Virulence factors and TEM-type β-lactamases produced by two isolates of an epidemic Klebsiella pneumoniae strain.

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Virulence Factors and TEM-Type

␤-Lactamases Produced by Two

Isolates of an Epidemic Klebsiella pneumoniae Strain

Frédéric Robin,a,b,c_{Claire Hennequin,}a,d_{Marek Gniadkowski,}e_{Racha Beyrouthy,}b,c,f_{Joanna Empel,}e_{Lucie Gibold,}a,b,c and Richard Bonneta,b,c

CHU Clermont-Ferrand, Centre de Biologie, Laboratoire de Bactériologie Clinique, Clermont-Ferrand, Francea_{; Clermont Université, Université d’Auvergne, Evolution des} Bactéries Pathogènes et Susceptibilité Génétique de l’Hôte, Clermont-Ferrand, Franceb_{; INRA, USC2018, Clermont-Ferrand, France}c_{; Clermont Université, Université} d’Auvergne, Laboratoire de Bactériologie, Faculté de Pharmacie, Clermont-Ferrand, Franced_{; National Medicines Institute, Warsaw, Poland}e_{; and Faculty of Public Health,} Lebanese University, Tripoli, Lebanonf

Two Klebsiella pneumoniae isolates of the same strain, identified in Poland, produced either TEM-47 or TEM-68, which differed

by the Arg275Leu substitution. They harbored a few virulence factors, including an iron-chelating factor and capsule

overpro-duction, suggesting that these factors were sufficient to enhance their nosocomial potency. TEM-68 and TEM-47 had similar

en-zymatic activities, but TEM-68 was less susceptible to inhibitors than TEM-47. These results confirm the role of the Arg275Leu

substitution in the evolution of TEM enzymes.

K

lebsiella pneumoniae strains are responsible for

community-acquired and nosocomial infections. Different virulence

fac-tors have been involved in the infective potency of the

community-acquired strains, such as K1 and K2 capsular

sero-types, types 3 and 1 fimbriae, capsule synthesis, production of

iron-chelating agents, hypermucoviscosity, biofilm formation

ability, and the presence of the genomic island encoding the

bio-synthesis of the colibactin (3, 10, 11, 14, 17, 18, 20, 25, 27, 33, 40).

However, data concerning the virulence factors expressed by

nos-ocomial strains of K. pneumoniae are scarce. The strains

respon-sible for nosocomial epidemics are usually multiresistant to

anti-biotics, and most of them produce extended-spectrum

␤-lactamases (ESBLs) (8, 26). The largest ESBL family is the TEM

family (5). However, another subgroup of enzymes has appeared

that combine the substitutions observed in the ESBLs and in the

inhibitor-resistant TEM (IRT): its members were designated

com-plex mutant TEMs (CMTs). They have been observed mainly in

Escherichia coli and mostly in France (16, 29, 31, 34–37, 39). The

enzyme TEM-68 (CMT-2) was identified by Fiett et al. in an

epi-demic strain of K. pneumoniae and appeared to have emerged

from the ESBL TEM-47 (16). The clinical isolate that produced

the ESBL TEM-47 was isolated from eight patients hospitalized in

the neonatal ward of the University Hospital of Wrocław, Poland.

During this spread, another isolate, which produced TEM-68, was

isolated from the respiratory tract of a ninth newborn. All of the

isolates were indistinguishable by pulsed-field gel electrophoresis

(15). TEM-68 and TEM-47 differed from TEM-1 by the

substitu-tions Gly238Ser, Glu240Lys, and Thr265Met. TEM-68 differed

from TEM-47 by the substitution Arg275Leu observed in the IRT

TEM-103 (IRT-28) (1, 16).

In this work, we studied the virulence factors that could have

allowed their nosocomial dissemination and characterized the

en-zymes TEM-47, TEM-68, and TEM-103 to understand the role of

the Arg275Leu substitution in TEM evolution.

