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1.  First Case of Liver Abscess in Scandinavia Due to the International Hypervirulent Klebsiella Pneumoniae Clone ST23 
This is the first case report from Scandinavia of a pyogenic liver abscess caused by a Klebsiella pneumoniae isolate belonging to the international hyper virulent clone ST23. The patient, an 85-year old Caucasian, had no history of foreign travel or any classical predisposing factors for infection. The isolate was hypermucoviscous of capsular serotype K1 and carried the virulence factors aerobactin, allS, kfu and rmpA.
doi:10.2174/1874285801408010022
PMCID: PMC3968548
Klebsiella pneumoniae; liver abscess; ST23.
2.  Prevalence and Characteristics of the Epidemic Multiresistant Escherichia coli ST131 Clonal Group among Extended-Spectrum Beta-Lactamase-Producing E. coli Isolates in Copenhagen, Denmark 
Journal of Clinical Microbiology  2013;51(6):1779-1785.
We report the characteristics of 115 extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli clinical isolates, from 115 unique Danish patients, over a 1-year study interval (1 October 2008 to 30 September 2009). Forty-four (38%) of the ESBL isolates represented sequence type 131 (ST13)1, from phylogenetic group B2. The remaining 71 isolates were from phylogenetic groups D (27%), A (22%), B1 (10%), and B2 (3%). Serogroup O25 ST131 isolates (n = 42; 95% of ST131) comprised 7 different K antigens, whereas two ST131 isolates were O16:K100:H5. Compared to non-ST131 isolates, ST131 isolates were associated positively with CTX-M-15 and negatively with CTX-M-1 and CTX-M-14. They also were associated positively with 11 virulence genes, including afa and dra (Dr family adhesins), the F10 papA allele (P fimbria variant), fimH (type 1 fimbriae), fyuA (yersiniabactin receptor), iha (adhesin siderophore), iutA (aerobactin receptor), kpsM II (group 2 capsules), malX (pathogenicity island marker), ompT (outer membrane protease), sat (secreted autotransporter toxin), and usp (uropathogenicity-specific protein) and negatively with hra (heat-resistant agglutinin) and iroN (salmochelin receptor). The consensus virulence gene profile (>90% prevalence) of the ST131 isolates included fimH, fyuA, malX, and usp (100% each), ompT and the F10 papA allele (95% each), and kpsM II and iutA (93% each). ST131 isolates were also positively associated with community acquisition, extraintestinal pathogenic E. coli (ExPEC) status, and the O25, K100, and H4 antigens. Thus, among ESBL E. coli isolates in Copenhagen, ST131 was the most prevalent clonal group, was community associated, and exhibited distinctive and comparatively extensive virulence profiles, plus a greater variety of capsular antigens than reported previously.
doi:10.1128/JCM.00346-13
PMCID: PMC3716056  PMID: 23554186
3.  Role of Enteroaggregative Escherichia coli Virulence Factors in Uropathogenesis 
Infection and Immunity  2013;81(4):1164-1171.
A multiresistant clonal Escherichia coli O78:H10 strain qualifying molecularly as enteroaggregative Escherichia coli (EAEC) was recently shown to be the cause of a community-acquired outbreak of urinary tract infection (UTI) in greater Copenhagen, Denmark, in 1991. This marks the first time EAEC has been associated with an extraintestinal disease outbreak. Importantly, the outbreak isolates were recovered from the urine of patients with symptomatic UTI, strongly implying urovirulence. Here, we sought to determine the uropathogenic properties of the Copenhagen outbreak strain and whether these properties are conferred by the EAEC-specific virulence factors. We demonstrated that through expression of aggregative adherence fimbriae, the principal adhesins of EAEC, the outbreak strain exhibited pronouncedly increased adherence to human bladder epithelial cells compared to prototype uropathogenic strains. Moreover, the strain was able to produce distinct biofilms on abiotic surfaces, including urethral catheters. These findings suggest that EAEC-specific virulence factors increase uropathogenicity and may have played a significant role in the ability of the strain to cause a community-acquired outbreak of UTI. Thus, inclusion of EAEC-specific virulence factors is warranted in future detection and characterization of uropathogenic E. coli.
doi:10.1128/IAI.01376-12
PMCID: PMC3639612  PMID: 23357383
4.  The Fimbriae of Enteroaggregative Escherichia coli Induce Epithelial Inflammation In Vitro and in a Human Intestinal Xenograft Model  
The Journal of Infectious Diseases  2012;206(5):714-722.
