Klebsiella Pneumoniae Associated with SHV-11 Hyperproduction

Annals of Microbiology, 58 (4) 727-730 (2008)

Cefotaxime and ceftriaxon resistant Klebsiella pneumoniae associated with SHV-11 hyperproduction

Nahed BEN ACHOUR1*, Paola Sandra MERCURI2, Chrifa BELHADJ1, Mohamed BEN MOUSSA3, Moreno GALLENI2, Omrane BELHADJ1

1Laboratoire de Biochimie et de Technobiologie, Faculté des Sciences de Tunis, Campus Universitaire, 2092 EL Manar II, Tunisie; 2Laboratoire de Macromolécules Biologiques, Centre d’Ingénierie des Protéines, Université de Liège, 4000 Sart-Tilman, Belgique; 3Service de Bactériologie, Hôpital Militaire de Tunis, Tunisie

Received 30 May 2008 / Accepted 23 September 2008

Abstract - Klebsiella pneumoniae ML0306 was isolated from a patient in the intensive care unit of the Military hospital of Tunis in Tunisia. The strain was resistant to β-lactams, aminoglycosides, quinolones, phenicols and tetracyclines. Crude extract from the isolate showed a broad-substrate profile by hydrolyzing benzylpenicillin, ampicillin, cloxacilline, cephalothin, cefotaxime and ceftriaxone but not ceftazidime, cefoxitin, cefpirome, imipinem and aztreonam. The isolate was identified as a producer of pI 7.6 β-lactamase. Sequencing of the β-lactamase gene showed that it coded for a penicillinase SHV-11. This is the first description of this enzyme in Tunisia. The gene encoding this enzyme was located on the chromosome and a 50 kb conjugative plasmid. The overproduction of SHV- 11 was involved in the wading of the hydrolytic spectrum of the strain.

Key words: β-lactamases; SHV-11; penicillinase; specific activity; overproduction.

INTRODUCTION Klebsiella pneumoniae ML0306 was identified using an API 20 E system (bio-Mérieux, Marcy l’Etoile, France). Biochemical Resistance to antibiotics may be caused by several mechanisms, and serological typing of this strain was done at the Laboratory but the most important of which is the production of β-lactamase of Bacteriology, Military Hospital, Tunisia. Disk diffusion assay that hydrolyzes the β-lactam ring (Lin et al., 2003; Liu et al., with Mueller-Hinton agar (Bio Rad, France) and MICs (CLSI, 2007). β-Lactamases (EC 3.5.2.6) are the primary agents of bac- 2006) indicated that the strain ML0306 was resistant to ampicil- terial resistance to penicillins and cephalosporins. The enzymes lin, amoxicillin, ticarcillin, cephaloridine, ceftazidime, aztreonam, are divided into four major molecular classes, classes A, B, C, kanamycin, chloramphenicol, ciprofloxacin, nalidixic acid, tetra- and D on the basis of their amino acid sequences (Nukaga et al., cycline and streptomycin (Table 1). The strain was moderately 2003; Wachino et al., 2004, 2006). All β-lactamases except the susceptible to cefoxitin, ceftriaxon, cefpirome, susceptible to zinc-containing class B enzymes are serine-reactive hydrolases imipenem and phenotypically negative for ESBL production by that act via an acyl intermediate (Nukaga et al., 2003). To date, double-disk diffusion testing with ceftazidime, cefotaxime, aztre- a large variety of β-lactamases have been documented in various onam, ceftriaxon, and amoxicillin-clavulanic acid (Livermore and Gram negative bacilli such as Pseudomonas spp. and members of Brawn, 2001). the family Enterobacteriaceae (Wachino et al., 2004). Resistance From K. pneumoniae ML0306, the chromosomal and a 50 to β-lactam antibiotics was demonstrated in Escherichia coli even kb plasmid DNA (pML0306) were extracted respectively with the before penicillin was released for clinical use. β-lactamases can kit illustra bacteria genomicPrep Mini Spin Kit and the GFX Micro be mediated by plasmid or chromosome (Nukaga et al., 2003). Plasmid Prep plasmid extraction kit (Amersham Biosciences, In the 1960s, the first plasmid-transferable β-lactamase was dis- UK), according to the manufacturer’s instructions. Transfer of covered and named TEM-1 (Blomberg et al., 2005). the resistance determinant was performed with E. coli DH5α In this study, we analysed the β-lactamase produced by as the recipient. Transformants were selected on Luria Bertani Klebsiella pneumoniae, a narrow-spectrum cephalosporins resist- Medium agar plates supplemented with ampicillin (100 g/ml). ant strain isolated on August 2005 from an intensive care unit In addition, conjugation experiments were carried out in liquid patient at the Tunisian Military Hospital. LB medium with E. coli HB101 as the recipient, as described by Ben-Hamouda et al. (2004). Transconjugants were selected on LB agar containing ampicillin (100 g/ml) and streptomycin (256 * Corresponding author. Address: Residence El-Yamama, g/ml). The frequency of conjugational transfer is 10-2/donor. Bloc A, App A61, Tunis El-Manar 2092, Tunisia; Phone: 0021622409959; Fax: 0021670860336; Analytical isoelectric focusing of crude extract of K. pneumo- E-mail: [email protected] niae ML0306 and its transconjugants and transformants, per- 728 N. BEN ACHOUR et al.

