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Virulence determinants of Klebsiella causing liver abscess in otherwise healthy hosts

L. Kristopher Siu

National Institute of Infectious Diseases and Vaccinology

National Health Research Institutes

&

KeMyth Biotech. Co., Ltd.,

Taiwan

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1. Pyogenic liver abscess is usually a polymicrobial infectionthat has ascended from the gastrointestinal tract.

2. In western countries, the most frequent aetiologic agentsof pyogenic liver abscess were Escherichia coli,streptococci and anaerobic bacteria.

3. Liver abscess has usually been caused by a singlemicroorganism, Klebsiella pneumoniae, presenting in50% to 88% of pyogenic liver abscesses in Taiwan.

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1. Approximately, 3% to 7.8% of cases involving this organism have distant metastasis to the eye via the bloodstream causing septic endophthalmitis

2. An extremely poor visual outcome seems unavoidable in septic K. pneumoniae endophthalmitis despite aggressive treatment

3. Previous studies from Taiwan demonstrated that diabetes mellitus is the most common underlying condition, with prevalence ranging from 45% to 75% in patients with K.pneumoniae liver abscess

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Global reports of K. pneumoniae liver abscess : High prevalence in Asia

KoreaChung et al. Emerging invasive liver abscess caused by K1 serotype Klebsiellapneumoniae in Korea. J Infect. 2007;54:578-83.

SingaporeTan TY et al., Hypermucoviscosity, rmpA, and aerobactin are associated with community-acquired Klebsiella pneumoniae bacteremic isolates causing liver abscess in Singapore. J Microbiol Immunol Infect. 2017

Hong KongWong et al., Septic metastatic endophthalmitis complicating Klebsiella pneumoniaeliver abscess in a non-diabetic Chinese man. Hong Kong Med J. 2001;7:303-6.

JapanKuramochi et al., Klebsiella pneumoniae liver abscess associated with septic spinal epidural abscess. Hepatol Res. 2005;31:48-52.

Nepal Karki et al., Liver abscess in the tropics: an experience from Nepal. Southeast Asian J Trop Med Public Health. 2004;35:425-9.

ThailandWiwanitkit et al., Causative agents of liver abscess in those with liver cirrhosis: a 10-year case review of hospitalized patients in Thailand. Ann Trop Med Parasitol. 2002;96:513-6.

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Global reports of K. pneumoniae liver abscess: An emerging disease in western countries

SpainCasanova et al., Klebsiella pneumoniae liver abscess associated with septic endophthalmitis. Arch Intern Med 1989;149:1467.

BelgiumKarama et al., Endogenous endophthalmitis complicating Klebsiella pneumoniaeliver abscess in Europe: case report. Int Ophthalmol. 2007.

ItalyGiobbia et al. Community-acquired Klebsiella pneumoniae bacteremia with meningitis and endophthalmitis in Italy. Int J Infect Dis 2003;7:234-5.

CanadaKeynan et al., Pyogenic liver abscess caused by hypermucoviscous Klebsiellapneumoniae. Scand J Infect Dis. 2007;39(9):828-30.

USASaccente M. Klebsiella pneumoniae liver abscess, endophthalmitis, and meningitis in a man with newly recognized diabetes mellitus. Clin Infect Dis1999;29:1570-1.

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Virulence determinants: Definition of Virulence?

Define virulence: cell model or animal model?

Host Bacteria

Environment

Diseases

Factors affect invasivenessin cell or animal model

Diabetes X

High prevalence Asia X

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Virulence factors

Cryz et al., Clin Microbiol Rev. 1998 ; 11(4): 589–603

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Virulence determinants associated with K. pneumoniae liver abscess in different literatures

Determinant Gene or type Function Reference

MagA (wzy_K1) K-antigen polymerase Fang et. al., 2004

Related to CPS rmpA & rmpA2 Transcriptional regulator Yu et., al 2008

Serotype K1 or K2 Capsular K antigen Fung et al., 2002

Related to LPS wbbo …. LPS O antigen Yeh et., al., 2016

Aerobactin Iron siderophore Yu et., al., 2008

Nutrition factors kfu Iron uptake system Yu et., al., 2008

allS associated with allantoin metabolism

Yu et., al., 2008

Type 6 secretion system

PLD 1 Phosapholipase D family Lery et al., 2015

Adhesins Type 1 fimbriae Mannose-sensitive Rosen et al., 2008

Type 3 fimbriae Mannose-reistance Wu et al., 2012

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Definitions of invasive liver abscess syndrome

