Imipenem Resistance in Klebsiella pneumoniae Is Associated to the Combination of Plasmid-Mediated CMY-4 AmpC β-Lactamase and Loss of an Outer Membrane Protein

Bacterial strains

A clinical isolate of multi-drug resistant K. pneumoniae named Kp16137 was isolated from a 50-year-old female patient in the University Hospital Sahloul of Sousse in Tunisia, a 550-bed university hospital. The patient was hospitalized in a surgical unit and then transferred to the intensive care unit in August 2008, where Kp16137 was isolated from peritoneal fluid.

The isolate was identified using the Api20E system (BioMérieux SA).

The rifampin-resistant Escherichia coli J53-2 was used as host in conjugation assays. The quality control strains used for the study of antimicrobial susceptibility was E. coli ATCC 25922.

Antibiotic susceptibility testing and screening for production of extended-spectrum β-lactamases and for metallo-β-lactamases (MBL)

The antibiotic susceptibility of the strain was determined by the disk diffusion method on Mueller-Hinton (MH) agar plates (Bio-Rad) with β-lactam and non β-lactam antibiotic-containing disks (Sanofi Diagnostics Pasteur) according to the Guidelines of the Clinical Laboratory Standards Institute. ⁷ The antibiotics used were: ampicillin (10 μg), piperacillin (100 μg), ticarcillin (75 μg), ampicillin-sulbactam (10/10 μg), piperacillin-tazobactam (100/10 μg), ticarcillin-clavulanic acid (75/10 μg), cephalotin (30 μg), cefuroxime (30 μg), cefepime (30 μg), cefoxitin (30 μg), cefotaxime (30 μg), ceftazidime (30 μg), moxalactam (30 μg), ertapenem (10 μg), meropenem (10 μg), imipenem (10 μg), aztreonam (30 μg), gentamicin (10 μg), amikacin (30 μg), kanamycin (30 μg), netilmicin (30 μg), tobramycin (10 μg), tetracycline (30 μg), ciprofloxacin (5 μg), levofloxacin (5 μg), ofloxacin (5 μg), norfloxacin (10 μg), nalidixic acid (30 μg), sulfonamides (250 μg), trimethoprim (5 μg), and chloramphenicol (30 μg).

The minimal inhibitory concentrations (MICs) of cefotaxime, cefazidime, cefoxitin, imipenem, meropenem, and ertapenem were determined using the E-test method (AB-Biodisk) in MH agar plates according to the recommendations of the manufacturer. Breakpoints were applied according to the guidelines of the Clinical Laboratory Standards Institute. ⁷

The presence of extended-spectrum-β-lactamases (ESBL) was evaluated by using a double-disk synergy test (DDST) ³² and the E-test strips (AB-Biodisk). The DDSTs were also performed on cloxacillin (250 μg/ml)-containing plates that inhibited cephalosporinase activity and might enhance the ability of those tests to detect ESBLs as described previously. ¹⁰ MBL production was evaluated by an ethylenediamine-tetra acetic acid (EDTA)-based screening technique test ²⁵ and by using E-test strips with imipenem and EDTA (AB Biodisk).

Characterization of β-lactamases genes by PCR experiments

Whole-cell DNA of Kp16137 was prepared by suspending one or two fresh colonies in 100 μl of sterile distilled water and heating at 95°C for 10 minutes. After centrifugation, the supernatant was stored at 4°C before an analysis. The detection of the β-lactamase gene was performed under standard PCR conditions, ²⁹ using a published set of primers. The primers were specific for bla_TEM, bla_SHV, bla_CTX-M, bla_OXA, bla_CMY, bla_ACC, bla_FOX, bla_MOX, bla_DHA, bla_ACT, bla_IMP, bla_KPC, and bla_VIM genes as described previously. ⁹

Amplicons were revealed by electrophoresis on a 1.2% agarose gel and stained with ethidium bromide. PCR products were used as templates for nucleotide sequence determination. The amplification products were purified on Qiaquick columns (QIAGEN) and sequenced on an ABI PRISM 310 automated sequencer (Applied Biosystems) according to the manufacturer's instructions. The nucleotide and the deduced amino-acid sequences were analyzed using the software available over the internet at the National Center of Biotechnology Information Web site (www.ncbi.nlm.nih.gov).

