Mutations in Genes Encoding Penicillin-Binding Proteins and Efflux Pumps Play a Role in β-Lactam Resistance in Helicobacter cinaedi

Abstract

β-Lactams are often used to treat Helicobacter cinaedi infections; however, the mechanism underlying β-lactam resistance is unknown. In this study, we investigated β-lactam resistance in an H. cinaedi strain, MRY12-0051 (MICs of amoxicillin [AMX] and ceftriaxone [CRO], 32 and 128 μg/ml; obtained from human feces). Based on a comparative whole-genome analysis of MRY12-0051 and the CRO-susceptible H. cinaedi strain MRY08-1234 (MICs of AMX and CRO, 1 and 4 μg/ml; obtained from human blood), we identified five mutations in genes encoding penicillin-binding proteins (PBPs), including two in pbpA, one in pbp2, and two in ftsI Transformation and penicillin binding assays indicated that CRO resistance was mainly associated with mutations in pbpA; mutations in ftsI also led to increased resistance to AMX. Knocking out cmeB and cmeD, which encode resistance-nodulation-division-type efflux pump components, in H. cinaedi type strain CCUG18818 (AMX MIC, 4 to 8 μg/ml) resulted in 8- and 64-fold decreases, respectively, in the AMX MIC. Hence, MICs of AMX in H. cinaedi become similar to those of Helicobacter pylori isolates in the absence of cmeD In conclusion, the difference in susceptibility to β-lactams between H. pylori and H. cinaedi is explained by differences in efflux pump components. Mutations in pbpA are the primary determinant of high resistance to β-lactams in H. cinaedi.

Keywords: Helicobacter cinaedi; amoxicillin; ceftriaxone; cmeB; cmeD; efflux pumps; penicillin-binding proteins; β-lactams.

FIG 1

Binding of ceftriaxone (A) and imipenem (B) to PBPA in H. cinaedi clinical isolates MRY08-1234 and MRY12-0051 and pbpA mutant strain MRY08-1234_pbpA. Membrane proteins were treated with various concentrations of ceftriaxone and imipenem and subsequently labeled with Bocillin FL. Arrows indicate the bands corresponding to PBPA. Images are representative of results from two independent experiments. Gel band quantitation is shown with the standard deviations from two independent experiments.

FIG 2

Expression of genes encoding efflux pump components and a porin in H. cinaedi isolates. Transcript levels of the specified target genes were normalized to the transcript level of the recA reference gene; the relative gene expression levels are reported as the fold change relative to the expression in the CCUG18818 strain, determined using the threshold cycle (C_T) method (33).

FIG 3

Predicted structures of H. cinaedi PBPA and FtsI. The structural predictions for PBPA (A) and FtsI (B) of the MRY08-1234 isolate were obtained using the Robetta server (http://robetta.bakerlab.org). The obtained structures were overlaid onto a crystal structure of Acinetobacter baumannii PBP1a in complex with penicillin G (PDB code 3UDI) and a crystal structure of Pseudomonas aeruginosa PBP3 in complex with ceftazidime (PDB code 3PBO), respectively, to visualize the β-lactam binding site. The ligands (penicillin G and ceftazidime) are shown in blue. Motifs 1, 2, and 3, shown in orange, are penicillin-binding motifs predicted based on the comparison with PBPA and FtsI of H. pylori, as appropriate (23). Mutations identified in MRY08-1234 are shown in pink. The amino acid numbers for PBPA and FtsI are based on the amino acid sequences of PBPA and FtsI in MRY08-1234.