ESBL-production is associated with an increase of cell invasion and fimbrial adhesins’

expression in Klebsiella pneumoniae

Running Title: ESBL and Klebsiella adhesion and invasion

H. Sahly1,2*

, S. Navon-Venezia3, L. Roesler

4, A. Hay

3, Y. Carmeli

3, R. Podschun

1, C.

Hennequin5, C. Forestier

5, I. Ofek

6

1. Institute for Infection Medicine, Faculty of Medicine, University of Kiel, Kiel

2. Institute of Immunology, Clinical Pathology and Molecular Medicine, Biotech,

Hamburg, Germany

3. Division of Epidemiology, Tel-Aviv Sourasky Medical Center, Sackler School of

Medicine, Tel-Aviv University, Israel

4. Department of Anaesthesiology and Intensive Care Medicine, University Hospital

Schleswig-Holstein, Campus Kiel, Germany

5. Laboratoire de Bactériologie, Faculté de Pharmacie, Université de Clermont 1,

Clermont-Ferrand, France

6. Department of Human Microbiology, University of Tel Aviv, Israel

Key words: Klebsiella, ESBL, fimbrial adhesins, adhesion, invasion, epithelial cells

*Corresponding footnote:

Institute of Immunology, Clinical Pathology and

Molecular Medicine, Biotech

Lademannbogen 61-63

22339 Hamburg

Germany

Tel. 00 49 40 53805104

Fax. 00 49 40 53805127

E-Mail sahly@labor-lademannbogen.de

ACCEPTED

Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Antimicrob. Agents Chemother. doi:10.1128/AAC.00010-08 AAC Accepts, published online ahead of print on 23 June 2008

at TE

L AV

IV U

NIV

on July 30, 2008 aac.asm

.orgD

ownloaded from

2

ABSTRACT

Extended-Spectrum-ß-lactamase- (ESBL) producing Klebsiella pneumoniae strains are

suggested to possess higher pathogenic potential than non-ESBL-producers. Microbial

adherence to and invasion of host cells are critical steps in the infectious process so we

examined the expression of type-1 and 3 fimbrial adhesins by 58 ESBL-producing and 152

non-producing isolates of K. pneumoniae and their ability to invade ileocecal and bladder

epithelial cells. Mannose-sensitive haemagglutination of guinea pig erythrocytes and

mannose-resistant haemagglutination of ox erythrocytes were evaluated for determining the

strains’ ability to express type-1 and type-3 fimbriae, respectively. Bacterial adhesion to and

invasion of epithelial cells were tested by ELISA and imipenem killing assay, respectively.

The adherence of ESBL and non-ESBL-producing strains to epithelial cells did not differ

significantly (p>0.05). In contrast, the proportion of strains capable of invading (>5% relative

invasion) ileocecal and bladder epithelial cells was significantly higher among ESBL-

producers (81%, n=47/58, and 27.6%, n=16/58, respectively) than among non-ESBL-

producers (61%, n=93/152, and 10%, n=15/152, respectively) (p=0.0084¸ OR=2.711; 95%

CI=1.302-5.643 and p=0.0021; OR=4.79; 95% CI 1.587-7.627); The mean invasion by

ESBL-producer (5.5±2.8% and 3.3±2.7%, respectively) was significantly higher than by non-

ESBL-producers (2.9±2.6% and 1.8±2%, respectively) (p<0.0001). Likewise, the proportion

of ESBL-producers co-expressing both fimbrial adhesins was significantly higher (79.3%,

n=46/58) than that of non-ESBL-producers (61.8%, n=94/152) (p=0.0214; OR=2,365; 95%

CI=1.157-4.834). Upon acquisition of SHV-12 encoding plasmids two transconjugants

switched on to produce type-3 fimbriae while expression of type-1 fimbriae was not affected.

The acquisition of an ESBL-plasmid appears to upregulate the phenotypic expression of one

or more genes resulting in higher invasion ability.

ACCEPTED

at TE

L AV

IV U

NIV

on July 30, 2008 aac.asm

.orgD

ownloaded from

3

INTRODUCTION

Klebsiella pneumoniae is one of the most common pathogens causing nosocomial infections

that affect mainly immunocompromized patients with severe underlying diseases. Its

frequency ranges from 3% to 17% of all nosocomial infections, placing it among the eight

most relevant pathogens in hospitals (15, 16, 19, 20, 27, 48, 51, 53).

Of particular interest is the dramatic worldwide spread of extended spectrum beta-lactamase-

producing Klebsiella strains (ESBL) that are resistant to third-generation cephalosporins and

that often display cross resistance also to aminogylcosides and quinolones (36, 37, 39, 41). It

is estimated that the proportion of ESBL-production among K. pneumoniae ranges from 12%

in the USA, 33% in Europe, 28% in the Western Pacific, and to 52% in Latin America (25).

Because ESBL-producing bacteria are often resistant to a variety of antibiotics, carbapenems

became the drug of choice against ESBL-producing strains (38).

