
cfu of the wild-type strain, K2044 (DORF7–9), or K2044 (DPTS); there were no significant differences in survival among these 3 groups (K2044 [DORF7–9] vs. wild-type strain, P p ; K2044 [DPTS] vs. wild-type strain, ; log-rank test)
.76 P p .67
(figure 4C). In contrast, all of the mice inoculated with K2044 (Dkfu) at the doses of 104–106cfu survived and appeared to be
healthy after 4 weeks. The survival of the mice inoculated with K2044 (Dkfu) differed significantly from that of the mice inoc-ulated the wild-type strain (P p .0067, log-rank test) (figure 4C). When inoculated with the wild-type strain, K2044 (DORF7– 9), or K2044 (DPTS) at the highest dose (106cfu), most of the
mice had died by 7 days after infection, with no obvious signs before death and with no pathological changes detected on histological examination; they were considered to have died of septic shock. At lower inoculation doses (104–105 cfu), most
mice died between day 12 and 21 after infection, with signs of
lethargy, labored breathing, or trembling 1–2 days before death. Large liver and/or brain abscesses were observed in this group, and septic shock with possible organ failure was considered to be the cause of death. However, there was no histological change in the livers and brains obtained from the mice inoculated with K2044 (Dkfu) at doses of 104
–106
cfu (figure 5). Because the manifestations of K. pneumoniae infection observed in BALB/ cByl mice were very similar to those observed in patients, we did not try other murine strains.
Functional analysis of the kfu system in K. pneumoniae.
K. pneumoniae kfuABC was found to be preceded by a putative
19-bp Fur box consensus sequence (figure 6A). The 3 genes of
kfu were transcribed in the same direction and had short
in-tergenic sequences (maximum 21 bp between kfuA and kfuB), suggesting that they were in a single transcriptional unit. RT-PCR analysis confirmed that the 3 genes were transcribed as a single operon (figure 6B).
Comparison of the growth rates of the wild-type strain, K2044 (Dkfu), MGH 78578, and a noninvasive strain, N3423, in an iron-chelated medium revealed slightly lower growth for K2044 (Dkfu) and MGH 78578 (figure 6C). The E. coli aroB mutant carrying kfuABC or sfuABC grew well under conditions of iron
Table 5. Distribution of the 3 chromosomal regions among tissue-invasive Klebsiella pneumoniae strains, according to the clinical disease of the patients.
Disease, no. of isolates with distribution pattern
Distribution pattern 20-kb region (kfu/PTS) 22-kb region (allS) 33-kb region (magA) PLA (n p 33) 22 + + + 3 ⫺ + + 1 + ⫺ + 1 + ⫺ ⫺ 6 ⫺ ⫺ ⫺ Meningitis (n p 4) 2 + ⫺ + 2 ⫺ ⫺ ⫺
PLA plus endophthalmitis (n p 8) + + +
PLA plus meningitis (n p 1) + + +
NOTE. Results were confirmed by both polymerase chain reaction and slot-blot hybridization (table 1). PLA, primary liver abscess; PTS, phospho-enolpyruvate sugar phosphotransferase system; +, region present;⫺, region absent.
limitation. However, the growth of H1443 with vector only (pBR322) was significantly inhibited (figure 6D).
Prevalence and correlation of the kfu/PTS, magA, and allS regions in K. pneumoniae clinical isolates. We compared the presence of the kfu/PTS, magA, and allS regions by PCR. Figure 7A shows the location of the primers used in this analysis. The criteria used to detect the prevalences of the allS and kfu/PTS regions were the same, because they both replaced a small frag-ment in the noninvasive strains. The PCR results were in com-plete agreement with those of slot-blot hybridization (figure 7B). When the data were combined with those on the kfu/PTS region, the presence of these 3 regions was strongly associated with the clinically invasive strains more than with the clinically noninvasive strains (for the kfu/PTS region, 35/46 vs. 19/98, [P!.0001, x2test]; for the allS region, 33/46 vs. 3/98 [P!.0001,
x2test]; and for the magA region, 37/46 vs. 6/98 [P!
.0001, x2
test]). All 3 chromosomal regions correlated well with one an-other, with coincidence in 39 (31 positive and 8 negative for all 3 regions) of 46 isolates (85%) (table 5).
The distribution of the 3 chromosomal regions was further analyzed according to the clinical disease of the patients (table 5). Strains from the patients with PLA plus endophthalmitis or meningitis were positive for all 3 regions. However, when strains from the 4 patients with meningitis but without PLA were examined, 2 of the strains were found to be negative for all 3 regions, and 2 of the strains were found to be positive for the kfu/PTS and magA regions but not for the allS region.
