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. 2015 Feb 11;59(3):1797–1801. doi: 10.1128/AAC.03894-14

Doripenem MICs and ompK36 Porin Genotypes of Sequence Type 258, KPC-Producing Klebsiella pneumoniae May Predict Responses to Carbapenem-Colistin Combination Therapy among Patients with Bacteremia

Ryan K Shields a,b,c, M Hong Nguyen a,b,c,, Brian A Potoski b,f, Ellen G Press a, Liang Chen d, Barry N Kreiswirth d, Lloyd G Clarke b, Gregory A Eschenauer b,e, Cornelius J Clancy a,c,g
PMCID: PMC4325815  PMID: 25534733

Abstract

Treatment failures of a carbapenem-colistin regimen among patients with bacteremia due to sequence type 258 (ST258), KPC-2-producing Klebsiella pneumoniae were significantly more likely if both agents were inactive in vitro, as defined by a colistin MIC of >2 μg/ml and the presence of either a major ompK36 porin mutation (guanine and alanine insertions at amino acids 134 and 135 [ins aa 134–135 GD], IS5 promoter insertion [P = 0.007]) or a doripenem MIC of >8 μg/ml (P = 0.01). Major ompK36 mutations among KPC-K. pneumoniae strains are important determinants of carbapenem-colistin responses in vitro and in vivo.

TEXT

Carbapenem-resistant Klebsiella pneumoniae (CR-Kp) strains have emerged worldwide, causing infections associated with significant mortality (13). Carbapenem resistance among strains in the United States is typically mediated by production of carbapenem-hydrolyzing enzymes, in particular Klebsiella pneumoniae carbapenemases (KPCs). Additional mechanisms include production of beta-lactamases and alterations in outer membrane porins (OMPs) (2). At our center, sequence type 258 (ST258), KPC-2-producing strains that harbor TEM-1 and SHV-12 beta-lactamases predominate (2). ST258, KPC-2-producing strains carry a mutant ompK35 gene that encodes a truncated porin (amino acid [aa] 89 STOP) and exhibit one of several ompK36 porin genotypes. The most active antimicrobial regimen against our strains in vitro is a carbapenem-colistin combination (4). However, two major ompK36 mutations (guanine and alanine insertions at aa 134 and 135 [ins aa134–135 GD] and an IS5 insertion sequence within the promoter) are associated with high-level carbapenem resistance (MIC, >8 μg/ml) and attenuated time-kill responses to carbapenem-colistin (4, 5).

Although retrospective studies have reported improved outcomes among patients with CR-Kp bacteremia treated with combination therapy rather than single agents (1, 6, 7), the response to carbapenem-based combination therapy was not consistent. To date, it is unclear which patients would benefit from this combination therapy. We hypothesized that carbapenem-colistin treatment failures are at least partially explained by the presence of major ompK36 mutations. The objective of this study was to evaluate the association between ompK36 genotypes, doripenem MICs, and responses to carbapenem-colistin therapy among patients with CR-Kp bacteremia.

We conducted a retrospective study of patients with CR-Kp bacteremia at our center between February 2010 and May 2013. CR-Kp was defined by nonsusceptibility to a carbapenem and all third-generation cephalosporins (8). Patients with bacteremia initially treated with carbapenem-colistin (colistimethate) for >3 days were included. For patients with normal renal function, standardized doses of colistin (5 mg/kg body weight loading, followed by 5 mg/kg/day divided into 3 doses) and doripenem (our formulary agent; 1 g every 8 h, infused over 3 h) were recommended. Among patients with renal impairment, doses were adjusted based on published recommendations (9). “Cure” was defined as resolution of symptoms and sterilization of blood cultures within 7 days. “Treatment failure” was defined as death within 7 days, persistent signs and symptoms of CR-Kp infection despite ≥7 days of therapy, recurrence of CR-Kp infection within 90 days, or breakthrough CR-Kp bacteremia while receiving carbapenem-colistin. Strains were tested for multilocus sequence type, KPC variant, beta-lactamases, and porin genotypes by standard methods (2). Doripenem and colistin MICs were determined using CLSI methods, and resistance was defined by MICs of >2 μg/ml (10). Based on our previously published in vitro studies and relevant pharmacokinetic-pharmacodynamic (PK-PD) data, carbapenems were considered inactive if the MIC of the agent used in treatment was >8 μg/ml (57). Comparisons between groups were made by Fisher's exact test for categorical variables and the Mann-Whitney U test for continuous variables. Significance was defined as P ≤ 0.05 (two tailed).