Multilocus sequence type analysis confirmed that all the

iso-lates belonged to the same new sequence type (ST), ST514 (9). The

clinical isolates 3144 and 3151, which produced TEM-47 and

TEM-68, respectively, were selected for further experiments. They

did not belong to the K1 or K2 genotypes (15). They were

inves-tigated for the presence of different virulence genes as

recom-mended by Brisse et al. (3), for the presence of the colibactin

genomic island (32), and for the production of the iron-chelating

factor aerobactin (28) and of type 1 fimbriae (19). They harbored

the genes wabG and uge, which are involved in lipopolysaccharide

synthesis and have been associated with virulence, the kfu gene,

which encodes an iron-chelating factor, and the gene mrkD, which

encodes a type 3 fimbria. They also expressed type 1 fimbriae.

Using a microtiter plate experimental model (30), we showed that

the two isolates produced no biofilm (Fig. 1). Since the capsule is

one of the main virulence factors of nosocomial K. pneumoniae

strains, we investigated the amount of capsular polysaccharides

produced by the two isolates. They could both be categorized as

high-level producers (⬎15

␮g of glucuronic acid/0.5 ml of

cul-ture) (18). This high level of capsule could be important in the

virulence because the capsule plays an important role in

protect-ing K. pneumoniae from intracellular killprotect-ing, complement,

surfac-tant, oxidative stress, and phagocytosis (12, 13, 38). Lawlor et al.

observed in an intranasal infection model that their

capsule-defective mutant was not able to grow in lung or tracheal tissues

and was unable to progress to a disseminated infection (24). Our

results suggest that a nosocomial strain does not need to be highly

virulent but only requires factors that allow its subsistence in the

human body, such as iron-chelating factors, and that protect it

from host defenses, such as high production of capsule, especially

when the strain is associated with a high resistance to antibiotics.

E. coli DH5

␣ isogenic clones producing TEM-68, TEM-47,

TEM-103, and TEM-1 were obtained using the vector pBK-CMV,

as previously described (34). The MICs of the clinical strain and

the different E. coli DH5

␣ clones were determined in duplicate by

Received 13 June 2011 Returned for modification 31 July 2011 Accepted 12 November 2011

Published ahead of print 21 November 2011

doi:10.1128/AAC.05079-11

0066-4804/12/$12.00 Antimicrobial Agents and Chemotherapy p. 1101–1104 aac.asm.org 1101

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a microdilution method and were interpreted according to the

guidelines of the CASFM (7) (Table 1). The clinical isolates 3144

and 3151 were highly resistant to penicillins and also

demon-strated resistance to cefuroxime, cefotaxime, ceftazidime, and

az-treonam. They were intermediate or resistant to cefepime but

re-mained susceptible to imipenem. K. pneumoniae 3144 was less

resistant to

␤-lactam–␤-lactamase inhibitor combinations than

K. pneumoniae 3151. Cefoxitin remained active only against

iso-late 3144. TEM-68-producing E. coli DH5␣ harbored high levels

of resistance to penicillins, close to those of the TEM-103- and

TEM-47-producing clones. However, although its MICs for

penicillin-clavulanate combinations were the same as those of the

TEM-47-producing clone, they were lower than those of the

TEM-103-producing clone. The piperacillin-tazobactam

combi-nation was active against the three clones. The cephalosporin and

aztreonam MICs of the TEM-68-producing clone were close to

those of the TEM-47-producing E. coli. Finally, as observed with

the TEM-47-producing clone, E. coli DH5␣(pBK-TEM-68) was

susceptible to all oxyimino

␤-lactam–clavulanate combinations.

TEM-68, TEM-47, TEM-103, and TEM-1 were overproduced in

E. coli BL21(DE3), as previously described (36). They were

puri-fied to homogeneity, and the level of purity was estimated to be

⬎95% by sodium dodecyl sulfate-polyacrylamide gel

electropho-resis (2, 23). Their kinetic parameters (Table 2) were determined

by computerized microacidimetry as previously described (22).

TEM-68 was slightly more active than TEM-47 against penicillins

but remained 6- to 52-fold less active than TEM-103, and TEM-1.