Infections caused by ESBL-producers are associated with severe adverse outcomes (28, 46,

47). This is related in part to delay in effective therapy and the failure to use an antibiotic

active against ESBL-producing K. pneumoniae. However, the high mortality rate observed

with infections caused by ESBL-producers may be related also to increased virulence of these

strains. Indeed, several studies have reported on an association between ESBL-production

and the higher expression of pathogenicity factors in Klebsiella. Significantly, it has been

shown that ESBL-producing Klebsiella strains exhibit increased adherence to human

epithelial cells, probably due to plasmid-coded production of fimbrial or non-fimbrial

adhesins (2, 5, 6, 29). Most K. pneumoniae strains express two types of fimbrial adhesins

(haemagglutinins) one of which is the mannose-sensitive (MS) type-1 and the other is the

mannose-resistant (MR) type-3 (26). Type-1 fimbriae mediate mannose-sensitive

haemagglutination (MSHA), are expressed by many enterobacterial species and play an

ACCEPTED

at TE

L AV

IV U

NIV

on July 30, 2008 aac.asm

.orgD

ownloaded from

4

important role in their pathogenesis (34, 35). Significantly, type-1 fimbriae of Klebsiella and

Escherichia coli have been shown to be important for urinary tract infections (1, 10, 11, 30,

31, 32). They can facilitate attachment of Klebsiella to a wide range of cells including those

of the urinary and respiratory tracts (12, 30). Type-3 fimbriae are capable of mediating

binding of Klebsiella sp. to various human cells, such as endothelial and epithelial cells of the

respiratory and urinary tracts (22, 23, 24, 49, 50, 52). Furthermore, they were shown to

facilitate adherence and biofilm formation on innert surfaces (4). Although K. pneumoniae is

considered to be an extracellular pathogen, several studies have revealed the ability of the

bacteria to invade and persist in epithelial cells (13, 14, 33, 45)

Because adherence of the microorganisms to and subsequent invasion into host cells are

considered a critical step in the infectious process (34), we sought to examine the ability of a

large number of ESBL-producing and non-producing strains of K. pneumoniae to express the

type-1 and 3 fimbrial adhesins and to adhere to and invade epithelial cell lines. It will be

shown that, the proportion of strains among the ESBL-producing Klebsiella isolates capable

of invading the ileocecal and bladder epithelial cells and of expressing the fimbrial adhesins is

significantly higher as compared to non ESBL-producing isolates.

ACCEPTED

at TE

L AV

IV U

NIV

on July 30, 2008 aac.asm

.orgD

ownloaded from

5

MATERIALS AND METHODS

Bacterial cells and culture conditions. A total of 58 ESBL-producing and 152 non-ESBL-

producing K. pneumoniae strains described elsewhere was employed in the study. The strains

were isolated from clinical specimens from hospitalized patients in different European

countries, tested for ESBL-production, serotyped with respect to O and K antigens, and

analysed for clonality as described earlier (42, 43). Production of extended-spectrum ß-

lactamases were detected according to recommendation of the CLSI (former NCCLS) as

described earlier (43).

Haemagglutination assays. The expression of type-1 (MSHA) and type-3 pili (MR/K-HA)

was examined as described previously (40). Briefly, after 4 passages (either serial 48 hour

passages in static brain-heart-infusion broth (BHI) for the detection of MSHA or passage in

nutrient broth (NB) for detection of MR/K-HA) the bacteria were allowed to grow statically

for 48 h at which time they were harvested and washed 3 times with PBS. 50 µl of bacterial

suspension (3 x109

CFU/ml) were mixed on WHO plates with 50 µl erythrocytes suspension

(5 x 108/ml). Agglutination was observed after 3 minutes of gentle shaking at room

temperature and after another 10 minutes at 4°C, and the intensity of reaction was graded

from – to +++. Positive mannose-sensitive haemagglutination (MSHA) of guinea pig

erythrocytes or haemagglutination of tanned ox erythrocytes indicate expression of type-1 or

type-3 MR/K-HA, respectively, fimbrial adhesins by at least 10% of the bacterial cell

population.

Detection of fimbriae encoding genes. The genes coding for type-1 (MSHA) and type-3 pili

(MR/K-HA) were detected by colony hybridization using as probes the insert of pGG101

(fimAE, SphI, 1,6 kb) and the insert of pFK10 (mrkBC, KpnI, 2.8 kb) , respectively (17, 18).

ACCEPTED

at TE

L AV

IV U

NIV

on July 30, 2008 aac.asm

.orgD

ownloaded from

6

For each strain, one microliter of overnight culture in Luria Bertani (LB) broth was spotted

onto Hybond-N nylon membranes (Amersham France, Les Ulis), placed on LB solid medium,

incubated for 6 h at 37°C and lysed according to the manufacturer's instructions. DNA probes

were labelled with [α-32

P]dATP by using random-primed DNA labelling kit (Boehringer

Mannheim, Meylan, France). Hybridization was performed with Rapid-hyb buffer

(Amersham) as recommended by the manufacturer under high-stringency conditions. In

addition, detection of type-3 encoding gene (mrkD) was performed by PCR as described

previously (21).

Cell lines and culture conditions. The human epithelial bladder cell line T24 and the

ileocecal cell line HCT-8 were cultivated as described previously (45). The T24 cells were

cultivated in McCoy´s 5A medium with 10% fetal bovine serum, the HCT-8 cells in RPMI

1640 medium with 1 mM pyruvate, 2mM glutamine, and 10% FCS and subcultivated at a

ratio of 1:8 twice a week. Human A549 adenocarcinoma cells were cultivated in F-12K

medium with 10% (v/v) fetal calf serum and 2mM glutamine as described elsewhere (44).

Invasion assay. The ability of the bacteria to invade epithelial cells was conducted as

described previously (45). The cells were cultured in 24-well cell culture clusters to

monolayers of approximately 7 x 104 cells/well. Two x 10

6 mid-log-phase bacteria were then

added to each well (approximately 30 bacteria/epithelial cell). After centrifugation at 200 x g

for 5 min, invasion was allowed to occur for 2 h at 37°C in an atmosphere of 94% air/6%

CO2. Before a second 2-h incubation (kill) period under the same conditions but with fresh

medium containing 100µg of imipenem/ml, monolayers were washed once with Earle´s

balanced salts solution (EBSS). All extracellular, but not internalized bacteria were killed by

the added imipenem during the kill period. The monolayers were then washed twice with

EBSS and lysed with 0.1% Triton X-100 before determination of viable counts of the released

ACCEPTED

at TE

L AV

IV U

NIV

on July 30, 2008 aac.asm

.orgD

ownloaded from

7

intracellular bacteria. The invasion ability was expressed as the percentage of inoculum

surviving imipenem treatment. The invasion was classified in two grades, grade I, invasion

<5% of inoculum, grade II > 5% of inoculum. The distribution of strains’ invasion and the

mean relative invasion were calculated.

