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Rapid detection of porins by matrix-assisted laser desorption/ionization-time of flight mass spectrometry.

Hu YY, Cai JC, Zhou HW, Zhang R, Chen GX - Front Microbiol (2015)

Bottom Line: The rapid and cost-efficient determination of carbapenem resistance is an important prerequisite for the choice of an adequate antibiotic therapy.A MALDI-TOF MS-based assay was set up to detect porins in the current study.A loss of the components of porin alone such as OmpK35/OmpK36 or together with the production of carbapenemases will augment the carbapenem resistance.

View Article: PubMed Central - PubMed

Affiliation: Second Affiliated Hospital of Zhejiang University, Zhejiang University, Hangzhou China.

ABSTRACT
The rapid and cost-efficient determination of carbapenem resistance is an important prerequisite for the choice of an adequate antibiotic therapy. A MALDI-TOF MS-based assay was set up to detect porins in the current study. A loss of the components of porin alone such as OmpK35/OmpK36 or together with the production of carbapenemases will augment the carbapenem resistance. Ten strains of Escherichia coli and eight strains of Klebsiella pneumoniae were conducted for both sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and MALDI-TOF MS analysis. MALDI-TOF/TOF MS analysis was then performed to verify the correspondence of proteins between SDS-PAGE and MALDI-TOF MS. The results indicated that the mass spectrum of ca. 35,000, 37,000, and 38,000-m/z peaks of E. coli ATCC 25922 corresponded to OmpA, OmpC, and OmpF with molecular weight of approximately ca. 38, 40, and 41 kDa in SDS-PAGE gel, respectively. The band of OmpC and OmpF porins were unable to be distinguished by SDS-PAGE, whereas it was easy to be differentiated by MALDI-TOF MS. As for K. pneumoniae isolates, the mass spectrum of ca. 36,000 and 38,600-m/z peaks was observed corresponding to OmpA and OmpK36 with molecular weight of approximately ca. 40 and 42 kDa in SDS-PAGE gel, respectively. Porin OmpK35 was not observed in the current SDS-PAGE, while a 37,000-m/z peak was found in K. pneumoniae ATCC 13883 and carbapenem-susceptible strains by MALDI-TOF MS which was presumed to be the characteristic peak of the OmpK35 porin. Compared with SDS-PAGE, MALDI-TOF MS is able to rapidly identify the porin-deficient strains within half an hour with better sensitivity, less cost, and is easier to operate and has less interference.

No MeSH data available.


Related in: MedlinePlus

Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of OMPs extracted from 8 K. pneumoniae and 10 E. coli isolates. M, protein molecular weight marker; KP1–KP6, carbapenem-resistant or reduced susceptible K. pneumoniae isolates; KP7, carbapenem-susceptible clinically isolated K. pneumoniae; EC1–EC8, carbapenem-resistant or reduced susceptible E. coli isolates; EC9, carbapenem-susceptible clinically isolated E. coli; 13883 and 25922 represent for ATCC strains of K. pneumoniae and E. coli, respectively. Twelve white arrowed bands were digested with trypsin for the following MALDI-TOF/TOF MS analysis.
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Figure 1: Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of OMPs extracted from 8 K. pneumoniae and 10 E. coli isolates. M, protein molecular weight marker; KP1–KP6, carbapenem-resistant or reduced susceptible K. pneumoniae isolates; KP7, carbapenem-susceptible clinically isolated K. pneumoniae; EC1–EC8, carbapenem-resistant or reduced susceptible E. coli isolates; EC9, carbapenem-susceptible clinically isolated E. coli; 13883 and 25922 represent for ATCC strains of K. pneumoniae and E. coli, respectively. Twelve white arrowed bands were digested with trypsin for the following MALDI-TOF/TOF MS analysis.

Mentions: The arrows observed in the ATCC strains between molecular weight of 36–48 kDa were assigned to 48 kDa-LamB, 44 kDa-putative OMP, 42 kDa-OmpK36, and 40 kDa-OmpA for K. pneumoniae isolates and 41 kDa-OmpC, 40 kDa-OmpF, and 38 kDa-OmpA for E. coli isolates, respectively (Figure 1). Forty-one kDa-OmpC and 40 kDa-OmpF bands in E. coli ATCC 25922 were in close proximity and were difficult to be distinguished (Figure 1). OmpK35 was not found in the current study. K. pneumoniae KP1–KP3 and KP6 failed to express OmpK36. E. coli EC1–EC3, EC6–EC8 failed to express OmpC and OmpF; E. coli EC4 and EC5 failed to express OmpF.