Its Km

values for these substrates were also slightly lower than

those of TEM-47. TEM-68’s catalytic efficiency against penicillins

was higher than that of TEM-47 but remained 3- to 10-fold lower

than those of TEM-103 and TEM-1. TEM-68 harbored hydrolytic

activity against cephalothin that was close to those of TEM-47 and

TEM-1 and 1.8-fold lower than that of TEM-103. The cephalothin

K

m

value was 1.6- to 7.6-fold lower for TEM-68 than for TEM-47,

TEM-103, and TEM-1. TEM-68’s catalytic efficiency against this

substrate was 1.7- to 6.3-fold higher than the catalytic efficiencies

of the three parental enzymes. TEM-68’s hydrolytic activities

against ceftazidime, cefotaxime, cefepime, and aztreonam were

close to that of TEM-47. Its Km

values for these substrates were

close to or even lower than those of TEM-47. Finally, its catalytic

efficiency against cefotaxime was close to that of TEM-47, and

against ceftazidime and cefepime, its catalytic efficiencies were

1.5- to 2.2-fold higher than those of TEM-47. TEM-68 was 2- to

2.6-fold less susceptible to clavulanate and tazobactam than

TEM-47 (50% inhibitory concentrations [IC

50

s], 0.02 and 0.08

versus 0.01 and 0.03

␮M), but it remained more susceptible than

TEM-1 and TEM-103 (IC

50

s, 0.02 and 0.08 versus 0.08 and 0.13

FIG 1 K. pneumoniae 3144 (TEM-47) and 3151 (TEM-68) biofilm formation.

Biofilms were developed in Dulbecco’s modified Eagle’s medium at 37°C after 6 h of incubation and stained with 0.5% crystal violet. (1) Positive control K.

pneumoniae LM21; (2) K. pneumoniae 3144; (3) K. pneumoniae 3151.

TABLE 1 MICs of␤-lactam antibiotics for K. pneumoniae 3144 (TEM-47), K. pneumoniae 3151 (TEM-68) and E. coli DH5␣ recombinants carrying

the indicated plasmids

␤-Lactama MIC (␮g/ml) for: K. pneumoniae 3144 (TEM-47) K. pneumoniae 3151 (TEM-68) E. coli DH5␣ carrying:

pBK-TEM-47 pBK-TEM-68 pBK-TEM-103 pBK-TEM-1 pBK-CMV

Amoxicillin ⬎2,048 ⬎2,048 ⬎2,048 ⬎2,048 ⬎2,048 ⬎2,048 4 Amoxicillin⫹ CLA 8 ⬎1,024 8 8 ⬎1,024 16 4 Ticarcillin ⬎2,048 ⬎2,048 ⬎2,048 ⬎2,048 ⬎2,048 ⬎2,048 2 Ticarcillin⫹ CLA 32 ⬎1,024 128 128 ⬎1,024 32 2 Piperacillin 1,024 1,024 ⬎2,048 ⬎2,048 ⬎2,048 512 2 Piperacillin⫹ TZB 4 1,024 2 1 2 2 2 Cephalothin ⬎256 ⬎256 ⬎256 ⬎256 64 4 4 Cefoxitin 4 16 4 4 4 4 4 Cefuroxime 32 256 256 256 0.06 4 4 Cefotaxime 8 32 32 16 0.06 0.06 0.06 Cefotaxime⫹ CLA 0.06 32 0.06 0.06 0.06 0.06 0.06 Ceftazidime 256 512 1,024 512 0.12 0.12 0.12 Ceftazidime⫹ CLA 0.25 256 1 0.5 0.06 0.12 0.12 Aztreonam 128 128 32 32 0.12 0.12 0.12 Aztreonam⫹ CLA 0.12 128 0.12 0.12 0.12 0.12 0.12 Cefepime 2 32 4 4 ⬍0.06 ⬍0.06 ⬍0.06 Cefepime⫹ CLA 0.25 32 ⬍0.06 ⬍0.06 ⬍0.06 ⬍0.06 ⬍0.06 Imipenem 0.12 0.25 0.25 0.25 0.25 0.25 0.25

a_{CLA, clavulanic acid at 2}_{␮g/ml; TZB, tazobactam at 4 ␮g/ml.} Robin et al.