Twenty-seven patients with CR-Kp bacteremia who received carbapenem-colistin therapy were identified. Seven patients were excluded due to receipt of a third agent (n = 4 [all received gentamicin]), death within 3 days (n = 2), or initiation of comfort measures only (n = 1). Twenty patients with CR-Kp bacteremia fulfilled inclusion criteria (Table 1). The median colistin and doripenem MICs were 0.25 and 64 μg/ml, respectively. All CR-Kp strains were ST258 strains that carried blaKPC-2, blaTEM1, blaSHV-12, and mutant gene ompK35 (aa 89 STOP). Sequence analysis revealed four ompK36 genotypes, including ins aa 134–135 GD (n = 10), wild type (n = 7), IS5 promoter insertions (IS5; n = 2), and an asparagine-asparagine-threonine-glutamic acid (NNTE) deletion at aa 84 to 87 (n = 1), which is a minor mutation that does not impact carbapenem-colistin responses in vitro (5). Median doripenem MICs were higher against major ompK36 mutant strains than wild-type or minor mutant strains (128 versus 16 μg/ml; P = 0.002).

TABLE 1.

Patient demographics, clinical characteristics, and outcomes of CR-Kp bloodstream infectionsa

Patient Age, yr (sex) Underlying disease(s) APACHE II scoreb Time to BSI, daysc Portal of entry Source control Duration of BSI, daysd Concomitant CR-Kp infection at time of BSI Time to initiation of therapy, he No. of days of therapy MIC (μg/ml)
ompK36 genotype Treatment outcome
Colistin Doripenem
1 72 (M) Metastatic pancreatic carcinoma 18 2 Abdominal Percutaneous drainage of bile duct (day 3) 5 None 81 17 0.25 8f WT Cure
2 75 (F) ESRD, CHF, CAD 16 33 Abdominal None 1 None 47 13 0.25 8f WT Cure
3 48 (M) Primary sclerosing cholangitis 7 1 Abdominal Cholecystectomy (day 18) 1 None 65 14 0.25 16 WT Cure
4 68 (M) Pancreatic cancer, cholecystitis 12 1 Abdominal Drainage via cholecystostomy tube (day 1), subtotal cholecystectomy (day 18) 2 IAI 62 14 0.25 8 WT Cure
5 50 (M) Kidney and liver transplant 16 51 Abdominal None 1 IAI 26 19 0.25 8 aa 84–87 (NNTE) del Cure
6 63 (F) Liver transplant 17 3 Abdominal Percutaneous drainage of abscess (day 2) 48 IAA 92 75 0.5 8 WT Failure due to persistent then recurrent BSI (day 90)
7 37 (F) Caroli disease 7 1 Abdominal None 1 None 77 19 0.25 16 WT Failure due to persistent IAI requiring liver resection and transplantation
8 54 (M) Liver transplant 14 122 Vascular catheter Catheter removal (day 2) 1 PNA Breakthrough BSI 44 64 16 WT Failure due to breakthrough BSI
9 56 (M) Liver transplant 20 21 Urine None 2 Cystitis 28 16 0.5 128 ins aa 134–135 DG Cure
10 55 (M) Polycythemia 33 44 Respiratory tract Chest tube insertion (day 6) 1 PNA, empyema Breakthrough BSI 19 0.25 128 ins aa 134–135 DG Failure due to breakthrough BSI
11 65 (F) Kidney transplant 24 81 Respiratory tract None 1 PNA Breakthrough BSI 59 0.5 128 ins aa 134–135 DG Failure due to breakthrough BSI
12 44 (M) Multivisceral transplant 21 22 Vascular catheter Catheter exchange (day 1), removal of TDC (day 2) 1 None Breakthrough BSI 54 64 64 ins aa 134–135 DG Failure due to breakthrough BSI and new site of infection (PNA, day 23)
13 55 (M) Polymyositis 30 102 Abdominal Surgical debridement and abdominal washout (days 3 and 5) 1 IAI 76 4 0.25 64 ins aa 134–135 DG Failure due to death (day 7)
14 57 (M) ESLD 30 58 Respiratory tract None 2 PNA 15 4 0.25 64 ins aa 134–135 DG Failure due to death (day 4)
15 60 (M) DM, necrotizing pancreatitis 28 24 Abdominal Abdominal washout (days 1, 9, and 11), surgical debridement (day 13) 27 IAI Breakthrough BSI 29 0.5 128 ins aa 134–135 DG Failure due to breakthrough and persistent BSI, death (day 28)
16 58 (F) Liver transplant 16 9 Abdominal Percutaneous drain of abscess (day 1), abdominal washout (days 3, 8, 19, 23, 33, and 39) 9 IAI 51 141 (gentamicin, 137)g 0.5 64 ins aa 134–135 DG Failure due to persistent BSI, then recurrent BSI (days 46 and 87), new site of infection (PNA, day 39)
17 61 (M) Liver transplant 14 30 Vascular catheter Catheter removed (day 3) 2 None 67 10 0.25 128 ins aa 134–135 DG Failure due to recurrent infection (PNA, day 31)
18 62 (M) Kidney transplant 23 6 Abdominal/urinary Removal of ureteral stent (day 3), nephrectomy (day 21) 42 Pyelonephritis 53 35 16 16 ins aa 134–135 DG Failure due to persistent BSI
19 26 (M) Gallstone pancreatitis 9 36 Abdominal Drainage of gallbladder and gall stone removal (day 2) 1 IAI 53 38 0.25 128 IS5 Failure due to recurrent IAI (day 16)
20 59 (M) DM, CRF 10 1 Abdominal None 9 IAI 69 5 0.5 128 IS5 Failure due to persistent BSI and death (day 9)
a