Rapid detection of porins by matrix-assisted laser desorption/ionization-time of flight mass spectrometry.

Hu YY, Cai JC, Zhou HW, Zhang R, Chen GX - Front Microbiol (2015)

Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of OMPs extracted from 8 K. pneumoniae and 10 E. coli isolates. M, protein molecular weight marker; KP1–KP6, carbapenem-resistant or reduced susceptible K. pneumoniae isolates; KP7, carbapenem-susceptible clinically isolated K. pneumoniae; EC1–EC8, carbapenem-resistant or reduced susceptible E. coli isolates; EC9, carbapenem-susceptible clinically isolated E. coli; 13883 and 25922 represent for ATCC strains of K. pneumoniae and E. coli, respectively. Twelve white arrowed bands were digested with trypsin for the following MALDI-TOF/TOF MS analysis.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4524100&req=5

Figure 1: Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of OMPs extracted from 8 K. pneumoniae and 10 E. coli isolates. M, protein molecular weight marker; KP1–KP6, carbapenem-resistant or reduced susceptible K. pneumoniae isolates; KP7, carbapenem-susceptible clinically isolated K. pneumoniae; EC1–EC8, carbapenem-resistant or reduced susceptible E. coli isolates; EC9, carbapenem-susceptible clinically isolated E. coli; 13883 and 25922 represent for ATCC strains of K. pneumoniae and E. coli, respectively. Twelve white arrowed bands were digested with trypsin for the following MALDI-TOF/TOF MS analysis.
Mentions: The arrows observed in the ATCC strains between molecular weight of 36–48 kDa were assigned to 48 kDa-LamB, 44 kDa-putative OMP, 42 kDa-OmpK36, and 40 kDa-OmpA for K. pneumoniae isolates and 41 kDa-OmpC, 40 kDa-OmpF, and 38 kDa-OmpA for E. coli isolates, respectively (Figure 1). Forty-one kDa-OmpC and 40 kDa-OmpF bands in E. coli ATCC 25922 were in close proximity and were difficult to be distinguished (Figure 1). OmpK35 was not found in the current study. K. pneumoniae KP1–KP3 and KP6 failed to express OmpK36. E. coli EC1–EC3, EC6–EC8 failed to express OmpC and OmpF; E. coli EC4 and EC5 failed to express OmpF.

Bottom Line: The rapid and cost-efficient determination of carbapenem resistance is an important prerequisite for the choice of an adequate antibiotic therapy.A MALDI-TOF MS-based assay was set up to detect porins in the current study.A loss of the components of porin alone such as OmpK35/OmpK36 or together with the production of carbapenemases will augment the carbapenem resistance.

View Article: PubMed Central - PubMed

Affiliation: Second Affiliated Hospital of Zhejiang University, Zhejiang University, Hangzhou China.

ABSTRACT
The rapid and cost-efficient determination of carbapenem resistance is an important prerequisite for the choice of an adequate antibiotic therapy. A MALDI-TOF MS-based assay was set up to detect porins in the current study. A loss of the components of porin alone such as OmpK35/OmpK36 or together with the production of carbapenemases will augment the carbapenem resistance. Ten strains of Escherichia coli and eight strains of Klebsiella pneumoniae were conducted for both sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and MALDI-TOF MS analysis. MALDI-TOF/TOF MS analysis was then performed to verify the correspondence of proteins between SDS-PAGE and MALDI-TOF MS. The results indicated that the mass spectrum of ca. 35,000, 37,000, and 38,000-m/z peaks of E. coli ATCC 25922 corresponded to OmpA, OmpC, and OmpF with molecular weight of approximately ca. 38, 40, and 41 kDa in SDS-PAGE gel, respectively. The band of OmpC and OmpF porins were unable to be distinguished by SDS-PAGE, whereas it was easy to be differentiated by MALDI-TOF MS. As for K. pneumoniae isolates, the mass spectrum of ca. 36,000 and 38,600-m/z peaks was observed corresponding to OmpA and OmpK36 with molecular weight of approximately ca. 40 and 42 kDa in SDS-PAGE gel, respectively. Porin OmpK35 was not observed in the current SDS-PAGE, while a 37,000-m/z peak was found in K. pneumoniae ATCC 13883 and carbapenem-susceptible strains by MALDI-TOF MS which was presumed to be the characteristic peak of the OmpK35 porin. Compared with SDS-PAGE, MALDI-TOF MS is able to rapidly identify the porin-deficient strains within half an hour with better sensitivity, less cost, and is easier to operate and has less interference.

No MeSH data available.


Related in: MedlinePlus