Background.Enteroaggregative Escherichia coli (EAEC) are increasingly recognized as an important agent of inflammatory and often persistent diarrhea. Although previous studies report on the inflammatory aspects of EAEC pathogenesis, the mechanisms by which EAEC trigger these events are not well understood.
Methods.EAEC strains harboring mutations in known EAEC virulence determinants were tested in an in vitro model of transepithelial migration of polymorphonuclear neutrophils (PMNs) and in human intestinal xenografts in severe-combined immunodeficient (SCID-HU-INT) mice, a novel model for studying EAEC disease in vivo.
Results.Expression of aggregative adherence fimbriae (AAFs), the principal adhesins of EAEC, was required for EAEC-induced PMN transepithelial migration in vitro. Moreover, constructed plasmids encoding AAF gene clusters demonstrated that the AAF adhesins are sufficient for triggering this event in a nonpathogenic E. coli background. Furthermore, with use of the SCID-HU-INT mouse model, severe tissue damage and infiltration of inflammatory cells was observed in the human tissue after EAEC infection. These pathological marks were strongly related to AAF expression, thus clearly confirming our in vitro findings.
Conclusions.The present work establishes EAEC as an important inflammatory pathogen and the AAF adhesins as inducers of potentially detrimental immune responses.
doi:10.1093/infdis/jis417
PMCID: PMC3491746  PMID: 22723643
5.  The Alkaloid Compound Harmane Increases the Lifespan of Caenorhabditis elegans during Bacterial Infection, by Modulating the Nematode’s Innate Immune Response 
PLoS ONE  2013;8(3):e60519.
The nematode Caenorhabditis elegans has in recent years been proven to be a powerful in vivo model for testing antimicrobial compounds. We report here that the alkaloid compound Harmane (2-methyl-β-carboline) increases the lifespan of nematodes infected with a human pathogen, the Shiga toxin-producing Escherichia coli O157:H7 strain EDL933 and several other bacterial pathogens. This was shown to be unrelated to the weak antibiotic effect of Harmane. Using GFP-expressing E. coli EDL933, we showed that Harmane does not lower the colonization burden in the nematodes. We also found that the expression of the putative immune effector gene F35E12.5 was up-regulated in response to Harmane treatment. This indicates that Harmane stimulates the innate immune response of the nematode; thereby increasing its lifespan during bacterial infection. Expression of F35E12.5 is predominantly regulated through the p38 MAPK pathway; however, intriguingly the lifespan extension resulting from Harmane was higher in p38 MAPK-deficient nematodes. This indicates that Harmane has a complex effect on the innate immune system of C. elegans. Harmane could therefore be a useful tool in the further research into C. elegans immunity. Since the innate immunity of C. elegans has a high degree of evolutionary conservation, drugs such as Harmane could also be possible alternatives to classic antibiotics. The C. elegans model could prove to be useful for selection and development of such drugs.
doi:10.1371/journal.pone.0060519
PMCID: PMC3609739  PMID: 23544153
6.  Novel screening assay for in vivo selection of Klebsiella pneumoniae genes promoting gastrointestinal colonisation 
BMC Microbiology  2012;12:201.
Background
Klebsiella pneumoniae is an important opportunistic pathogen causing pneumonia, sepsis and urinary tract infections. Colonisation of the gastrointestinal (GI) tract is a key step in the development of infections; yet the specific factors important for K. pneumoniae to colonize and reside in the GI tract of the host are largely unknown. To identify K. pneumoniae genes promoting GI colonisation, a novel genomic-library-based approach was employed.
Results
Screening of a K. pneumoniae C3091 genomic library, expressed in E. coli strain EPI100, in a mouse model of GI colonisation led to the positive selection of five clones containing genes promoting persistent colonisation of the mouse GI tract. These included genes encoding the global response regulator ArcA; GalET of the galactose operon; and a cluster of two putative membrane-associated proteins of unknown function. Both ArcA and GalET are known to be involved in metabolic pathways in Klebsiella but may have additional biological actions beneficial to the pathogen. In support of this, GalET was found to confer decreased bile salt sensitivity to EPI100.
Conclusions
The present work establishes the use of genomic-library-based in vivo screening assays as a valuable tool for identification and characterization of virulence factors in K. pneumoniae and other bacterial pathogens.
doi:10.1186/1471-2180-12-201
PMCID: PMC3463446  PMID: 22967317
Klebsiella pneumoniae; Genomic library; Mouse model of gastrointestinal colonisation
7.  Characterization of a novel chaperone/usher fimbrial operon present on KpGI-5, a methionine tRNA gene-associated genomic island in Klebsiella pneumoniae 
BMC Microbiology  2012;12:59.