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␮M for TEM-1 and 0.72 and 0.22 ␮M for TEM-103). The

stan-dard deviations for the IC

50

results were lower than 15%. As

sug-gested by the MIC results, TEM-68 and TEM-47 have closely

re-lated kinetic parameters. However, TEM-68 was more active

against some substrates, including ceftazidime and aztreonam,

and harbored lower Km

values for cefotaxime and cefepime, which

allowed higher catalytic efficiencies against most of the oxyimino

␤-lactams tested. Increases in catalytic efficiencies against most

substrates, especially cephalothin, were also observed for the

Arg275Leu-harboring IRT TEM-103 in comparison to the

cata-lytic efficiencies of TEM-1. Brown et al. also reported that the

substitution Arg275Glu affected the catalytic efficiency of TEM

enzymes (4); the presence of the Glu275 residue decreased the

catalytic efficiency through an increase in K

m

. In contrast, the

catalytic efficiencies of the Arg275Leu-harboring enzymes

TEM-68 and TEM-103 against most substrates were higher than

those of the parental enzymes, TEM-47 and TEM-1, respectively,

suggesting a role for Leu275 in the increase in catalytic efficiency.

There is little structural information about the Arg275Leu

substi-tution. Most authors consider that the Arg275Leu/Glu

substitu-tions probably modify the position of Arg244, as observed for the

substitutions at position 276 (6). Recently, Kather et al. and

Brown et al. observed that the Arg275Leu/Glu substitutions have a

stabilizing action in TEM enzymes (4, 21).

In conclusion, our study suggests that the production of a high

quantity of capsule and a few other virulence factors could be

sufficient to enhance the dissemination potency of a K.

pneu-moniae strain in a nosocomial setting, especially when associated

with the production of an ESBL. We also confirmed the role of the

Arg275Leu substitution in TEM-68 as a catalytic efficiency

en-hancer.

ACKNOWLEDGMENTS

We thank Marlene Jan and Rolande Perroux for their technical assistance, Sophie Quevillon-Cheruel for providing the modified pET9a plasmid, and Patrice Courvalin for providing us the clinical E. coli strain BM4511. This work was supported in part by grants from the Ministère de l’Enseignement Supérieur et de la Recherche (JE2526), INRA (USC2018), the Centre Hospitalier Régional Universitaire de Clermont-Ferrand, France, and the Ministère de la Santé, de la Jeunesse et des Sports.

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extended-TABLE 2 Kinetic parameters of␤-lactamases TEM-47, TEM-68, TEM-103, and TEM-1a

␤-Lactam

TEM-68 (Ser238, Lys240, Met265, Leu275)

TEM-47 (Ser238, Lys240, Met265, Arg275)

TEM-103 (Gly238, Glu240, Thr265, Leu275)

TEM-1 (Gly238, Glu240, Thr265, Arg275) kcat (s⫺1₎ Km (␮M) kcat/Km (␮M⫺1_{· s}⫺1₎ kcat (s⫺1₎ Km (␮M) kcat/Km (␮M⫺1_{· s}⫺1₎ kcat (s⫺1₎ Km (␮M) kcat/Km (␮M⫺1_{· s}⫺1₎ kcat (s⫺1₎ Km (␮M) kcat/Km (␮M⫺1_{· s}⫺1₎ Benzylpenicillin 61.2 17.9 3.4 30.8 24.8 1.2 3,190 89.5 36 1,500 34 44 Amoxicillin 156.5 22.4 7 66.5 27.5 2.4 2,563 69.7 37 1,125 15 75 Ticarcillin 20.9 17.3 1.2 17.1 34.3 0.5 411 47.9 8.6 135 36 4 Piperacillin 172.7 45.3 3.8 150.1 52.6 2.8 1,228 30.15 41 1,250 55 23 Cephalothin 139.3 31.9 4.4 127.1 48 2.6 246 148 1.7 165 242 0.7 Ceftazidime 22.7 29.3 0.77 19.8 56.3 0.35 ⬍0.1 ND ⬍0.1 ND Cefotaxime 178.1 100.4 1.8 212.7 110.9 1.9 ⬍0.1 ND ⬍0.1 ND Aztreonam 0.9 17.2 0.05 ⬍0.2 40.5b _⬍0.1 _ND _⬍0.1 _ND Cefepime 18 30.5 0.59 34.8 89.5 0.39 ⬍0.1 ND ⬍0.1 ND

a_{The standard deviation for analysis was}_{ⱕ15%. ND, not determined.} b_K

mvalues were determined as the Kivalues by substrate competition with benzylpenicillin.

␤-Lactamases and Virulence of a K. pneumoniae Strain

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