Adhesion assay. The adhesion of six strains that harbour the ESBL-coding plasmid and of

their plasmid-cured derivatives (see below) to epithelial cells was tested as described

previously (44). Briefly, 50 µl of the bacteria (ca. 108 CFU/ml) were added to wells of

microtiter flat bottom plates containing confluent monolayers of T24, HCT-8, or A549 cells.

After 30 min at room temperature, the non-adherent bacteria were removed by repeated

washing with EBSS and the cell-monolayers were fixed with 2.5% glutaraldehyde in HBS for

5 min. The plates were then washed and incubated in blocking buffer overnight at 4ºC. The

bound bacteria were quantified using anti-Klebsiella serum at 1:1000 in ELISA.

Preparation of plasmid DNA and plasmid curing. To investigate the contribution of the

ESBL-coding plasmid in the ability of the bacteria to produce type-1 and type-3 pili and to

adhere to and invade the epithelial cells, five strains harbouring the ESBL-coding plasmid,

expressing type-1 and type-3 pili and positive in the adhesion and invasion assays were

subjected to a plasmid-curing procedure as described elsewhere (43). The obtained plasmid-

cured derivatives were tested for ESBL-production and for their ability to express the

pathogenicity factors as described above.

Transconjugation and the effect on pili expression. Transconjugation experiments were

performed by filter-mating (45) using two ESBL-producing K. pneumoniae isolates (isolates

1054 and 1147) as donors and one non-ESBL-producing K. pneumoniae a recipient strain

(KPTA 29).

ACCEPTED

at TE

L AV

IV U

NIV

on July 30, 2008 aac.asm

.orgD

ownloaded from

8

Transconjugants were selected on LB agar plates containing nalidixic acid (128µg/ml) and

ceftriaxone (4µg/ml), and subjected to the Vitek-2 susceptibility testing. ESBL-production

was confirmed as described above. Pulse Field Gel Electrophoresis (PFGE) was used to

confirm the genetical identity of the transconjugants and the recipient parental strain. The

acquisition of ESBL-encoding plasmids was verified by comparative plasmid analysis of the

donor and recipient strains. Polymerase Chain Reaction (PCR) confirmed the presence of the

expected ESBL genes. Two confirmed transconjugants, T1147 and T1054, were assayed for

their ability to express type-1 and type-3 pili.

To further verify the influence of ESBL-producing plasmid on type-3 fimbrial expression a

real-time RT-PCR was conducted. For this purpose RNA was extracted from 1 ml of a 5 h

culture using the NucleoBond® NucleoSpin® NucleoTrap® kit from Machery-Nagel (Düren,

Germany) according to the manufacturer’s instructions. The real-time RT-PCR step was

carried out with the LightCycler® RNA Master SYBR Green I kit (Roche Diagnostics,

Mannheim, Germany) on LightCycler® systems (Roche Diagnostics) with primers MrkD 2 F:

5’-CCACCAACTATTCCCTCGAA-3’ and MrkD 2 R: 5’-ATGGAACCCACATCGACATT-

3’ (GenBank accession number: AY225462). As an internal control, the 16S rRNA gene was

amplified with universal primers TM1, 5’-ATGACCAGCCACACTGGAAC-3’ and TM2, 5’-

CTTCCTCCCCGCTGAAAGTA-3’. A 20 µl mixture containing 1 µl of RNA and 2 µl of

primers was prepared and amplified according to the manufacturer’s instructions (the RNA

preprations were checked for absence of DNA contamination). To compare relative gene

expression between the wild-type strain and its transconjugants, the ratio =

2[(CPtransconjugant-CPwild-type)TM-(CPtransconjugant-CPwild-type)MrkD] was

calculated (CP: crossing point). The experiment was made twice.

Statistical analysis. The significance of differences between groups of isolates was evaluated

by Yate’s corrected Chi square test. For tables showing values of n < 5 within a cell, Fisher’s

ACCEPTED

at TE

L AV

IV U

NIV

on July 30, 2008 aac.asm

.orgD

ownloaded from

9

exact test was used. The odds ratio (OR) and 95% confidence intervals (CI) were determined

and values were taken as a gauge of association. The significance of differences between the

relative invasion of the tested bacteria was evaluated by the unpaired t-test with Welch

correction.

ACCEPTED

at TE

L AV

IV U

NIV

on July 30, 2008 aac.asm

.orgD

ownloaded from

10

RESULTS

Clonality of the strains and ESBL types. After exclusion of clonal strains, a total of 152

non-ESBL-producing and 58 ESBL-producing strains were defined. Sixteen ESBL types were

identified, among which SHV-5 was the most common ESBL type (23%; 12/58), followed by

TEM-3 (15.6%; 9/58 isolates), SHV-4 and SHV-12 (each 10% and 6/58 isolates), and TEM-

24 (8.6%; 5/58 isolates). SHV-2, 2A, 36, 40 and TEM-5, 8, 12, 15 and 16 were detected in

one strain each.

Expression of type-1 and type-3 fimbriae. The percentage of strains that do not express

either type-1 or type-3 fimbrial adhesin was very low (i.e. 3.5% and 5.2 % of ESBL-

producing and non-ESBL-producing strains, respectively) (Table 1), suggesting that at least

one of these adhesins is required for colonization irrespective of the presence of the ESBL-

plasmid. Non-ESBL-producing strains produced more often as compared to ESBL-producers

either one of the fimbrial adhesins: 50 out of the 152 non-ESBL-producing strains (32.8%)

expressed either type-1 or type-3 fimbrial adhesins and only 10 out of the 58 (17.2%) ESBL-

producers did so (p = 0.0268; OR = 2.353; 95% CI = 1.099 to 5.036). In contrast, the

frequency of ESBL-producing strains co-expressing both type-1 and type-3 fimbrial adhesins

was significantly higher than that of the non-ESBL-producers (79.3% and 61.8%,

respectively) (p = 0.0214; OR = 2,365; 95% CI 1.157 to 4.834).