Background
Several strain-specific Klebsiella pneumoniae virulence determinants have been described, though these have almost exclusively been linked with hypervirulent liver abscess-associated strains. Through PCR interrogation of integration hotspots, chromosome walking, island-tagging and fosmid-based marker rescue we captured and sequenced KpGI-5, a novel genomic island integrated into the met56 tRNA gene of K. pneumoniae KR116, a bloodstream isolate from a patient with pneumonia and neutropenic sepsis.
Results
The 14.0 kb KpGI-5 island exhibited a genome-anomalous G + C content, possessed near-perfect 46 bp direct repeats, encoded a γ1-chaperone/usher fimbrial cluster (fim2) and harboured seven other predicted genes of unknown function. Transcriptional analysis demonstrated expression of three fim2 genes, and suggested that the fim2A-fim2K cluster comprised an operon. As fimbrial systems are frequently implicated in pathogenesis, we examined the role of fim2 by analysing KR2107, a streptomycin-resistant derivative of KR116, and three isogenic mutants (Δfim, Δfim2 and ΔfimΔfim2) using biofilm assays, human cell adhesion assays and pair-wise competition-based murine models of intestinal colonization, lung infection and ascending urinary tract infection. Although no statistically significant role for fim2 was demonstrable, liver and kidney CFU counts for lung and urinary tract infection models, respectively, hinted at an ordered gradation of virulence: KR2107 (most virulent), KR2107∆fim2, KR2107∆fim and KR2107∆fim∆fim2 (least virulent). Thus, despite lack of statistical evidence there was a suggestion that fim and fim2 contribute additively to virulence in these murine infection models. However, further studies would be necessary to substantiate this hypothesis.
Conclusion
Although fim2 was present in 13% of Klebsiella spp. strains investigated, no obvious in vitro or in vivo role for the locus was identified, although there were subtle hints of involvement in urovirulence and bacterial dissemination from the respiratory tract. Based on our findings and on parallels with other fimbrial systems, we propose that fim2 has the potential to contribute beneficially to pathogenesis and/or environmental persistence of Klebsiella strains, at least under specific yet-to-be identified conditions.
doi:10.1186/1471-2180-12-59
PMCID: PMC3419637  PMID: 22520965
8.  Origins of the E. coli Strain Causing an Outbreak of Hemolytic–Uremic Syndrome in Germany 
The New England journal of medicine  2011;365(8):709-717.
BACKGROUND
A large outbreak of diarrhea and the hemolytic–uremic syndrome caused by an unusual serotype of Shiga-toxin–producing Escherichia coli (O104:H4) began in Germany in May 2011. As of July 22, a large number of cases of diarrhea caused by Shiga-toxin–producing E. coli have been reported — 3167 without the hemolytic–uremic syndrome (16 deaths) and 908 with the hemolytic–uremic syndrome (34 deaths) — indicating that this strain is notably more virulent than most of the Shiga-toxin–producing E. coli strains. Preliminary genetic characterization of the outbreak strain suggested that, unlike most of these strains, it should be classified within the enteroaggregative pathotype of E. coli.
METHODS
We used third-generation, single-molecule, real-time DNA sequencing to determine the complete genome sequence of the German outbreak strain, as well as the genome sequences of seven diarrhea-associated enteroaggregative E. coli serotype O104:H4 strains from Africa and four enteroaggregative E. coli reference strains belonging to other serotypes. Genomewide comparisons were performed with the use of these enteroaggregative E. coli genomes, as well as those of 40 previously sequenced E. coli isolates.
RESULTS
The enteroaggregative E. coli O104:H4 strains are closely related and form a distinct clade among E. coli and enteroaggregative E. coli strains. However, the genome of the German outbreak strain can be distinguished from those of other O104:H4 strains because it contains a prophage encoding Shiga toxin 2 and a distinct set of additional virulence and antibiotic-resistance factors.
CONCLUSIONS
Our findings suggest that horizontal genetic exchange allowed for the emergence of the highly virulent Shiga-toxin–producing enteroaggregative E. coli O104:H4 strain that caused the German outbreak. More broadly, these findings highlight the way in which the plasticity of bacterial genomes facilitates the emergence of new pathogens.
doi:10.1056/NEJMoa1106920
PMCID: PMC3168948  PMID: 21793740
9.  Heat Resistance Mediated by a New Plasmid Encoded Clp ATPase, ClpK, as a Possible Novel Mechanism for Nosocomial Persistence of Klebsiella pneumoniae 
PLoS ONE  2010;5(11):e15467.