Abbreviations: BSI, bloodstream infection (bacteremia); CAD, coronary artery disease; CHF, congestive heart failure; CRF, chronic renal failure; DM, diabetes mellitus; ESLD, end-stage liver disease; F, female; HIV, human immunodeficiency virus; IAI, intra-abdominal infection; IAA, intra-abdominal abscess; M, male; PNA, pneumonia; TDC, tunneled dialysis catheter; WT, wild type.

b

At the onset of bloodstream infection.

c

Time from hospital admission to positive blood culture.

d

Days of positive blood cultures.

e

Time from collection of blood culture to first dose of combination therapy.

f

Patients 1 and 2 were treated with meropenem. The meropenem MIC was 8 μg/ml in each case. All other patients were treated with doripenem.

g

Gentamicin was added to the doripenem-colistin combination due to persistent infection.

Ninety percent (18/20) of patients received doripenem, and 10% (2/20) received meropenem. Seventy percent (14/20) of patients experienced treatment failures; the 28-day mortality rate was 20% (4/20). All three patients infected with colistin-resistant strains (MIC, >2 μg/ml) failed treatment. Eighty-six percent (12/14) and 33% (2/6) of patients infected with strains that exhibited doripenem MICs of >8 μg/ml and ≤8 μg/ml, respectively, experienced treatment failure (P = 0.04). Ninety-two percent (11/12) of patients infected with strains harboring major ompK36 mutations failed therapy compared to 38% (3/8) of patients infected with wild-type or minor ompK36 mutant strains (P = 0.02). Clinical cures occurred in 80% (4/5) of patients who received two active agents (as defined by MICs), compared to 13% (2/15) of patients who received one or no active agent (P = 0.01). If carbapenem resistance was defined by the presence of a major ompK36 mutation, cures occurred in 71% (5/7) of patients who received two active agents compared to 8% (1/13) who received one or no active agent (Table 2 [P = 0.007]). No other clinical factors were significantly associated with treatment failure (Table 3). The four patients who died within 28 days were infected with colistin-susceptible, major ompK36 mutant strains that exhibited doripenem MICs of ≥64 μg/ml (ins aa 134–135 GD, n = 3; IS5, n = 1).

TABLE 2.

Rates of clinical cure by number of active agents in the treatment combination

Definition of resistance Regimen Rate of clinical cure, % (no. cured/total) P valuea
Doripenem MIC of >8 μg/ml and colistin MIC of >2 μg/ml Both agents active 80 (4/5) 0.01
1 agent active 17 (2/12)
No agent active 0 (0/3)
Major ompK36 mutation and colistin MIC of >2 μg/ml Both agents active 71 (5/7) 0.007
1 agent active 9 (1/11)
No agent active 0 (0/2)
a

Comparing both agents active versus one or no agent active.

TABLE 3.

Factors associated with carbapenem-colistin treatment failure

Factor Value for patients with indicated outcome
P value
Cure (n = 6) Failure (n = 14)
Solid organ transplant recipient, no. (%) 2 (33) 9 (64) 0.36
Renal replacement therapy, no. (%) 1 (17) 6 (43) 0.35
Residence in intensive care unit, no. (%) 2 (33) 11 (79) 0.12
Median APACHE II score (range) 16 (7–20) 19 (7–33) 0.28
Median time from admission to BSI,a days (range) 12 (1–51) 27 (1–122) 0.23
Concomitant CR-Kp infection, no. (%) 3 (50) 11 (79) 0.30
Median time to initiation of combination therapy, h (range)b 55 (26–81) 52 (15–92) 0.71
Appropriate colistin dosing, no. (%)c 4 (67) 9 (64) 1.00
Major ompK36 mutation, no. (%) 1 (17) 11 (79) 0.02
a

BSI, bloodstream infection.

b

Excludes patients with breakthrough bacteremia.

c

Colistin dosing regimens were considered to be appropriate if loading doses were given and maintenance doses were prescribed according to institutional guidelines and published recommendations (9).