The magnitude of the haemagglutination of guinea pig erythrocytes and of tanned ox

erythrocytes did not differ significantly between the ESBL-producing and non-ESBL-

producing strains (p>0.05) (Figure 1 A and B), suggesting that once the adhesin genes are

turned on, the phenotypic expression of the adhesins is not affected by the presence of ESBL-

plasmids. Colony hybridization revealed the presence of the type-1 encoding genes in all but

one strain and only two did not harbor the type-3 encoding genes

ACCEPTED

at TE

L AV

IV U

NIV

on July 30, 2008 aac.asm

.orgD

ownloaded from

11

Adhesion and Invasion ability. The adherence of ESBL and non-ESBL-producing strains to

ileocecal, bladder and alveolar epithelial cells did not differ significantly (p>0.05) (Figure 2).

In contrast, the frequency of isolates that efficiently invaded the ileocecal epithelial cells

(grade II invasion, >5% of inoculum) was higher among ESBL-producers than among non-

ESBL-producers (Table 2). (81%, 47of 58 and 61%, 93/152, respectively; p=0.0084; OR =

2.711; 95% CI = 1.302 to 5.643). Likewise, 27.7% (n = 16/58) of the ESBL-producers

invaded the bladder T24 cells efficiently whereas only 10% (n = 15/152) of the non-ESBL-

producer did so (p = 0.0021; OR = 3.479; 95% CI 1.587 to 7.627). The mean % relative

invasion of HCT8 and T24 cells by ESBL-producing strains ( 5.5 ± 2.8% and 3.3 ± 2.7%) was

significantly higher than that of non-ESBL-producer (2.9 ± 2.6% and 1.8 ± 2%) (p< 0.0001)

(Figure 3). The invasion of the A549 lung epithelial cells by both ESBL and non-ESBL-

producer did not exceed 1%.

Haemagglutination, adhesion and invasion ability of plasmid cured strains. Five strains

were cured from their ESBL-plasmid. The lack of ESBL-producing phenotype in all plasmid

cured derivatives was confirmed using the CLSI guidelines. The loss of the ESBL-coding

plasmids in the strains was demonstrated by agarose gel electrophoresis (data not shown). In

all cured strains the MICs to third-generation cephalosporins, e.g. cefotaxime, ceftriaxone,

and ceftazidime were <2 µg/ml. There was no difference between the five plasmid-cured

derivatives and their parental strains in the ability of the strains to express type-1 and type-3

fimbriae, and to adhere to and invasion into tissue culture cell lines (Figure 4).

Haemagglutination of transconjugants. The parental recipient strain KPTA 29 expressed

type-1 pili (e.g. induced MSHA of guinea pig erythrocytes), but not type-3 fimbriae (e.g did

not induce mannose-resistant haemagglutination of ox erythrocytes). Two transconjugates,

T1147 and T1054, of this strain that acquired the ESBL-plasmid were obtained and both

ACCEPTED

at TE

L AV

IV U

NIV

on July 30, 2008 aac.asm

.orgD

ownloaded from

12

switched on to express type-3 fimbia as shown in haemagglutination assay of tanned ox

erythrocytes. Specific PCR indicated that both the wild-type strain KPTA 29 and its two

transconjugants, T1147 and T1054, harbored the type-3 encoding genes (data not shown).

Differences in type-3 fimbrial expression was further studied in the parental strain and both

transconjogants by real-time RT-PCR. A 226 bp fragment of the mrkD-encoding gene was

targeted. When considering the reference, the comparative CP values between the parental

strain and the transconjugants T1054 and T1147 were respectively 3702 and 5595 (average

CP values) indicating that type-3 fimbrial expression was upregulated in both transconjugants

consistent with the haemagglutination results.

ACCEPTED

at TE

L AV

IV U

NIV

on July 30, 2008 aac.asm

.orgD

ownloaded from

13

DISCUSSION.

A number of findings support the notion that infections caused by ESBL-producing Klebsiella

strains are associated with severe adverse outcomes by virtue of the higher ability of these

strains to express pathogenicity factors. The latter include (i) ESBL-plasmid mediate adhesive

properties in Klebsiella (2, 5, 6, 29) (ii) ESBL-producers exhibit increased ability to resist the

bactericidal activity of serum (43) and to evade the opsonin-mediated phagocytosis and

killing by polymorphonuclear granulocytes (42), and (iii) the present study shows that the

proportion of ESBL-producing strains that simultaneously express mannose-sensitive and

mannose-resistant pili and capable of invading ileocecal and bladder epithelial cells is

significantly higher among ESBL-producers than non-ESBL-producers.

It has been shown that invasion of non-phagocytic cells by the bacteria requires the co-

expression of numerous components to involve signal transduction via receptors different

from those requested for the adhesion process (3, 7, 8, 9). It can be speculated that the higher

invasive ability of the ESBL-producing strains might be due to the higher frequency of strains

that co-expressed both the type-1 and type-3 fimbriae, supporting the notion that both fimbrial

types are important virulence factors by virtue of their ability to adhere to epithelial cells and

extracellular matrix. Indeed, the percentage of Klebsiella isolates from different clinical

sources expressing both adhesins was found to be significantly higher than that of strains

isolated from sewage (40). Likewise, in the present study most of the clinical isolates

produced at least one adhesin and many produced both adhesins. . Because the assessment of

adhesion by heamagglutiantion is qualitative, we cannot speculate whether the invasion

process requires expression of both type-1 and type-3 fimbriae or if only one fimbrial adhesin

expressed in higher numbers per cell suffices. Furthermore, using the impenem killing assay

to distinguish between intracellular and extracellular bound bacteria assumes that impenem

ACCEPTED

at TE

L AV

IV U

NIV

on July 30, 2008 aac.asm

.orgD

ownloaded from

14

neither diffuses into the cells nor acts to inhibit bacteria exposed to the antibiotic before entry

into the cells. These assumptions require further validation.