Klebsiella pneumoniae is an important opportunistic pathogen and a frequent cause of nosocomial infections. We have characterized a K. pneumoniae strain responsible for a series of critical infections in an intensive care unit over a two-year period. The strain was found to be remarkably thermotolerant providing a conceivable explanation of its persistence in the hospital environment. This marked phenotype is mediated by a novel type of Clp ATPase, designated ClpK. The clpK gene is encoded by a conjugative plasmid and we find that the clpK gene alone renders an otherwise sensitive E. coli strain resistant to lethal heat shock. Furthermore, one third of a collection of nosocomial K. pneumoniae isolates carry clpK and exhibit a heat resistant phenotype. The discovery of ClpK as a plasmid encoded factor and its profound impact on thermal stress survival sheds new light on the biological relevance of Clp ATPases in acquired environmental fitness and highlights the challenges of mobile genetic elements in fighting nosocomial infections.
doi:10.1371/journal.pone.0015467
PMCID: PMC2976762  PMID: 21085699
10.  Role of type 1 and type 3 fimbriae in Klebsiella pneumoniae biofilm formation 
BMC Microbiology  2010;10:179.
Background
Klebsiella pneumoniae is an important gram-negative opportunistic pathogen causing primarily urinary tract infections, respiratory infections, and bacteraemia. The ability of bacteria to form biofilms on medical devices, e.g. catheters, has a major role in development of many nosocomial infections. Most clinical K. pneumoniae isolates express two types of fimbrial adhesins, type 1 fimbriae and type 3 fimbriae. In this study, we characterized the role of type 1 and type 3 fimbriae in K. pneumoniae biofilm formation.
Results
Isogenic fimbriae mutants of the clinical K. pneumoniae isolate C3091 were constructed, and their ability to form biofilm was investigated in a flow cell system by confocal scanning laser microscopy. The wild type strain was found to form characteristic biofilm and development of K. pneumoniae biofilm occurred primarily by clonal growth, not by recruitment of planktonic cells. Type 1 fimbriae did not influence biofilm formation and the expression of type 1 fimbriae was found to be down-regulated in biofilm forming cells. In contrast, expression of type 3 fimbriae was found to strongly promote biofilm formation.
Conclusion
By use of well defined isogenic mutants we found that type 3 fimbriae, but not type 1 fimbriae, strongly promote biofilm formation in K. pneumoniae C3091. As the vast majority of clinical K. pneumoniae isolates express type 3 fimbriae, this fimbrial adhesin may play a significant role in development of catheter associated K. pneumoniae infections.
doi:10.1186/1471-2180-10-179
PMCID: PMC2911432  PMID: 20573190
11.  Identification of a Conserved Chromosomal Region Encoding Klebsiella pneumoniae Type 1 and Type 3 Fimbriae and Assessment of the Role of Fimbriae in Pathogenicity▿  
Infection and Immunity  2009;77(11):5016-5024.
Type 3 fimbriae are expressed by most clinical Klebsiella pneumoniae isolates and mediate adhesion to host structures in vitro. However, the role of type 3 fimbriae in K. pneumoniae virulence has not been evaluated by use of in vivo infection models. In this study, the type 3 fimbrial gene cluster (mrk) of the clinical isolate C3091 is described in detail. The mrk gene cluster was revealed to be localized in close proximity to the type 1 fimbrial gene cluster. Thus, a 20.4-kb fimbria-encoding region was identified and found to be highly conserved among different K. pneumoniae isolates. Interestingly, a homologue to PecS, known as a global regulator of virulence in Erwinia chrysanthemi, was identified in the fimbria-encoding region. Comparison to the previously characterized plasmid encoded mrk gene cluster revealed significant differences, and it is established here that the putative regulatory gene mrkE is not a part of the chromosomally encoded type 3 fimbrial gene cluster. To evaluate the role of type 3 fimbriae in virulence, a type 3 fimbria mutant and a type 1 and type 3 fimbria double mutant was constructed. Type 3 fimbria expression was found to strongly promote biofilm formation. However, the fimbria mutants were as effective at colonizing the intestine as the wild type, and their virulence was not attenuated in a lung infection model. Also, in a urinary tract infection model, type 3 fimbriae did not influence the virulence, whereas type 1 fimbriae were verified as an essential virulence factor. Thus, type 3 fimbriae were established not to be a virulence factor in uncomplicated K. pneumoniae infections. However, since type 3 fimbriae promote biofilm formation, their role in development of infections in catheterized patients needs to be elucidated.
doi:10.1128/IAI.00585-09
PMCID: PMC2772557  PMID: 19703972
12.  Comparative Structure-Function Analysis of Mannose-Specific FimH Adhesins from Klebsiella pneumoniae and Escherichia coli▿  
Journal of Bacteriology  2009;191(21):6592-6601.