DISCUSSION
In the present study, microarray comparison of the genomic DNA of 4 tissue-invasive K. pneumoniae strains and 3 non-invasive strains identified 12 clones with significantly different hybridization signals. Two clones that matched the sequence in MGH 78578 were probably the result of experimental var-iations, because both were present in the 2 groups of strains. Another 2 clones were located in a large plasmid. However, the prevalences of the large plasmid were invariant in the 2 groups of strains. The remaining 8 clones connected together and fur-ther extended to encompass a 20-kb fragment (table 3). Only the deletion mutant NTUH-K2044 (Dkfu) showed decreased vir-ulence in vivo. Functional studies confirmed the operon’s in-volvement in iron uptake. Because our array was estimated to cover only 88% of the entire K. pneumoniae genome, we could miss some regions specific to tissue-invasive strains; hence, the
allS and magA regions were not identified by this method.
The 20-kb kfu/PTS region was markedly more prevalent among tissue-invasive strains. The kfu/PTS region encoded an iron-uptake system involved in virulence and fit the size range (10–200 kb) of a pathogenicity island. However, the GC content of this region was similar to the remainder of the whole genome, and no mobile elements or insertion sequences were noted [42]. Thus, the fundamental nature of the heterogeneity of this spe-cific region in K. pneumoniae awaits further study [43].
Acquisition of nutrients such as iron to sustain growth in the host environment is essential for bacterial pathogens to establish an infection. Iron uptake is also critical to pathogenesis as a vital cofactor for many components of microbial antiox-idative stress defense, including such components as superoxide dismutase, catalase, and peroxidase [44]. We have shown here that the kfu operon was present in most of the genomes of the tissue-invasive K. pneumoniae strains we examined and was absent from most of the genomes of the noninvasive strains. It is possible that the presence of a functional kfu operon might principally or secondarily modulate virulence in vivo and so provide a strong competitive advantage to those strains that harbor it.
There was a significant difference in the prevalence of the
kfu/PTS region between noninvasive control strains from NTUH
and FEMH. Strains from FEMH were all isolated from patients with nosocomial infection, whereas strains from NTUH were all isolated from patients with community-acquired infection. This could be the reason for the genetic difference. In addition, there would be an epidemiological difference between strains from a medical center (NTUH) and a those from a community hospital (FEMH).
How K. pneumoniae enter into the bloodstream and liver has not yet been documented. However, bacterial cells would enter the bloodstream through M cells [45] or as a result of
minor mucosal injuries of the gastrointestinal tract. Larger ab-scesses were found in the mice infected via ig inoculation, whereas, in the mice infected via ip inoculation, microabscesses were found. This finding supports the hypothesis that K.
pneu-moniae gains entry into the bloodstream via the gastrointestinal
tract, because all venous returns in the gastrointestinal tract were collected via a portal vein into liver. Many bacteria were likely trapped in the liver by Kupffer cells; however, the tissue-invasive strains were resistant to phagocytosis and serum killing. This may result in the subsequent formation of liver abscesses. The present study reinforces our awareness of the vital role that bacterial virulence factors play in the pathogenesis of PLA and metastatic infection. According to the prevalences of the 3 specific chromosomal regions in tissue-invasive strains, we were able to classify the 54 invasive strains into 3 groups: group 1 strains contained all 3 regions, group 2 strains contained 1– 2 regions, and group 3 strains contained none of the regions. When all 3 regions were absent, the strains were only occa-sionally capable of causing PLA. The most interesting finding was that all of the strains from patients with PLA plus metastatic infection contained all 3 regions. Most of the group 3 strains were noninvasive (table 1), suggesting that they might be less virulent than the group 1 strains. That the group 1 strains had an increased ability to invade tissue may well relate to their enhanced survival and ability to compete for nutrients. The
kfu/PTS region could enrich the ability of bacteria to secure
iron, even in the relatively iron-deficient conditions of the hu-man host. The allS region could help bacteria to compete for nitrogen sources via the allantoin-utilizing ability [15]. More-over, the magA region could render bacteria resistant to phago-cytosis by polymorphonuclear leukocytes and serum killing [16]. Therefore, the group 1 strains might be classified as a specific genotype that is associated with PLA and metastatic infection. Two strains from 4 patients with meningitis but without PLA were negative for all 3 regions, and the remaining 2 strains were negative for the allS region. These results suggest that different pathogenic mechanisms could be at work in patients with men-ingitis plus PLA and in those with menmen-ingitis only.
In conclusion, we have identified a 20-kb chromosomal kfu/ PTS region in K. pneumoniae that is associated with iron ac-quisition and virulence. This region is more prevalent in tissue-invasive strains. Strains containing the kfu/PTS, allS, and magA regions are strongly associated with PLA and metastatic infec-tion and may be classified as a specific genotype. However, a different virulence mechanism seems to be at work in patients with meningitis but without PLA. Therefore, the kfu/PTS re-gion, as well as the allS and magA regions, can be exploited as a genetic marker for rapid molecular diagnosis and for tracing the source of these emergent tissue-invasive strains.