Interestingly, the elimination of the plasmid that encoded for the ESBL-production did not

influence the ability of strains to adhere to or invade the epithelial cells, indicating that the

invasion potential in Klebsiella is chromosomal rather than plasmid mediated, contradicting

previous reports indicating a R-plasmid restricted increase of adhesive properties (2, 5, 6, 29).

Alternatively, the adhesion/invasion of the strains in the above cited studies is mediated by

adhesins that differ from those expressed by the strains of the present study.

The expression of fimbrial adhesins is under tight phase variation control involving the

switching of the fim gene translation from off to on and vice versa at a defined frequency (34).

The presence of ESBL-plasmid could increase the conversion frequency of type-3 fimbrial

gene translation from off to on, without changing the phenotypic expression of type-1 fimbria

encoding genes. Such increased and specific conversion may result in significantly higher

proportion of strains among ESBL-producers that phenotypically express both type-1 and

type-3 fimbrial adhesins and as a consequence better invade epithelial cells. It is tempting to

speculate that upon acquisition of the ESBL-plasmid, a number of chromosomal gene

rearrangements occurs, involving the turn-on of some of them. Indeed, most strains were

shown to harbour the genes coding for both fimbrial types, but only a part of them

phenotypically expressed one or both adhesion phenotypes.

Taken together, the data suggest that the increase in mortality and severity of infections

caused by ESBL-producers is not due to appearance of new virulence factors but rather to

increase in the ability of the ESBL strains to simultaneously express several virulence factors.

This notion is supported by our findings showing that the acquisition of ESBL-plasmid

rendered the transconjugates phenotypically capable of expressing fimbrial genes which were

not expressed in the parent recipient strain. Thus, while the proportion of strains exhibiting

ACCEPTED

at TE

L AV

IV U

NIV

on July 30, 2008 aac.asm

.orgD

ownloaded from

15

simultaneously several virulence factors and therefore causing severe infections was relatively

small in the pre-ESBL era, it has been significantly increased in the post–ESBL era.

Unravelling the complex intracellular events that promote the expression of virulence factors

upon acquisition of ESBL-plasmid may provide a knowledgeable approach for better

management of nosocomial infections caused by ESBL-producers.

ACCEPTED

at TE

L AV

IV U

NIV

on July 30, 2008 aac.asm

.orgD

ownloaded from

16

References

1. Connell, I., Agace, W., Klemm, P., Schembri, M., Mărild, S., and C. Svanborg.

1996. Type-1 fimbrial expression enhances Escherichia coli virulence for the urinary

tract.

Proc. Natl. Acad. Sci. U S A. 93:9827-32.

2. Darfeuille-Michaud, A., C. Jallat, D. Aubel, D. Sirot, C. Rich, J. Sirot, and B. Joly.

1992. R-plasmid-encoded adhesive factor in Klebsiella pneumoniae strains responsible

for human nosocomial infections. Infect. Immun. 60:44-45.

3. De Vries, F. P., van der Ende, A., van Putten, J. P. M., and J. Dankert. 1996.

Invasion of primary nasopharyngeal epithelial cells by Neisseria meningitidis is

controlled by phase variation of multiple surface antigens. Infect. Immun. 64:2998-

3006.

4. Di Martino, P., Cafferini, N., Joly, B., and A. Darfeuille-Michaud. 2003. Klebsiella

pneumoniae type-3 pili facilitate adherence and biofilm formation on abiotic surfaces.

Res. Microbiol. 154:9-16.

5. Di Martino, P., D. Sirot, B. Joly, C. Rich, and A. Darfeuille-Michaud. 1997.

Relationship between adhesion to intestinal Caco-2 cells and multidrug resistance in

Klebsiella pneumoniae clinical isolates. J. Clin. Microbiol. 35:1499-1503.

6. Di Martino, P., V. Livrelli, D. Sirot, B. Joly, and A. Darfeuille-Michaud. 1996. A

new fimbrial antigen harbored by CAZ-5/SHV-4-producing Klebsiella pneumoniae

strains involved in nosocomial infections. Infect. Immun. 64:2266-2273.

7. Donnenberg, M. S. 1999. Interactions between enteropathogenic Escherichia coli and

epithelial cells. Clin. Infect. Dis. 28:451-455.

8. Donnenberg, M. S., Kaper, J. K., and B. B., Finlay. 1997. Interaction between

enteropathogenic Escherichia coli and host epithelial cells. Trends Microbiol. 5:109-

114.

ACCEPTED

at TE

L AV

IV U

NIV

on July 30, 2008 aac.asm

.orgD

ownloaded from

17

9. Duncan, M. J., Mann, E. L., Cohen, M. S., Ofek, I., Sharon, N., and S. N. Abraham.

2005. The distinct binding specificities exhibited by enterobacterial type-1 fimbriae are

determined by their fimbrial shafts. J. Biol. Chem. 280:37707-37716.

10. Fader, R. C., and C. P. Davis. 1980. Effect of piliation on Klebsiella pneumoniae

infection in rat bladders. Infect. Immun. 30:554-561.

11. Fader, R. C., and C. P. Davis. 1982. Klebsiella pneumoniae induced experimental

pyelitis: the effect of piliation on infectivity. J. Urol. 128:197-201.