FimH, the adhesive subunit of type 1 fimbriae expressed by many enterobacteria, mediates mannose-sensitive binding to target host cells. At the same time, fine receptor-structural specificities of FimH from different species can be substantially different, affecting bacterial tissue tropism and, as a result, the role of the particular fimbriae in pathogenesis. In this study, we compared functional properties of the FimH proteins from Escherichia coli and Klebsiella pneumoniae, which are both 279 amino acids in length but differ by some ∼15% of residues. We show that K. pneumoniae FimH is unable to mediate adhesion in a monomannose-specific manner via terminally exposed Manα(1-2) residues in N-linked oligosaccharides, which are the structural basis of the tropism of E. coli FimH for uroepithelial cells. However, K. pneumoniae FimH can bind to the terminally exposed Manα(1-3)Manβ(1-4)GlcNAcβ1 trisaccharide, though only in a shear-dependent manner, wherein the binding is marginal at low shear force but enhanced sevenfold under increased shear. A single mutation in the K. pneumoniae FimH, S62A, converts the mode of binding from shear dependent to shear independent. This mutation has occurred naturally in the course of endemic circulation of a nosocomial uropathogenic clone and is identical to a pathogenicity-adaptive mutation found in highly virulent uropathogenic strains of E. coli, in which it also eliminates the dependence of E. coli binding on shear. The shear-dependent binding properties of the K. pneumoniae and E. coli FimH proteins are mediated via an allosteric catch bond mechanism. Thus, despite differences in FimH structure and fine receptor specificity, the shear-dependent nature of FimH-mediated adhesion is highly conserved between bacterial species, supporting its remarkable physiological significance.
doi:10.1128/JB.00786-09
PMCID: PMC2795292  PMID: 19734306
13.  Primary Klebsiella pneumoniae Liver Abscess with Metastatic Spread to Lung and Eye, a North-European Case Report of an Emerging Syndrome 
A syndrome of community acquired liver abscess caused by highly virulent hypermucoviscous, rmpA positive Klebsiella pneumoniae strains occurs with high incidence in Asia. We here report a case complicated by metastatic infection, to our knowledge, the first one described in Northern Europe. This and other recently reported cases indicate the emergence of this severe syndrome outside of Asia.
doi:10.2174/1874285801004010005
PMCID: PMC2864426  PMID: 20448814
Klebsiella pneumoniae; metastatic infection; hypermucoviscous; rmpA.
14.  Population Variability of the FimH Type 1 Fimbrial Adhesin in Klebsiella pneumoniae▿  
Journal of Bacteriology  2009;191(6):1941-1950.
FimH is an adhesive subunit of type 1 fimbriae expressed by different enterobacterial species. The enteric bacterium Klebsiella pneumoniae is an environmental organism that is also a frequent cause of sepsis, urinary tract infection (UTI), and liver abscess. Type 1 fimbriae have been shown to be critical for the ability of K. pneumoniae to cause UTI in a murine model. We show here that the K. pneumoniae fimH gene is found in 90% of strains from various environmental and clinical sources. The fimH alleles exhibit relatively low nucleotide and structural diversity but are prone to frequent horizontal-transfer events between different bacterial clones. Addition of the fimH locus to multiple-locus sequence typing significantly improved the resolution of the clonal structure of pathogenic strains, including the K1 encapsulated liver isolates. In addition, the K. pneumoniae FimH protein is targeted by adaptive point mutations, though not to the same extent as FimH from uropathogenic Escherichia coli or TonB from the same K. pneumoniae strains. Such adaptive mutations include a single amino acid deletion from the signal peptide that might affect the length of the fimbrial rod by affecting FimH translocation into the periplasm. Another FimH mutation (S62A) occurred in the course of endemic circulation of a nosocomial uropathogenic clone of K. pneumoniae. This mutation is identical to one found in a highly virulent uropathogenic strain of E. coli, suggesting that the FimH mutations are pathoadaptive in nature. Considering the abundance of type 1 fimbriae in Enterobacteriaceae, our present finding that fimH genes are subject to adaptive microevolution substantiates the importance of type 1 fimbria-mediated adhesion in K. pneumoniae.
doi:10.1128/JB.00601-08
PMCID: PMC2648365  PMID: 19151141
15.  Characterization of Klebsiella pneumoniae Type 1 Fimbriae by Detection of Phase Variation during Colonization and Infection and Impact on Virulence▿  
Infection and Immunity  2008;76(9):4055-4065.