Clinical definitions Definite invasive syndrome: Klebsiella pneumoniae liver

abscess with extrahepatic complications, especially CNSinvolvement, necrotising fasciitis, or endophthalmitis

Probable invasive syndrome: K pneumoniae liver abscess asthe sole presenting clinical manifestation

Microbiological definitions Definite invasive syndrome: K pneumoniae liver abscess

caused by the K1 or K2 serotype Probable invasive syndrome: the hypermucoviscous

phenotype is defined by the string test, which monitors theformation of a viscous string of greater than 0.5 cm in lengthstretched by the inoculation loop

Lancet Infect Dis 2012;12: 881–87

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Serotype rmpA aerobactin Resistance Virulence＊

phagocytic serum

K1 + + + + +++

+ + + − V(+++,+)

+ + − − ++ − + + ++ − + − ++ + ND ND V(+++,+)− − ND ND −

K2 + + + + ++++ + + − V(+++,+)

+ + − + V(+++,+)+ − + − ++ + ND ND V(+++,+)

− − ND ND V (+,−)Non K1/K2 + + ND ND V(+++,+)

− − ND ND −

Microbiological features associated with virulence

*(+++) Hypervirulent strains with LD50 < 1×103 CFU are more likely to induce complications in mice. (+)

Virulent strains with LD50 ≥ 1×103 CFU and < 1×106 CFU are less likely to induce complications in mice. (−)

non virulent strains with LD50 ≥ 1×106 CFU and do not cause complications. V, variable; ND, no dataLancet Infect Dis 2012;12: 881–87

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Virulence determinants contributed to mice lethality (LD50)

The tested liver abscess isolates in serotypes K1/K2/K20 with rmpA and aerobactin could show a hypervirulence (LD50, <103 CFU). The hypervirulent strains could be negative for kfu and allSgenes (LD50, <103 CFU). Those strains without rmpA were avirulent.

Yu et. al., Diagn Microbiol Infect Dis. 2008; 62(1):1-6.

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Fung CP et al., Gut. 2002. 50(3):420-4

Correlation between definite invasive K. pneumoniae liver abscess syndrome and serotypes distribution

Only serotypes K1 and K2 were found in patients with septic endophthalmitis, and especially K1,

which was found in 85.7% of all septic endophthalmitis cases (12/14).

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Virulence determinants associated with K. pneumoniaeliver abscess

1. K. pneumoniae strains with hypervirulence (LD50 <103

CFU) are mostly carrying rmpA and aerobactin withproperties of highly phagocytic resistance to neutrophiland serum killing

2. Patients with definite invasive syndrome, K.pneumoniae strains were exclusively caused byserotype K1 or K2 strains and these strains are alsodefinitely carrying rmpA and aerobactin with highlyphagocytic resistance to neutrophil and serum killing

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Serotype O (LPS) and K (CPS) antigen contributed differently in the pathophysiological features

Serum bactericidal assay of Klebsiella pneumoniae wildtype strains and their mutants.

(A) STL43, serotype O1:K1 wild type; STL43Δwzy, K-antigen-deficient mutant;

STL43ΔwbbO, O-antigen-deficient mutant. ＊STL43 vs STL43ΔwbbO (P=.004).

(B) TSGH69, serotype O1:K2 wild type; TSGH69Δwzy, K-antigen-deficient mutant;

TSGH69ΔwbbO, O-antigen-deficient mutant. ＊TSGH69 vs TSGH69Δwzy, and

TSGH69 vs TSGH69Δwbbo (for both, P=.006). Yeh et al. Gut Pathog (2016) 8:4

Serotype K2 Serotype K1

Serum Killing

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STL43, serotype O1:K1 wild type; STL43Δwzy, K-antigen-deficient mutant;STL43ΔwbbO O-antigen-deficient mutant; TSGH69, serotype O1:K2 wild type;TSGH69Δwzy , K-antigen deficient mutant; TSGH69ΔwbbO. O-antigen-deficientmutant. Yeh et al. Gut Pathog (2016) 8:4

Association of phagocytosis rate and glucuronic acid contents of wildtype strains and O/K antigen mutants

CPS knockout

LPS knockout

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A STL43, serotype O1:K1 wild type; STL43Δwzy, K-antigen-deficient mutant, STL43ΔwbbO O-antigen deficient mutant. Sublethal dose injection of each strains, <10 cfu for wild type; 3 × 105 cfu for ΔwbbO and 3 × 106 cfu for Δwzy.