TABLE 1 - MICs of various antimicrobial agents obtained for the clinical isolate Klebsiella pneumoniae ML0306, transformants, transconjugants and the Escherichia coli recipients Antibiotics MIC (g/ml) K. pneumoniae E. coli DH5α/ E. coli DH5α E. coli HB 101 X E. coli HB 101 ML0306 pML0306 pML0306 Ampicillin > 512 > 512 8 > 512 4 Amoxicillin > 512 > 512 8 > 512 4 Ticarcillin > 512 > 512 2 > 512 2 Cephaloridine 32 32 0.13 32 1 Cefoxitin 32 8 4 8 2 Cefotaxime 32 16 0.13 16 1 Ceftazidime 64 16 0.13 16 1 Ceftriaxone 8 8 0.13 8 1 Cefpirome 16 16 0.13 16 2 Aztreonam 256 64 0.13 64 1 Chloramphenicol > 256 2 2 2 2 Nalidixic acid > 512 1 0.06 1 1 Ciprofloxacin 128 1 0.06 1 1 Tetracyclin > 512 2 0.25 2 2 Streptomycin 32 0.25 0.25 256 > 256 Imipenem 0.38 0.06 0.06 0.06 0.25

formed as described by Bellaaj et al. (2002), revealed the pres- Amplification of the blaSHV gene by PCR was carried out using ence of an identical β-lactamase band of pI 7.6. The E. coli trans- the chromosome and the plasmid pML0306 as the template and formants and transconjugants were resistant to penicillins and the primers SHV-F (5’-ATGCGTTATATTCGCCTGTGTATT-3’) and intermediary susceptible to expanded-spectrum cephalosporins SHV-R (5’-TTAGCGTTGCCAGTGCTCGATCAG-3’). PCR analyses but were susceptible to imipenem (Table 1). These results rule confirmed the presence of blaSHV on the chromosomal and plas- out the plasmid-mediated production of an ESBL enzyme. mid DNA of K. pneumoniae ML0306 (Fig. 1), indicating that this gene is located on both chromosome and plasmid. Sequence data indicate an open reading frame of 868 bp, corresponding to 286 amino acid residues. Sequence analysis of the deduced amino acid sequences showed the presence of SHV-11. This β-lactamase differs from SHV-1 by a Leu to Gln, change at positions 35 (Fig. 2). This is the first report of SHV-11-type β-lactamases in Tunisia. Although this enzyme is a penicillinase, the enzyme activity measured by a spectrophotometer as previ- ously described (Ross and O’Callaghan, 1975) showed a hydrolytic activities against cloxacilline, cephalotin and oxymino cephalosporins such as cefotaxime and ceftriaxon (Table 2). The specific activity varies from 0.09 to 2.4 U/mg of proteins. Ampicillin was hydrolyzed best compared to all antibiotics tested. Indeed, the ampicillin was hydrolyzed respectively at a speed 5 and 19 times higher than cefotaxime and ceftriaxon, respectively. No hydrolytic activity was detected against cefoxitin, ceftazidime, cefpirome, aztreonam and imipenem. Inhibition studies showed that SHV-11 was strongly inhibited

by clavulanic acid, with IC50 = 5.1 M. An eight-fold higher con- centration of sulbactam was needed to obtain inhibition at 50% (Table 2). The activity wasn’t inhibited by EDTA at 1 mM, ruling out the presence of group 3 metallo-β-lactamases (Bush et al., 1995). The hydrolysis of the extended spectrum cephalosporins by the crude extract of K. pneumoniae ML0306 is due to the hyper production of SHV-11 coded simultaneously by plasmidic and chromosomal genes. In fact, modifications of outer membrane proteins (Rice et al., 2000), and hyperprodution of SHV-1 FIG. 1 - PCR for blaSHV gene in Klebsiella pneumoniae ML0306 enzyme due to high gene copy number (Podbielski et al., 1991), isolate. Lane 1: PCR product specific for blaSHV from or a single base pair change in the promoter sequence can the plasmid of K. pneumoniae ML0306, lane 2: PCR compensate for the low specific activity of the β-lactamase to product specific for blaSHV from the chromosome of K. an extended spectrum β-lactams and monobactams (Jacoby and pneumoniae ML0306, lane 3: DNA O’GeneRuler™ 1 kb Han, 1996; Rice et al., 2000). In the same way penicillinase over- molecular size markers. production increases simultaneously amoxicillin-clavulanate and Ann. Microbiol., 58 (4), 727-730 (2008) 729

FIG. 2 - Amino acid substitution in SHV-11 compared with parental β-lactamase SHV-1. L to Q in position 35 (Ambler numbering: four amino acids are shifted).

TABLE 2 - Specific activities (U/mg of protein) and effect of inhibitors upon β-lactamase activity of the clinical isolate Klebsiella pneumoniae ML0306 β Antibiotics Specific activity (U/mg) -Lactamase inhibitors IC50 (M) Ampicillin 2.4 Clavulanic Acid 5.1 Benzylpenicillin 0.88 Sulbactam 40 Ticarcillin 1.9 EDTA - Cephalotin 0.69 Cefoxitin ND Cefotaxime 0.52 Ceftazidime ND Ceftriaxon 0.13 Cefpirome ND Aztreonam ND Cloxacilline 0.09 Imipenem ND ND: not detected, (-): not inhibited.

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