β-Lactam resistance transfer assays

Tests were carried out in trypticase soy broth agar with E. coli J53-2 as recipient strain. Mating broths were incubated at 37°C for 16 hours. Transconjugants were selected on MH agar plates containing rifampin (250 μg/ml) and cefoxitin (10 μg/ml).

Hybridization

Plasmid DNA extraction was performed by using the Kado et liu method as previously described, ¹¹ followed by a Southern transfer of an agarose gel containing plasmid DNA of the CMY-positive isolate onto a nylon membrane (Hybond N⁺; GE Healthcare). Plasmid DNA was hybridized as described by Sambrook et al. ²⁸ The probe specific for the bla_CMY gene consisted of the PCR product generated from the CMY-positive isolate. Labeling of this probe and signal detection were carried out using the electrochemical luminescence (ECL) nonradioactive labeling and detection kit, according to the manufacturer's instructions (GE Healthcare).

Outer membrane protein analysis

A K. pneumoniae isolate resistant to imipenem by producing an AmpC gene (ACC-1), in addition to a loss of an outer membrane protein, ³ and a wild-type K. pneumoniae isolate were used as positive and negative controls, respectively. Bacterial cell membranes were isolated as described previously. ³⁰ Outer membrane samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) in a running buffer of 0.025 M Tris, 0.19 M glycine, and 0.1% SDS (pH 8.3) with a 12% acrylamide, 0.12% bisacrylamide gel and were visualized after staining with Coomassie blue.

Antibiotic susceptibility and MIC determination

K. pneumoniae Kp16137 was resistant to all tested β-lactams and to gentamicin, amikacin, tobramycin, kanamycin, sulfonamides, trimethoprim, chloramphenicol, and tetracycline, but susceptible to nalidixic acid, ciprofloxacin, ofloxacin, and norfloxacin. The MIC values of cefotaxime, cefazidime, cefoxitin, imipenem, meropenem, and ertapenem were reported in Table 1.

This isolate was negative by the EDTA disk synergy test and the DDST.

Characterization of β–lactamases

The strain was positive for bla_TEM, bla_SHV, and bla_CMY. Sequencing identified a narrow spectrum β-lactamase TEM-1 SHV-61 and an AmpC cephalosporinase CMY-4, respectively.

Genetic studies

The donor strain harbored five plasmids. His transconjugant TC 16137SH harbored a single plasmid of about 80 kb in size. Hybridization shows that the AmpC gene CMY-4 was located on to the 80-kb plasmid (data not shown).

Outer membrane protein analysis

Analysis of the outer membrane proteins by SDS-PAGE, as shown in Fig. 1, reveals that the susceptible strain of K. pneumoniae expressed two outer membrane proteins, one band of approximately 42 kDa and the other band of 40 kDa. Kp16137 and the K. pneumoniae imipenem-resistant strain expressing ACC-1 enzyme and loss of an outer membrane protein were missing the major 42 kDa protein band. The loss of this outer membrane protein band correlated with imipenem resistance in these strains.

OPEN IN VIEWER

FIG. 1.

Outer membrane protein profiles of Klebsiella pneumoniae strains. Outer membrane proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis in 12% acrylamide gel. Lane 1, K. pneumoniae Kp16137 imipenem resistant; lane 2, K. pneumoniae wild-type (imipenem susceptible); lane 3, K. pneumoniae imipenem resistant expressing ACC-1 enzyme and loss of an outer membrane protein and lane 4, molecular size standard. The closed arrowhead indicates the position of the 42 kDa outer membrane protein (OMP).