B TSGH69, serotype O1:K2 wild type; TSGH69Δwzy, K-antigen-deficient mutant;TSGH69ΔwbbO, O-antigen-deficient mutant. The values are the mean ± standarddeviation. Yeh et al. Gut Pathog (2016) 8:4

Liver bacterial clearance study of K. pneumoniaewildtype strains and O/K antigen mutants

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3 hours Day 3 Day 3 Day 3

Serotype O (LPS) and K (CPS) antigen contributed differently in the pathophysiological features

Yeh et al. Gut Pathog (2016) 8:4

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Serotype O and K antigen contributed differently in the pathophysiological features

For K antigen (related to CPS)1. Both K1 and K2 antigen mutants lost their wildtype resistance to phagocytosis ,

increasing hepatic clearance and failed to cause abscess formation. 2. K2 antigen mutant became susceptible to serum bactericidal assay while K1-

antigen maintained their serum resistance. 3. The amount of glucuronic acid (amount of CPS related) was inversed proportional

to the rate of neutrophil phagocytosisFor O antigen (related LPS)1. O-antigen mutants of serotype K1 had significant more amount of CPS and more

resistant to neutrophil phagocytosis that its wild-type.2. O-antigen mutants of serotype K1 and K2 lost their wildtype serum resistance and

kept resistant to phagocytosis .3. O antigen mutants of serotype K1 became susceptible to liver clearance and cause

mild abscess formation but serotype K2 mutant maintained these wild type virulence

There is a various role of CPS and LPS antigens in serotype K1 and K2 strains on virulence and also contribut a different pathophysiological on liver abscess.

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Capsular polysaccharide synthesis gene cluster in serotypes K1 and K20 of K. pneumoniae

1. The capsule synthesis gene cluster in the different serotypes has 2 distinct regions. One region consists of highly conserved genes (>50% nucleotide sequence similarity) while the other region is hypervariable.

2. Each of these structural genes is serotype specific, and may or may not be replaceable with the homologous gene from another serotype

Virulence. 2017 Jul 4;8(5):487-493.

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Switching conserved genes from serotype K1 into K20 and their complemented mutants

(1) DgalF, DacidPPc, D20D-acidPPc: (K1)acidPPc and restoration of DacidPPc::acidPPc in mutant acidPPc, with low effect (L) in LD50; (2) Dwzi, K20Dwzi::(K1)wzi and restoration of wzi in mutant Dwzi, with moderate effect (M) in LD50; (3) Dwza, K20Dwza::(K1)wza, Dwzb and Dwzb and restoration of K20 Dwza:: wza with high effect on virulence (H) Virulence. 2017 Jul 4;8(5):487-493.

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Summary the effects on virulence of switching the genes acidPPc, wzi, wza, wzb or wzc from K1 into K20

1. Six genes in the CPS conserved region in serotype K20, including galF, acidPPc, wzi, wza, wzb and wzc, were knocked out and replaced by the homologous genes from serotype K1.

2. Knockout mutants showed a decline in lethality (LD50) in mice from 10-fold to >105-fold and were categorized in terms of the effect on virulence as low (L) for galF and acidPPC, moderate (M) for wzi, and high (H) for wza, wzb and wzc.

3. Variable improvement in serum killing, phagocytosis and mice lethality (LD50) was observed when the knockout mutants were compared with the gene-switched strains.

Virulence. 2017 Jul 4;8(5):487-493.

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Summary the effects on virulence of switching the genes acidPPc, wzi, wza, wzb or wzc from K1 into K20

Low virulence effect group (L)The switching of K1-acidPPc into K20 restored virulence in terms of serum resistance, phagocytic resistance and the LD50

compared to the K20 parent strain.

Moderate viurlence effect group (M) The substitution of K1-wzi into K20 caused a 10-fold decrease in LD50 compared to the K20 parent strain.

High virulence effect group (H)Three exchange mutants, K20::K1-wza,-wzb and -wzc, did not show restored virulence compared to the K20 parent strain, and no change was noted in the anti-K20 agglutination assay.

