Characterization of porin expression in Klebsiella pneumoniae Carbapenemase (KPC)-producing K. pneumoniae identifies isolates most susceptible to the combination of colistin and carbapenems

Abstract

We characterized carbapenem resistance mechanisms among 12 Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (referred to here as KPC K. pneumoniae) clinical isolates and evaluated their effects on the activity of 2- and 3-drug combinations of colistin, doripenem, and ertapenem. All isolates were resistant to ertapenem and doripenem; 75% (9/12) were resistant to colistin. Isolates belonged to the ST258 clonal group and harbored blaKPC-2, blaSHV-12, and blaTEM-1. As determined by time-kill assays, doripenem (8 μg/ml) and ertapenem (2 μg/ml) were inactive against 92% (11/12) and 100% (12/12) of isolates, respectively. Colistin (2.5 μg/ml) exerted some activity (range, 0.39 to 2.5 log10) against 78% (7/9) of colistin-resistant isolates. Colistin-ertapenem, colistin-doripenem, and colistin-doripenem-ertapenem exhibited synergy against 42% (5/12), 50% (6/12), and 67% (8/12) of isolates, respectively. Expression of ompK35 and ompK36 porins correlated with each other (R(2) = 0.80). Levels of porin expression did not correlate with colistin-doripenem or colistin-ertapenem synergy. However, synergy with colistin-doripenem-ertapenem was more likely against isolates with high porin expression than those with low expression (100% [8/8] versus 0% [0/4]; P = 0.002). Moreover, bactericidal activity (area under the bacterial killing curve) against isolates with high porin expression was greater for colistin-doripenem-ertapenem than colistin-doripenem or colistin-ertapenem (P ≤ 0.049). In conclusion, colistin-carbapenem combinations may provide optimal activity against KPC K. pneumoniae, including colistin-resistant isolates. Screening for porin expression may identify isolates that are most likely to respond to a triple combination of colistin-doripenem-ertapenem. In the future, molecular characterization of KPC K. pneumoniae isolates may be a practical tool for identifying effective combination regimens.

Figures

Fig 1

Outer membrane protein analysis of representative KPC K. pneumoniae isolates by 12% SDS-PAGE. The isolate number and relative expression of ompK36 by real-time RT-PCR are provided for each lane. The lane marked “Ladder” contains size markers (sizes are in kDa).

Fig 2

Time-kill curve of single drugs and 2- and 3-drug combinations against a representative KPC K. pneumoniae isolate. Doripenem and colistin MICs against this isolate (isolate 178) were 256 μg/ml and 16 μg/ml, respectively. Times on the x axis are incubation times.

Fig 3

Bactericidal activity and degree of killing by single drugs and 2- and 3-drug combinations against 12 KPC K. pneumoniae isolates. The line graph represents the median degree of killing, and the bar graph represents the percentage of bactericidal activity of each drug regimen against the 12 KPC K. pneumoniae isolates. There was a stepwise improvement in bactericidal activity from colistin only and colistin-ertapenem to colistin-doripenem to colistin-doripenem-ertapenem. Bars (left y axis) show bactericidal activity, and the line (right y axis) shows the degree of killing by single drugs and 2- and 3-drug combinations.

Fig 4

Area under the bacterial killing curves (AUBC) of single drugs and 2- and 3-drug combinations against 12 KPC K. pneumoniae isolates. The median AUBC was significantly lower for colistin-doripenem-ertapenem than colistin with a carbapenem (colistin-ertapenem, P = 0.0002; colistin-doripenem, P = 0.05) and colistin only (P < 0.0001).

Fig 5

AUBCs of representative KPC K. pneumoniae isolates with high porin expression (isolate 25 [left]) and low porin expression (isolate 121 [right]). The dotted line, dashed line, and solid line represent time-kill curves of colistin, colistin-doripenem, and colistin-doripenem-ertapenem, respectively. Black, AUBC of colistin-doripenem-ertapenem; dark gray, difference in AUBC between colistin-doripenem and colistin-doripenem-ertapenem; light gray, difference in AUBC between colistin and colistin-doripenem.