Exploration of the role of the penicillin binding protein 2c (Pbp2c) in inducible β-lactam resistance in Corynebacteriaceae

Abstract

Six genes encoding putative high molecular weight penicillin-binding proteins (Pbp) are present in the genome of the β-lactam-resistant strain Corynebacterium jeikeium K411. In this study, we show that pbp2c, one of these six genes, is present in resistant strains of Corynebacteriaceae but absent from sensitive strains. The molecular study of the pbp2c locus from C. jeikeium and its heterologous expression in Corynebacterium glutamicum allowed us to show that Pbp2c confers high levels of β-lactam resistance to the host and is under the control of a β-lactam-induced regulatory system encoded by two adjacent genes, jk0410 and jk0411. The detection of this inducible resistance may require up to 48 h of incubation, particularly in Corynebacterium amycolatum. Finally, the Pbp2c-expressing strains studied were resistant to all the β-lactam antibiotics tested, including carbapenems, ceftaroline, and ceftobiprole.

Keywords: Corynebacteria; Corynebacteriaceae; Corynebacterium jeikeium; Pbp; Pbp2c; β-lactam.

Figure 1

Antibiogram by the disk diffusion assay performed on MH-F agar for (A) Corynebacterium jeikeium strains CIP82.51 (CjkS), (B) CIP103337 (CjkR) incubated 24 h, (C) CIP103337 (CjkR) incubated 48 h, and (D) CIP103337 (CjkR) cultivated in the presence of clavulanate (Clav) at 10 μg/mL and incubated 24 h. Disks contain 1: cefoxitin (30 μg), 2: benzylpenicillin (1 U), 3: ticarcillin (75 μg), 4: ampicillin (10 μg), 5: cefalexin (30 μg), 6: amoxicillin/clavulanate (20 μg/ 10 μg), 7: ceftaroline (5 μg), 8: piperacillin (30 μg), 9: piperacillin/tazobactam (30 μg/6 μg), 10: cefotaxime (5 μg), 11: ticarcillin/clavulanate (75 μg/10 μg), 12: cefepime (30 μg), 13: clavulanate (10 μg), 14: meropenem (10 μg), 15: imipenem (10 μg), or 16: ertapenem (10 μg).

Figure 2

Double disk diffusion assay for C. jeikeium strains CIP82.51 (CjkS) (A,C) and CIP103337 (CjkR) (B,D). Plates were incubated for 24 h on MH-F agar. Disk 1: clavulanate (Clav, 10 μg), disk 2: meropenem (Mem, 10 μg), disk 3: ampicillin (Ap, 10 μg).

Figure 3

Growth curves of C. jeikeium K411 in the absence or presence of meropenem. An overnight culture of strain K411 diluted to 0.1 OD₆₀₀ was grown in the absence (Ap 0) or presence (Ap 20) of ampicillin at 20 μg/mL for 4 h (OD₆₀₀ of approximately 0.4). Meropenem (32 μg/mL, MEM 32) was added to the indicated cultures. OD₆₀₀ values are the mean ± standard error from three determinations.

Figure 4

Induction of meropenem resistance by the disk diffusion assay for strain C. jeikeium K411. Plates were incubated for 48 h. The assays were performed on BHI agar containing 0.5% Tween 80 and meropenem at 128 μg/mL. The disks contained 1: ampicillin (10 μg), 2: oxacillin (5 μg), 3: piperacillin (30 μg), 4: cephalothin (30 μg), 5: cefoxitin (30 μg), 6: ceftazidime (30 μg), 7: cefepime (30 μg), 8: colistin (30 μg), 9: chloramphenicol (30 μg), 10: benzylpenicillin (10 U), 11: amoxicillin (25 μg), 12: amoxicillin/clavulanate (20 μg/10 μg), 13: gentamicin (10 μg), 14: erythromycin (15 μg), 15: norfloxacin (10 μg), 16: vancomycin (5 μg), 17: teicoplanin (30 μg), 18: tetracycline (30 μg), 19: imipenem (10 μg), 20: meropenem (10 μg), or 21: ertapenem (10 μg).

Figure 5

Impact of ampicillin (A) or clavulanate (B) on the level of Pbp2c production. C. jeikeium strains were grown for 24 h in the presence of ampicillin (Ap) or clavulanate (Clav). Crude extracts (10 μg) were analyzed by Western blotting with polyclonal antibodies raised against the Pbp2c protein encoded by jk0412. The extracts were prepared from strain C. jeikeium CIP82.51 (CjkS), a β-lactam-sensitive strain that does not harbor pbp2c, and from the β-lactam resistant strains CIP103337 (CjkR) and K411. Purified Pbp2c (3 ng) was used as a control.

Figure 6

Formation of adducts between Pbp2c and β-lactams. Pbp2c was incubated without or with antibiotics. Peaks at m/z 1,016.7 and 1,019.6 correspond to the [M + 62H]⁶²⁺ of the native protein (deduced mass average of 62,975 Da) and a spontaneous α-N-6-phosphogluconoylation of the poly histidine tag (Geoghegan et al., 1999). ND: not detected.

Figure 7

Schematic organization and G + C content of the pbp2c locus. (A) Map of the pbp2c locus in C. jeikeium K411 and comparison with the homologous region of the C. glutamicum ATCC 13032 locus. (B) G + C content of relevant genes and comparison with that of whole genome.

Figure 8

(A) Schematic representation of the constructs pML1, pML2, and pLM3. (B) Western blot of total proteins extracted from C. glutamicum RES167 transformed with pML1, pML2, or pML3. Strains were cultivated overnight without antibiotics, diluted to an OD₆₀₀ of 0.1 in fresh BHI media complemented (+) or not (−) with clavulanate (clav, 1 μg/mL), and cultivated under agitation for 24 h. Protein extracts (10 μg) were analyzed by Western blotting with polyclonal antibodies raised against the Pbp2c protein encoded by jk0412. Line 1: purified Pbp2C; Line 2–3: RES167 harboring pMM36 (#3278); Line 4–5 and 10–11: RES167 harboring pMM36Ωjk0410-jk0411-jk0412 (#3815); Line 6–7: RES167 harboring pMM36ΩP_trc-jk0412 (#3279); Line 8–9: RES167 harboring pMM36Ωjk0412 (#3812).