Virulence. 2017 Jul 4;8(5):487-493.

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Phospholipase D familyprotein (PLD1) as a virulence factor

1. Type VI secretion system (T6SS) putative genes were located in atleast three different loci of the K. pneumoniae genomes, inaccordance with a previous in-silico study.

2. T6SS has been suggested to assist colonization and infection.Indeed, in a screen that identified K. pneumoniae mutants failing tocolonize mice.

3. It has been hypothesize that PLD1 alters the membranecomposition and charge, affecting bacterial interaction with thehost environment.

4. In 2014, Lery et al. performed an analysis on PLD1 and suggestedPLD1 is a factor contributed to virulence

Lery et al., BMC Biol. 2014 May 29;12:41

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PLD1 involvement in K. pneumoniae virulence

Lery et al., BMC Biol. 2014 May 29;12:41

Mice survival after infection (108

CFU) with Kp52.145 wild-type (K2), pld1 mutant and complemented strains.

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Functions of type 1 fimbriae during K. pneumoniae infection and biofilm formation

Red sentences indicate possibly detrimental roles on K. pneumoniae virulence in vivo.

Paczosa et al., Microbiol. Mol. Biol. Rev. 2016; 80(3):629-61

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Functions of type 3 fimbriae during K. pneumoniae infection and biofilm formation

Red sentence indicates a possibly detrimental role on K. pneumoniae virulence in vivo.


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Functions of fimbriae in K. pneumoniae


3. Other infections: The role of fimbriae remains to be determined.

1. The most important clinically significant roles for fimbriae maybe in biofilm formation and binding to abiotic surfaces.

• Type 3 fimbriae can bind to bladder epithelial cells grown in culture,but in mouse model systems, they do not seem to contribute to UTIs.

• In a mouse lung model of infection, a K. pneumoniae mutant lackingthe type 1 fimbriae was capable of not only colonizing the lungs butalso disseminating to the spleen and liver at WT level.

2. Urinary tract infections: Type 1 fimbriae contribute to UTIs.

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Other factor contributed to the virulence in K. pneunminae but not normally identified in liver

abscess patients:Outer membrane Proteins

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Effect of OmpK35 and OmpK36 on bacterial growth (A1 and A2), susceptibility to normal human serum (B1 and B2)

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The association of OmpK loss and virulence

K. pneumoniae ΔompK36Growth rate (generation time)

Serum resistance

Resistance to Phagocytosis

K. pneumoniae ΔompK35/36Growth rate (generation time)

Serum resistance

Resistance to Phagocytosis

Porin deficiency in K. pneumoniae could increase antimicrobialresistance but decrease virulence at the same time.

Tsai et. al., Antimicrob Agents Chemother. 2011

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summary1. All virulence factors described in this presentation are contributing to

the virulence in Klebseila pneumoniae infections. However, there is nosingle determinant could contribute to the hypervirulence in micelethality.

2. Liver abscess patients with extrahepatic complications are frequentlyinvolved with hypervirulent serotype K1 and K2 strains and these strainsnormally contained rmpA and arobactin with properties of highlyphagocytic and serum resistance.

3. Non-serotype K1/K2 contained rmpA and arobactin with properties ofhighly phagocytic and serum resistance could achieve similarhypervirulence as serotype K1/K2 but rarely not seen in liver abscesspatients with complication like CNS involvement, necrotising fasciitis, orendophthalmitis (definite invasive)

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4. Lost of capsule render the great effect on virulence and acapsulatedstrains normally become avirulent.

5. Lost of a gene in CPS synthesis contributed differently in virulence andgene switching even in conserved region of CPS cluster cannot restorevirulence as parental strain.

6. All other determinants, rmpA, aerobactin, PDL1, and phagocytic orserum resistance showed different degree of effect on virulence.

7. Type 1 and 3 fimibriae involved in biofilm formation but the effect onvirulence in liver abscess due to the lost of fimibriae has yet to bedetermined and fimibriae may contributing the dissemination the K.pneumoniae into liver.

8. In comparing the reported factors, as there are various LD50 observed,there are unknown factors that have yet to be found and shouldcontribute to the virulence in K. pneumoniae.

summary

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Four well-characterized virulence factors in classical and hypervirulent K. pneumoniae strains

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