12. Fader, R. C., Gondesen, K., Tolley, B., Ritchie, D.G., and P. Moller. 1988. Evidence

that in vitro adherence of Klebsiella pneumoniae to ciliated hamster tracheal cells is

mediated by type-1 fimbriae. Infect. Immun. 11:3011-3013.

13. Favre-Bonte, S., Joly, B., and C. Forestier. 1999. Consequences of reduction of

Klebsiella pneumoniae capsule expression on interaction of this bacterium with

epithelial cells. Infect. Immun. 67:554-561.

14. Fumagalli, O., Tall, B., Schipper, C., and T. A. Oelschlaeger. 1997. N-Glycosylated

proteins are involved in efficient internalization of Klebsiella pneumoniae by cultured

human epithelial cells. Infect. Immun. 65:4445-4451.

15. Gaynes R., and J. R. Edwards. 2005. National Nosocomial Infections Surveillance

System.Overview of nosocomial infections caused by gram-negative bacilli. Clin.

Infect. Dis. 41:848-854.

16. Geffers, C., Zuschneid, I., Sohr, D., Ruden, H., and P. Gastmeier. 2004.

Microbiological isolates associated with nosocomial infections in intensive care units:

Data of 274 intensive care units participating in the German Nosocomial Infections

Surveillance System (KISS). Anaesthesiol. Intensivmed. Notfallmed. Schmerzther.

39:15-19.

17. Gerlach, G. F., and S. Clegg. 1988. Characterization of two genes encoding distinct

type-1 fimbriae of Klebsiella pneumoniae. Gene. 64:231-240

ACCEPTED

at TE

L AV

IV U

NIV

on July 30, 2008 aac.asm

.orgD

ownloaded from

18

18. Gerlach, G. F., Clegg, S., and B.L. Allen. 1989. Identification and characterization

ofthe genes encoding the type-3 and type-1 fimbrial adhesins of Klebsiella pneumoniae.

J. Bacteriol. 171:1262-1270.

19. Gikas, A., Samonis, G., Christidou, A., Papadakis, J., Kofteridis, D., Tselentis, Y.

and N. Tsaparas. 1998. Gram-negative bacteremia in non-neutropenic patients: a 3-

year review. Infection. 26:155-159.

20. Harbarth, S., Ruef, C., Francioli, P., Widmer, A., and D. Pittet. 1999. Nosocomial

infections in Swiss university hospitals: a multi-centre survey and review of the

published experience. Swiss-Noso Network. Schweiz. Med. Wochenschr. 129:1521-

1528.

21. Hennequin, C., and C. Forestier. 2007. Influence of capsule and extended-spectrum

beta-lactamases encoding plasmids upon Klebsiella pneumoniae adhesion. Research in

Microbiology. 158:339-347.

22. Hornick, D. B., Allen, B. l., Horn, M. A., and S. Clegg. 1991. Fimbrial types among

respiratory isolates belonging to the familiy Enterobacteriaceae. J. Clin. Microbiol.

29:1795-1800.

23. Hornick, D. B., Allen, B. l., Horn, M. A., and S. Clegg. 1992. Adherence to

respiratory epithelia by recombinant Escherichia coli expressing Klebsiella pneumoniae

type-3 fimbrial gene products. Infect. Immun. 60:1577-1588

24. Hornick, D. B., Thommandru, J., Smits, W., and S. Clegg. 1995. Adherence

properties of an mrkD-negative mutant of Klebsiella pneumoniae. Infect. Immun.

63:2026-2032.

25. Jones, R. N., Mendes, C., Turner, P. J., and R. Masterton. 2005. An overview of the

Meropenem Yearly Susceptibility Test Information Collection (MYSTIC) Program:

1997-2004. Diagn. Microbiol. Infect. Dis. 53:247-256.

ACCEPTED

at TE

L AV

IV U

NIV

on July 30, 2008 aac.asm

.orgD

ownloaded from

19

26. Klemm, P., and M. A. Schembri. 2000. Bacterial adhesins: function and structure. Int.

J. Med. Microbiol. 290:27-35.

27. Kohlenberg, A., Schwab, F., Geffers, C., Behnke, M., Rüden, H., Gastmeier, P.

2007. Time-trends for Gram-negative and multidrug-resistant Gram-positive bacteria

associated with nosocomial infections in German intensive care units between 2000 and

2005. Clin. Microbiol. Infect. 14: 82-96.

28. Lee, S. Y. , Kotapati, S., Kuti, J. L., Nightingale, C. H., and D. P. Nicolau. 2006.

Impact of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella

species on clinical outcomes and hospital costs: a matched cohort study. Infect. Control

Hosp. Epidemiol. 27:1226-1232.

29. Livrelli, V., De Champ, C. Di Martino, P., Darfeuille-Michaud, A, Forestier, C.,

and B. Joly. 1996. Adhesive properties and antibiotic resistance of Klebsiella,

Enterobacter, and Serratia clinical isolates involved in nosocomial infections. J. Clin.

Microbiol. 34:1963-1969.

30. Maayan, M. C., Ofek, I., Medalia, O., and M. Aronson. 1985. Population shift in

mannose-specific fimbriated phase of Klebsiella pneumoniae during experimental

urinary tract infection in mice. Infect. Immun. 49:785-789.

31. Mulvey, M. A., Lopez-Boado, Y. S., Wilson, C. L., Roth, R., Parks, W. C., Heuser,

J. and S. J. Hultgren. 1998. Induction and evasion of host defenses by type-1-piliated

uropathogenic Escherichia coli. Science. 282:1494–1497.

32. Mulvey, M. A., Schilling, J. D., Martinez, J. J., and S. J. Hultgren. 2000. Bad bugs

and beleaguered bladders: interplay between uropathogenic Escherichia coli and innate

host defenses. Proc Natl Acad Sci U S A. 97:8829-8835.