Acknowledgment
We thank Dr. Volkmar Braun, Universitat Tubingen (Tubingen, Ger-many), for providing Escherichia coli strain H1443 and pSZ1 plasmid.
References
1. Podschun R, Ullmann U. Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev 1998; 11:589–603.
2. Chang SC, Fang CT, Hsueh PR, Chen YC, Luh KT. Klebsiella
pneu-moniae isolates causing liver abscess in Taiwan. Diagn Microbiol Infect
Dis 2000; 37:279–84.
3. Cheng DL, Liu YC, Yen MY, Liu CY, Wang RS. Septic metastatic lesions of pyogenic liver abscess: their association with Klebsiella pneumoniae bacteremia in diabetic patients. Arch Intern Med 1991; 151:1557–9. 4. Chiu CT, Lin DY, Liaw YF. Metastatic septic endophthalmitis in
pyo-genic liver abscess. J Clin Gastroenterol 1988; 10:524–7.
5. Fung CP, Chang FY, Lee SC, et al. A global emerging disease of Klebsiella
pneumoniae liver abscess: is serotype K1 an important factor for
com-plicated endophthalmitis? Gut 2002; 50:420–4.
6. Ko WC, Paterson DL, Sagnimeni AJ, et al. Community-acquired
Kleb-siella pneumoniae bacteremia: global differences in clinical patterns.
Emerg Infect Dis 2002; 8:160–6.
7. Liu YC, Cheng DL, Lin CL. Klebsiella pneumoniae liver abscess associ-ated with septic endophthalmitis. Arch Intern Med 1986; 146:1913–6. 8. Wang JH, Liu YC, Lee SS, et al. Primary liver abscess due to Klebsiella
pneumoniae in Taiwan. Clin Infect Dis 1998; 26:1434–8.
9. Saccente M. Klebsiella pneumoniae liver abscess, endophthalmitis, and meningitis in a man with newly recognized diabetes mellitus. Clin In-fect Dis 1999; 29:1570–1.
10. Cahill M, Chang B, Murray A. Bilateral endogenous bacterial endo-phthalmitis associated with pyogenic hepatic abscess [letter]. Br J Oph-thalmol 2000; 84:1436.
11. Casanova C, Lorente JA, Carrillo F, Perez-Rodriguez E, Nunez N.
Kleb-siella pneumoniae liver abscess associated with septic endophthalmitis
[letter]. Arch Intern Med 1989; 149:1467.
12. Lee KH, Hui KP, Tan WC, Lim TK. Klebsiella bacteraemia: a report of 101 cases from National University Hospital, Singapore. J Hosp In-fect 1994; 27:299–305.
13. Ohmori S, Shiraki K, Ito K, et al. Septic endophthalmitis and meningitis associated with Klebsiella pneumoniae liver abscess. Hepatol Res 2002; 22:307–12.
14. Assantachai P, Luengrojanakul P, Phimolsarnti R, Charoenlarp K, Ra-tanarapee S. Liver abscess in polycystic liver disease. J Med Assoc Thai 1995; 78:210–6.
15. Chou HC, Lee CZ, Ma LC, Fang CT, Chang SC, Wang JT. Isolation of a chromosomal region of Klebsiella pneumoniae associated with allantoin metabolism and liver infection. Infect Immun 2004; 72:3783–92. 16. Fang CT, Chuang YP, Shun CT, Chang SC, Wang JT. A novel virulence
gene in Klebsiella pneumoniae strains causing primary liver abscess and septic metastatic complications. J Exp Med 2004; 199:697–705. 17. Fitzgerald JR, Musser JM. Evolutionary genomics of pathogenic
bac-teria. Trends Microbiol 2001; 9:547–53.
18. Kim CC, Joyce EA, Chan K, Falkow S. Improved analytical methods for microarray-based genome-composition analysis. Genome Biol 2002; 3:RESEARCH0065.
19. Abbott SL. Klebisella, Enterobacter, Citrobacter, Serratia, Plesiomonas, and other Enterobacteriaceae. In: Murray PR, Baron EJ, Jorgensen JH, Pfaller M, Yolken RH, eds. Manual of clinical microbiology. 8th ed. Washington, DC: American Society for Microbiology, 2003:684–700. 20. Fang CT, Chen HC, Chuang YP, Chang SC, Wang JT. Cloning of a
cation efflux pump gene associated with chlorhexidine resistance in
Klebsiella pneumoniae. Antimicrob Agents Chemother 2002; 46:2024–8.
iso-lating differentially expressed genes by cDNA microarray system with colorimetry detection. Genomics 1998; 51:313–24.
22. Ang S, Lee CZ, Peck K, et al. Acid-induced gene expression in
Heli-cobacter pylori: study in genomic scale by microarray. Infect Immun 2001; 69:1679–86.