Klebsiella pneumoniae is recognized as an important gram-negative opportunistic pathogen. The ability of bacteria to adhere to host structures is considered essential for the development of infections; however, few studies have examined the influence of adhesion factors on K. pneumoniae virulence. In this study, we cloned and characterized the type 1 fimbria gene cluster of a clinical K. pneumoniae isolate. Although this cluster was not identical to the Escherichia coli type 1 fimbria gene cluster, an overall high degree of structural resemblance was demonstrated. Unique to the K. pneumoniae fim gene cluster is the fimK gene, whose product contains an EAL domain, suggesting that it has a role in regulation of fimbrial expression. Like expression of type 1 fimbriae in E. coli, expression of type 1 fimbriae in K. pneumoniae was found to be phase variable, and an invertible DNA element (fim switch) was characterized. An isogenic type 1 fimbria mutant was constructed and used to evaluate the influence of type 1 fimbriae in different infection models. Type 1 fimbriae did not influence the ability of K. pneumoniae to colonize the intestine or infect the lungs, but they were determined to be a significant virulence factor in K. pneumoniae urinary tract infection. By use of a PCR-based assay, the orientation of the fim switch during colonization and infection was investigated and was found to be all “off” in the intestine and lungs but all “on” in the urinary tract. Our results suggest that during colonization and infection, there is pronounced selective pressure in different host environments for selection of either the type 1 fimbriated or nonfimbriated phenotype of K. pneumoniae.
doi:10.1128/IAI.00494-08
PMCID: PMC2519443  PMID: 18559432
16.  New Adhesin of Enteroaggregative Escherichia coli Related to the Afa/Dr/AAF Family▿  
Infection and Immunity  2008;76(7):3281-3292.
Enteroaggregative Escherichia coli (EAEC) is an important cause of diarrhea worldwide. We analyzed 17 Danish EAEC strains, isolated in the course of a case control study, for phenotypic and genotypic properties. The strains belonged to at least 14 different serotypes. Using PCR to investigate the prevalence of various putative virulence genes, we found that all but two strains were typical EAEC, as they harbored all or part of the previously described AggR regulon. The majority of the strains harbored genes encoding aggregative adherence fimbriae (AAF). The most common was AAF/I, found in nine strains; eight strains carried no known AAF-related genes. We utilized TnphoA mutagenesis to localize the aggregative adherence (AA) adhesin from one typical EAEC strain, C1010-00, which lacked a known AAF. We identified a TnphoA insertion in a hypothetical Dr-related pilin deposited in GenBank as HdaA. Four additional Danish strains harbored HdaA, and all but one displayed AA to HEp-2 cells. By using PCR primers derived from the pilins and ushers from the three AAF and Hda, we found that 16 of 17 strains exhibited evidence of one of these factors; importantly, the one negative strain also lacked the aggR gene. Cloning of the complete Hda gene cluster and expression in E. coli DH5α resulted in AA and complementation of the C1010-00 nonadherent mutant. Four related adhesins have now been found to confer AA in typical EAEC strains; our data suggest that, together, these variants may account for AA in the large majority of strains.
doi:10.1128/IAI.01646-07
PMCID: PMC2446688  PMID: 18443096
17.  Glycogen and Maltose Utilization by Escherichia coli O157:H7 in the Mouse Intestine▿  
Infection and Immunity  2008;76(6):2531-2540.
Mutant screens and transcriptome studies led us to consider whether the metabolism of glucose polymers, i.e., maltose, maltodextrin, and glycogen, is important for Escherichia coli colonization of the intestine. By using the streptomycin-treated mouse model, we found that catabolism of the disaccharide maltose provides a competitive advantage in vivo to pathogenic E. coli O157:H7 and commensal E. coli K-12, whereas degradation of exogenous forms of the more complex glucose polymer, maltodextrin, does not. The endogenous glucose polymer, glycogen, appears to play an important role in colonization, since mutants that are unable to synthesize or degrade glycogen have significant colonization defects. In support of the hypothesis that E. coli relies on internal carbon stores to maintain colonization during periods of famine, we found that by providing a constant supply of a readily metabolized sugar, i.e., gluconate, in the animal's drinking water, the competitive disadvantage of E. coli glycogen metabolism mutants is rescued. The results suggest that glycogen storage may be widespread in enteric bacteria because it is necessary for maintaining rapid growth in the intestine, where there is intense competition for resources and occasional famine. An important implication of this study is that the sugars used by E. coli are present in limited quantities in the intestine, making endogenous carbon stores valuable. Thus, there may be merit to combating enteric infections by using probiotics or prebiotics to manipulate the intestinal microbiota in such a way as to limit the availability of sugars preferred by E. coli O157:H7 and perhaps other pathogens.
