A low-affinity penicillin-binding protein 2x variant is required for heteroresistance in Streptococcus pneumoniae.
Heteroresistance to penicillin in Streptococcus pneumoniae is the ability of subpopulations to grow at a higher antibiotic concentration than expected from the MIC. This may render conventional resistance testing unreliable and lead to therapeutic failure. We investigated the role of the primary β-lactam resistance determinants, penicillin-binding protein 2b (PBP2b) and PBP2x, and the secondary resistance determinant PBP1a in heteroresistance to penicillin. Transformants containing PBP genes from the heteroresistant strain Spain(23F) 2349 in the nonheteroresistant strain R6 background were tested for heteroresistance by population analysis profiling (PAP). We found that pbp2x, but not pbp2b or pbp1a alone, conferred heteroresistance to R6. However, a change of pbp2x expression was not observed, and therefore, expression does not correlate with an increased proportion of resistant subpopulations. In addition, the influence of the CiaRH system, mediating PBP-independent β-lactam resistance, was assessed by PAP on ciaR disruption mutants but revealed no heteroresistant phenotype. We also showed that the highly resistant subpopulations (HOM*) of transformants containing low-affinity pbp2x undergo an increase in resistance upon selection on penicillin plates that partially reverts after passaging on selection-free medium. Shotgun proteomic analysis showed an upregulation of phosphate ABC transporter subunit proteins encoded by pstS, phoU, pstB, and pstC in these highly resistant subpopulations. In conclusion, the presence of low-affinity pbp2x enables certain pneumococcal colonies to survive in the presence of β-lactams. Upregulation of phosphate ABC transporter genes may represent a reversible adaptation to antibiotic stress.
Institute for Infectious Diseases, Faculty of Medicine, University of Bern, Bern, Switzerland Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland.
Hansjürg
H
Engel
Institute for Infectious Diseases, Faculty of Medicine, University of Bern, Bern, Switzerland Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland.
Moana
M
Mika
Department of Microbiology, University of Kaiserslautern, Kaiserslautern, Germany.
Dalia
D
Denapaite
Department of Microbiology, University of Kaiserslautern, Kaiserslautern, Germany.
Regine
R
Hakenbeck
Institute for Infectious Diseases, Faculty of Medicine, University of Bern, Bern, Switzerland Department of Infectious Diseases, University Hospital and University of Bern, Bern, Switzerland.
Kathrin
K
Mühlemann
Laboratory of Thrombosis Research, Department of Clinical Research, University of Bern, Bern, Switzerland.
Manfred
M
Heller
Institute for Infectious Diseases, Faculty of Medicine, University of Bern, Bern, Switzerland.
Lucy J
LJ
Hathaway
Institute for Infectious Diseases, Faculty of Medicine, University of Bern, Bern, Switzerland Department of Infectious Diseases, University Hospital and University of Bern, Bern, Switzerland markus.hilty@ifik.unibe.ch.
Markus
M
Hilty
D004269:DNA, Bacterial / Q000235:genetics; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D008826:Microbial Sensitivity Tests; D010403:Penicillin Resistance / Q000235:genetics*; D046915:Penicillin-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D011008:Pneumococcal Infections / Q000382:microbiology; D040901:Proteomics / Q000379:methods; D060888:Real-Time Polymerase Chain Reaction; D013296:Streptococcus pneumoniae / Q000187:drug effects* / Q000235:genetics* / Q000254:growth & development; D014169:Transformation, Bacterial
D004269:DNA, Bacterial; D046915:Penicillin-Binding Proteins; C072223:PBP 2x protein, Streptococcus