33. Oelschlaeger, T., and B. Tall. 1997. Invasion of cultured human epithelial cells by

Klebsiella pneumoniae isolated from the urinary tract. Infect. Immun. 65:2950-2958.

ACCEPTED

at TE

L AV

IV U

NIV

on July 30, 2008 aac.asm

.orgD

ownloaded from

20

34. Ofek, I., D. Hasty, and R. J. Doyle. 2003. Bacterial adhesion to animal cells and

tissues. ASM press, Washington, D.C.

35. Ofek, I., Hasty, D. L., and N. Sharon. 2003. Anti-adhesion therapy of bacterial

diseases: prospects and problems. FEMS Immunol Med Microbiol. 38:181-191.

36. Paterson, D. L. Resistance in gram-negative bacteria: Enterobacteriaceae. 2006. Am. J.

Med. 119:20-28

37. Paterson, D. L., and R. A. Bonomo. 2005. Extended-spectrum beta-lactamases: a

clinical update. Clin. Microbiol. Rev. 18:657-686.

38. Paterson, D. L., Ko, W. C., Von Gottberg, A., Mohapatra, S., Casellas, J. M.,

Goossens, H., Mulazimoglu, L., Trenholme, G., Klugman, K. P., Bonomo, R. A.,

Rice, L. B., Wagener, M. M., McCormack, J. G., and V. L. Yu. 2004. Antibiotic

therapy for Klebsiella pneumoniae bacteremia: implications of production of extended-

spectrum beta-lactamases. Clin. Infect. Dis. 39:31-37.

39. Paterson, D. L., Rossi, F., Baquero, F., Hsueh, P. R., Woods, G. L., Satishchandran,

V., Snyder, T. A., Harvey, C. M., Teppler, H., Dinubile, M. J., and J. W. Chow.

2005. In vitro susceptibilities of aerobic and facultative Gram-negative bacilli isolated

from patients with intra-abdominal infections worldwide: the 2003 Study for Monitoring

Antimicrobial Resistance Trends (SMART). J. Antimicrob. Chemother. 55:965-973.

40. Podschun, R., and H. Sahly. 1991. Haemagglutinins of Klebsiella pneumoniae and K.

oxytoca isolated from different sources. Int. J. Hyg. Environ. Health. 191:46-52.

41. Rossi, F., Baquero, F., Hsueh, P. R., Paterson, D. L., Bochicchio, G. V., Snyder, T.

A., Satishchandran, V., McCarroll, K., DiNubile, M. J., and J. W Chow. 2006. In

vitro susceptibilities of aerobic and facultatively anaerobic Gram-negative bacilli

isolated from patients with intra-abdominal infections worldwide: 2004 results from

SMART (Study for Monitoring Antimicrobial Resistance Trends). J. Antimicrob.

Chemother. 58:205-210.

ACCEPTED

at TE

L AV

IV U

NIV

on July 30, 2008 aac.asm

.orgD

ownloaded from

21

42. Sahly, H., Aucken, H., Benedi, V. J., Forestier, C., Fussing, V., Hansen, D. S., Ofek,

I., Podschun, R., Sirot, D., Tomás, J. M. and U. Ullmann. 2004. Impairment of

respiratory burst in polymorphonuclear leukocytes by extended-spectrum beta-

lactamase-producing strains of Klebsiella pneumoniae. Eur. J. Clin. Microbiol. Infect.

Dis. 23:20-26.

43. Sahly, H., Aucken, H., Benedi, V. J., Forestier, C., Fussing, V., Hansen, D. S., Ofek,

I., Podschun, R., Sirot, D., Tomás, J. M. and U. Ullmann. 2004. Increased serum

resistance properties in Klebsiella pneumoniae strains producing extended spectrum ß-

lactamases. Antimicrob. Agents Chemother. 48: 3477-3482.

44. Sahly, H., Ofek, I., Podschun, R., Brade, H., He, Y., and E. Crouch. 2002. Surfactant

protein D binds selectively to Klebsiella pneumoniae lipopolysaccharides containing

mannose-rich-O-antigens. J. Immunol. 169:3267-3274.

45. Sahly, H., Podschun, R., Oelschlaeger, T. A., Greiwe, M., Parolis, H., Hastey, D.,

Kekow, J., Ullmann, U., Ofek, I., and S. Sela. 2000. Capsule impedes adhesion and

invasion of epithelial cells by Klebsiella pneumoniae. Infect. Immun. 68:6744-6749.

46. Schwaber, M. J., and Y. Carmeli. 2007. Mortality and delay in effective therapy

associated with extended-spectrum {beta}-lactamase production in Enterobacteriaceae

bacteraemia: a systematic review and meta-analysis. J. Antimicrob. Chemother. 60:913-

920.

47. Schwaber, M. J., Navon-Venezia, S., Kaye, K. S., Ben Ami, R., Schwartz, D., and

Y. Carmeli. 2006. Clinical and Economic Impact of Bacteremia with Extended-

Spectrum-β-Lactamase-Producing Enterobacteriaceae. Antimicrob. Agents Chemother.

50:1257-1262.

48. Spencer, R. C. 1996. Predominant pathogens found in the European Prevalence of

Infection in Intensive Care Study. Eur. J. Clin. Microbiol. Infect. Dis. 15:281-285.

ACCEPTED

at TE

L AV

IV U

NIV

on July 30, 2008 aac.asm

.orgD

ownloaded from

22

49. Tarkkanen, A. M., Allen, B. L., Westerlund, B., Holthofer, H., Kuusela, P., Risteli,

L., Clegg, S., and T. K. Korhonen. 1990. Type V collagen as the target for type-3

fimbriae enterobacterial adherence organelles. Mol. Microbiol. 4:1353-1361.