doi:10.1128/IAI.00096-08
PMCID: PMC2423072  PMID: 18347038
18.  Transfer of antimicrobial resistance plasmids from Klebsiella pneumoniae to Escherichia coli in the mouse intestine 
Objectives and methods
Klebsiella pneumoniae is a nosocomial pathogen and is considered the most common Gram-negative bacterium that exhibits multiple antimicrobial resistances. In this study, the transfer of antimicrobial resistance genes from the clinical multiresistant K. pneumoniae MGH75875 isolate was assessed in vitro and in vivo in an intestinal colonization animal model. The ability to colonize and transfer was tested under different antimicrobial treatments. The frequency of the horizontal gene transfer was also examined in vitro.
Results
The clinical isolate of K. pneumoniae colonized the intestine of mice at levels up to 109 cfu/g faeces in antimicrobial-treated mice. In mice without antimicrobial treatment, the strain quickly decreased to below the detection limit due to competitive exclusion by the indigenous mouse flora. Onset of antimicrobial treatment gave immediate rise to detectable levels of the strain in the faeces of up to 109 cfu/g faeces. The experiment clearly shows that the treatment selects resistant strains and gives advantages to colonize the gastrointestinal tract. Furthermore, high transfer frequency of different plasmids was observed during colonization of the mouse intestine. The blaSHV and blaTEM genotypes were transferred to both an indigenous recipient in the in vivo setting and to an MG1655 Escherichia coli recipient strain in vitro.
Conclusions
K. pneumoniae is an excellent colonizer of the intestine and is extremely promiscuous with respect to the transferability of its numerous plasmids. Antimicrobial treatment enhances the selection of resistant strains and results in an increase in the resistance gene pool, which ultimately raises the risk of spreading resistance genes.
doi:10.1093/jac/dkn323
PMCID: PMC2566516  PMID: 18703526
MGH78578; horizontal gene transfer; streptomycin-treated mice; gastrointestinal tract; indigenous flora; selective pressure
19.  Complete Genome Sequence of the N2-Fixing Broad Host Range Endophyte Klebsiella pneumoniae 342 and Virulence Predictions Verified in Mice 
PLoS Genetics  2008;4(7):e1000141.
We report here the sequencing and analysis of the genome of the nitrogen-fixing endophyte, Klebsiella pneumoniae 342. Although K. pneumoniae 342 is a member of the enteric bacteria, it serves as a model for studies of endophytic, plant-bacterial associations due to its efficient colonization of plant tissues (including maize and wheat, two of the most important crops in the world), while maintaining a mutualistic relationship that encompasses supplying organic nitrogen to the host plant. Genomic analysis examined K. pneumoniae 342 for the presence of previously identified genes from other bacteria involved in colonization of, or growth in, plants. From this set, approximately one-third were identified in K. pneumoniae 342, suggesting additional factors most likely contribute to its endophytic lifestyle. Comparative genome analyses were used to provide new insights into this question. Results included the identification of metabolic pathways and other features devoted to processing plant-derived cellulosic and aromatic compounds, and a robust complement of transport genes (15.4%), one of the highest percentages in bacterial genomes sequenced. Although virulence and antibiotic resistance genes were predicted, experiments conducted using mouse models showed pathogenicity to be attenuated in this strain. Comparative genomic analyses with the presumed human pathogen K. pneumoniae MGH78578 revealed that MGH78578 apparently cannot fix nitrogen, and the distribution of genes essential to surface attachment, secretion, transport, and regulation and signaling varied between each genome, which may indicate critical divergences between the strains that influence their preferred host ranges and lifestyles (endophytic plant associations for K. pneumoniae 342 and presumably human pathogenesis for MGH78578). Little genome information is available concerning endophytic bacteria. The K. pneumoniae 342 genome will drive new research into this less-understood, but important category of bacterial-plant host relationships, which could ultimately enhance growth and nutrition of important agricultural crops and development of plant-derived products and biofuels.