23. Herrero M, de Lorenzo V, Timmis KN. Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chro-mosomal insertion of foreign genes in gram-negative bacteria. J Bac-teriol 1990; 172:6557–67.
24. Zimmermann L, Angerer A, Braun V. Mechanistically novel iron (III) transport system in Serratia marcescens. J Bacteriol 1989; 171:238–43. 25. Angerer A, Klupp B, Braun V. Iron transport systems of Serratia
mar-cescens. J Bacteriol 1992; 174:1378–87.
26. Okamoto M, Nakane A, Minagawa T. Host resistance to an intragastric infection with Listeria monocytogenes in mice depends on cellular im-munity and intestinal bacterial flora. Infect Immun 1994; 62:3080–5. 27. Reed LJ, Muench H. A simple method of estimating fifty percent
end-points. Am J Hyg 1938; 27:493–7.
28. Winnacker E-L. Genomic libraries. In: Weller MG, ed. From genes to clones: introduction to gene technology. Weinheim, Germany: VCH Verlagsgesellschaft mbH, 1987:383–5.
29. Chen YT, Chang HY, Lai YC, Pan CC, Tsai SF, Peng HL. Sequencing and analysis of the large virulence plasmid pLVPK of Klebsiella
pneu-moniae CG43. Gene 2004; 337:189–98.
30. Tomii K, Kanehisa M. A comparative analysis of ABC transporters in complete microbial genomes. Genome Res 1998; 8:1048–59. 31. Angerer A, Gaisser S, Braun V. Nucleotide sequences of the sfuA, sfuB,
and sfuC genes of Serratia marcescens suggest a periplasmic-binding-protein-dependent iron transport mechanism. J Bacteriol 1990; 172: 572–8.
32. Adhikari P, Kirby SD, Nowalk AJ, Veraldi KL, Schryvers AB, Mietzner TA. Biochemical characterization of a Haemophilus influenzae peri-plasmic iron transport operon. J Biol Chem 1995; 270:25142–9. 33. Gong S, Bearden SW, Geoffroy VA, Fetherston JD, Perry RD.
Char-acterization of the Yersinia pestis Yfu ABC inorganic iron transport system. Infect Immun 2001; 69:2829–37.
34. Chin N, Frey J, Chang CF, Chang YF. Identification of a locus involved in the utilization of iron by Actinobacillus pleuropneumoniae. FEMS Microbiol Lett 1996; 143:1–6.
35. Adhikari P, Berish SA, Nowalk AJ, Veraldi KL, Morse SA, Mietzner TA. The fbpABC locus of Neisseria gonorrhoeae functions in the per-iplasm-to-cytosol transport of iron. J Bacteriol 1996; 178:2145–9. 36. Mao Y, Varoglu M, Sherman DH. Molecular characterization and
anal-ysis of the biosynthetic gene cluster for the antitumor antibiotic mi-tomycin C from Streptomyces lavendulae NRRL 2564. Chem Biol 1999; 6:251–63.
37. Postma PW, Lengeler JW, Jacobson GR. Phosphoenolpyruvate:car-bohydrate phosphotransferase systems of bacteria. Microbiol Rev 1993; 57:543–94.
38. Kok M, Bron G, Erni B, Mukhija S. Effect of enzyme I of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) on vir-ulence in a murine model. Microbiology 2003; 149:2645–52. 39. Tan MW, Rahme LG, Sternberg JA, Tompkins RG, Ausubel FM.
Pseu-domonas aeruginosa killing of Caenorhabditis elegans used to identify P. aeruginosa virulence factors. Proc Natl Acad Sci USA 1999; 96:2408–13.
40. Turner AK, Lovell MA, Hulme SD, Zhang-Barber L, Barrow PA. Iden-tification of Salmonella typhimurium genes required for colonization of the chicken alimentary tract and for virulence in newly hatched chicks. Infect Immun 1998; 66:2099–106.
41. Escolar L, Perez-Martin J, de Lorenzo V. Opening the iron box: tran-scriptional metalloregulation by the Fur protein. J Bacteriol 1999; 181: 6223–9.
42. Hacker J, Kaper JB. Pathogenicity islands and the evolution of mi-crobes. Annu Rev Microbiol 2000; 54:641–79.
43. Lai YC, Yang SL, Peng HL, Chang HY. Identification of genes present specifically in a virulent strain of Klebsiella pneumoniae. Infect Immun 2000; 68:7149–51.
44. Griffiths E. The iron-uptake systems of pathogenic bacteria. In: Bullen JJ, Griffiths E, eds. Iron and infection. New York: John Wiley & Sons, 1987:69–137.
45. Lu L, Walker WA. Pathologic and physiologic interactions of bacteria with the gastrointestinal epithelium. Am J Clin Nutr 2001; 73:S1124–30.