50. Tarkkanen, A. M., Virkola, R., Clegg, S., and T. K. Korhonen. 1997. Binding of the

type-3 fimbriae of Klebsiella pneumoniae to human endothelial and urinary bladder

cells. Infect. Immun. 1997. 65:1546-1549

51. Vincent, J. L. 2003. Nosocomial infections in adult intensive-care units. Lancet. 361:

2068-2077.

52. Würker, M., Beuth, J., Ko, H.L., Przondo-Mordarska, A., and G. Pulverer. 1990.

Type of fimbriation determines adherence of Klebsiella bacteria to human epithelial

cells. Zbl. Bakt. 274: 239-245.

53. Yinnon, A. M., Schlesinger, Y., Gabbay, D., and B. Rudensky. 1997. Analysis of 5

years of bacteraemias: importance of stratification of microbial susceptibilities by

source of patients. J. Infect. 35:17-23.

ACCEPTED

at TE

L AV

IV U

NIV

on July 30, 2008 aac.asm

.orgD

ownloaded from

23

Table 1. Distribution of type-1 and type-3 pili expressing isolates among ESBL and non-

ESBL-producing K. pneumoniae strains.

Hemagglutination % of strains (n)

pattern no type-1 & 3 only type-1 only type-3 type-1 & 3*

ESBL 3.5 (2/58) 12 (7/58) 5.1 (3/58) 79.3 (46/58)

Non-ESBL 5.2 (8/152) 20 (32/152) 11.8 (18/152) 61.8 (94/152)

* Significantly higher frequency of ESBL-producing strains co-expressing both type-1 and

type-3 fimbrial adhesins than non-ESBL-producers (p = 0.0214; OR 2,365, CI 1.157 – 4.834).

ACCEPTED

at TE

L AV

IV U

NIV

on July 30, 2008 aac.asm

.orgD

ownloaded from

24

Table 2. Invasion of HCT8 ileocecal, T24 bladder, and A549 alveolar epithelial cells by

ESBL and non-ESBL-producing Klebsiella isolates.

Invasion ESBL producing strains Non-ESBL-producing strains

% (n) % (n)

HCT8

Grade I 19 (11/58) 39 (59/152)

Grade II 81 (47/58)a 61 (93/152)

T24

Grade I 72.4 (42/58) 90 (137/152)

Grade II 27.6 (16/58)a 10 (15/152)

A549

Grade I 100 (47/47)b 100 (50/50)

b

a significantly higher frequency of ESBL-producing strains that effectively invade the HCT8

ileocecal (p=0.0084; OR = 2.711; 95% CI = 1.302 to 5.643) and T24 bladder epithelial cells

(p = 0.0021; OR = 3.479; 95% CI 1.587 to 7.627) as compared to the frequency among non-

ESBL-producers. Grade I, relative invasion <5%; grade II, relative invasion >5% of

inoculum.

binvasion did not exceed 1% in all strains

ACCEPTED

at TE

L AV

IV U

NIV

on July 30, 2008 aac.asm

.orgD

ownloaded from

25

LEGENDS TO FIGURES

Figure 1. Mannose-sensitive hemagglutination of guinea-pig erythrocytes(A) and mannose-

resistant (Klebsiella-like hemagglutination) of tanned ox erythrocytes (B) by ESBL- and non-

ESBL-producing K. pneumoniae isolates. –: no hemagglutination; +: weak hemagglutination,

++: mediate hemagglutination; +++: strong hemagglutination

Figure 2. Adhesion of ESBL and non-ESBL-producing strains to ileocecal, bladder and

alveolar epithelial cells.

Figure 3. Mean relative invasion of T24 bladder and HCT8 ileocecal epithelia cells and

distribution of invasive strains among ESBL and non ESBL-producers. * Significantly higher

invasive ability of the cells by ESBL-producing strains (p< 0.0001).

Figure 4. Invasion and adhesion ability of five ESBL-producing strains and their

corresponding plasmid-cured derivatives.

ACCEPTED

at TE

L AV

IV U

NIV

on July 30, 2008 aac.asm

.orgD

ownloaded from

26

- + ++ +++

non-ESBL

ESBL

mannose-sensitive hemagglutination

0

10

20

30

40

50

60%

ofis

ola

tes

- + ++ +++

non-ESBL

ESBL

0

10

20

30

40

50

60

%of

isola

tes

A

B

mannose-resistant hemagglutination

Figure 1

ACCEPTED

at TE

L AV

IV U

NIV

on July 30, 2008 aac.asm

.orgD

ownloaded from

27

non-ESBL

ESBL

0.1

0.3

0.5

0.4

0.2

0.7

0.6

Adhes

ion

(Abso

rban

ce4

90nm

)

HCT8 T24 A549

Figure 2

ACCEPTED

at TE

L AV

IV U

NIV

on July 30, 2008 aac.asm

.orgD

ownloaded from

28

Figure 3

18

16

14

12

10

8

6

4

2

0

T24 HCT8

Mean

rela

tive invasio

n(%

of

inoculu

m)

*

*

ESBL Non-ESBL ESBL Non-ESBL

18

16

14

12

10

8

6

4

2

0

T24 HCT8

Mean

rela

tive invasio

n(%

of

inoculu

m)

*

*

*

*

ESBL Non-ESBL ESBL Non-ESBLACCEPTED

at TE

L AV

IV U

NIV

on July 30, 2008 aac.asm

.orgD

ownloaded from

29

7

6

5

4

3

2

1

0

plasmid-cured derivativesESBL-producing parental strains

mea

nin

vas

ion

(%of

ino

culu

m)

0.1

0.3

0.5

0.4

0.2

0.7

0.6

Adhes

ion

(Abso

rban

ce490nm

)

HCT8 T24 A549HCT8 T24

Figure 4

ACCEPTED

at TE

L AV

IV U

NIV

on July 30, 2008 aac.asm

.orgD

ownloaded from