Author Summary
Bacterial endophytes are capable of inhabiting the living tissues of plants without causing them significant harm. Klebsiella pneumoniae 342 (Kp342) is a model for this plant host-bacterial association, in part due to its capacity to colonize in high numbers the interior of plants including wheat and maize, two of the most important crops in the world. Kp342 possesses the ability to capture atmospheric nitrogen gas and turn it into an organic form (a process known as nitrogen fixation), of which part may be used as fertilizer by its plant host. Here, we describe the genome sequence and analysis of this model endophyte. When the Kp342 genome is compared to the genome of a closely related pathogenic relative, we can begin to surmise that its preference to engage in a harmonious relationship with plants is a result of many interacting factors. These include differences in its protein secretion systems, the manner in which its genes are regulated, and its ability to sense and respond to its environment. The study of endophytes is increasing in intensity due to the roles they may play in multiple biotechnological applications, including enhancing crop growth and nutrition, bioremediation, and development of plant-derived products and biofuels.
doi:10.1371/journal.pgen.1000141
PMCID: PMC2453333  PMID: 18654632
20.  Development of a Long-Term Ascending Urinary Tract Infection Mouse Model for Antibiotic Treatment Studies 
A model of ascending unobstructed urinary tract infection (UTI) in mice was developed to study the significance of the antibiotic concentration in urine, serum, and kidney tissue for efficacy of treatment of UTI in general and pyelonephritis in particular. Outbred Ssc-CF1 female mice were used throughout the study, and Escherichia coli was used as the pathogen. The virulence of 11 uropathogenic E. coli isolates and 1 nonpathogenic laboratory E. coli strain was examined. Strain C175-94 achieved the highest counts in the kidneys, and this strain was subsequently used as the infecting organism. The model gave reproducible bladder infections, i.e., bacteria were recovered from 22 of 23 control mice after 3 days, and histological examination of kidney tissue showed that of 14 infected kidneys, 7 (50%) showed major histological changes, whereas 3 of 36 uninfected kidneys showed major histological changes (P = 0.018). Once the model was established, the efficacies of different doses of cefuroxime and gentamicin, corresponding to active concentrations in urine only or in urine, serum, and kidney tissue simultaneously, were examined. All cefuroxime doses resulted in significantly lower counts in urine than control treatments, but the dose which produced concentrations of cefuroxime only in urine and not in serum or kidney tissue had no effect on kidney infection. Even low doses of gentamicin (0.05 mg/mouse) resulted in concentrations in renal tissue for prolonged times due to accumulation. All gentamicin doses had a significant effect (compared to the effect of the control treatment) on bacterial counts in urine and kidneys. The antibiotic effect on bacterial counts in bladders was negligible for unknown reasons. Use of the mouse UTI model is feasible for study of the effect of an antibiotic in the urinary system, although the missing antibacterial effect in the bladder needs further evaluation.
PMCID: PMC89643  PMID: 10602738
21.  Biofilm formation of Klebsiella pneumoniae on urethral catheters requires either type 1 or type 3 fimbriae 
Urinary catheters are standard medical devices utilized in both hospital and nursing home settings, but are associated with a high frequency of catheter-associated urinary tract infections (CAUTI). In particular, biofilm formation on the catheter surface by uropathogens such as Klebsiella pneumoniae causes severe problems. Here we demonstrate that type 1 and type 3 fimbriae expressed by K. pneumoniae enhance biofilm formation on urinary catheters in a catheterized bladder model that mirrors the physico-chemical conditions present in catheterized patients. Furthermore, we show that both fimbrial types are able to functionally compensate for each other during biofilm formation on urinary catheters. In situ monitoring of fimbrial expression revealed that neither of the two fimbrial types is expressed when cells are grown planktonically. Interestingly, during biofilm formation on catheters, both fimbrial types are expressed, suggesting that they are both important in promoting biofilm formation on catheters. Additionally, transformed into and expressed by a nonfimbriated Escherichia coli strain, both fimbrial types significantly increased biofilm formation on catheters compared with the wild-type E. coli strain. The widespread occurrence of the two fimbrial types in different species of pathogenic bacteria stresses the need for further assessment of their role during urinary tract infections.
doi:10.1111/j.1574-695X.2012.00965.x
PMCID: PMC3410544  PMID: 22448614
CAUTI; type 1 fimbriae; biofilm; Klebsiella pneumoniae; type 3 fimbriae

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