Four clinical Helicobacter pylori isolates with high-level resistance to β-lactams exhibited low- to moderate-level resistance to the structurally and functionally unrelated antibiotics ciprofloxacin, chloramphenicol, metronidazole, rifampin, and tetracycline. This pattern of multidrug resistance was transferable to susceptible H. pylori by natural transformation using naked genomic DNA from a clinical multidrug-resistant isolate. Acquisition of the multidrug resistance was also associated with a change in the genotype of the transformed multidrug-resistant H. pylori. DNA sequence analyses of the gene encoding penicillin binding protein 1A (PBP 1A) showed 36 nucleotide substitutions resulting in 10 amino acid changes in the C-terminal portion (the putative penicillin binding domain). Acquisition of β-lactam resistance was consistently associated with transfer of a mosaic block containing the C-terminal portion of PBP 1A. No changes of genes gyrA, rpoB, rrn16S, rdxA, and frxA, and nine other genes (ftsI, hcpA, llm, lytB, mreB, mreC, pbp2, pbp4, and rodA1) encoding putative PBPs or involved in cell wall synthesis were found among the transformed resistant H. pylori. Antibiotic accumulations of chloramphenicol, penicillin, and tetracycline were all significantly decreased in the natural and transformed resistant H. pylori compared to what was seen with susceptible H. pylori. Natural transformation also resulted in the outer membrane protein profiles of the transformed resistant H. pylori becoming similar to that of the clinical resistant H. pylori isolates. Overall, these results demonstrate that high-level β-lactam resistance associated with acquired multidrug resistance in clinical H. pylori is mediated by combination strategies including alterations of PBP 1A and decreased membrane permeability.