To our knowledge, this is the first study to demonstrate a correlation between molecular mechanisms of carbapenem resistance and outcomes of combination antimicrobial therapy among patients with CR-Kp bacteremia. The presence of a major ompK36 mutation was a highly sensitive marker for a doripenem MIC of >8 μg/ml and accurately predicted carbapenem-colistin treatment failures. Moreover, carbapenem-colistin therapy was significantly more likely to be effective when both agents were active in vitro, as defined by colistin MICs of ≤2 μg/ml and the presence of either a major ompK36 mutation or a doripenem MIC of ≤8 μg/ml. Our clinical experience supports in vitro data from our center, which demonstrated that major ompK36 mutations and a doripenem MIC of >8 μg/ml predicted a lack of KPC-Kp responsiveness to doripenem-colistin during time-kill assays (5).

Previous studies reported decreased mortality among patients with carbapenemase-producing K. pneumoniae bacteremia who were treated with carbapenem-containing combinations (1, 6, 7). In two of these studies, survival was higher if carbapenem MICs were ≤8 μg/ml (6, 7). The mortality rate in our study was only 20%, but all patients who died were infected with major ompK36 mutant strains that exhibited extremely high-level doripenem resistance. Our study differs from earlier reports by focusing specifically on colistin as the second agent in combination and by linking results to underlying mechanisms of carbapenem resistance. Positive interactions between carbapenems and colistin are consistent with a model in which membrane permeabilization by the latter facilitates increased access of the former to target sites, thereby overcoming carbapenemase hydrolysis (11). Major ompK36 mutations constrict the porin channel (ins aa 134–135 GD mutations) or attenuate gene expression and porin levels (IS5 promoter insertions) (12).

Our findings highlight the clinical importance of quantifying carbapenem MICs beyond current CLSI resistance breakpoints (doripenem, imipenem, and meropenem MICs of >2 μg/ml; ertapenem MIC of >1 μg/ml). Modeling data indicate that high-dose and prolonged-infusion carbapenem regimens, as recommended at our center, can increase the probability of achieving serum PK-PD targets of free-drug concentrations above the MIC (fT > MIC) for ≥35% of the dosing interval for inhibition of resistant Enterobacteriaceae if MICs are ≤8 μg/ml (13, 14). Along these lines, emerging clinical data suggest that optimized carbapenem dosing can successfully treat at least some infections due to Enterobacteriaceae with lower-level resistance (6, 7). Our study design precludes any conclusions about the benefits of carbapenem-colistin therapy over carbapenem monotherapy or definitive assessments of colistin monotherapy. It is notable that treatment failures were observed among the overwhelming majority of patients who received a regimen in which only colistin was active. A major shortcoming of many clinical studies has been the use of suboptimal colistin regimens, which confounds interpretations of the drug's effectiveness. However, 70% (7/10) of treatment failures among our patients receiving colistin as the sole active agent occurred despite recommended loading and maintenance doses (9). The poor clinical responses are in keeping with our previous in vitro time-kill data showing that colistin was merely bacteriostatic against colistin-susceptible KPC-Kp strains at concentrations achievable in human serum (5).

There are several limitations to this study. The retrospective design limited us to existing clinical data. It is possible that our experience was skewed by clinician biases in choosing a carbapenem-colistin regimen to treat these particular patients. Since all except two patients received doripenem, we cannot comment about other carbapenems. Indeed, it is important to acknowledge that our clinical experience may have differed if meropenem or another carbapenem was used instead of doripenem. Three patients infected with wild-type or minor mutant ompK36 strains exhibited doripenem MICs of 16 μg/ml; two of these patients failed treatment. Therefore, we cannot comment on the significance of discrepancies between genotypic and phenotypic definitions of carbapenem inactivity. The validity of our definitions requires future studies. Finally, our results may not be relevant at all centers since our sample size was small and strain resistance phenotypes and genotypes may differ. Nevertheless, our findings are likely to be broadly representative, as it is biologically plausible that antimicrobial responses in vitro and in patients are influenced by porin mutations in conjunction with other resistance mechanisms.

In conclusion, this pilot study indicates that carbapenem-colistin regimens are viable options against a subset of CR-Kp infections, but overall outcomes remain suboptimal. Our data suggest that antimicrobial MICs and/or molecular markers of resistance, such as the presence of particular carbapenemases and porin mutations, may be useful for identifying strains most likely to respond to carbapenem-colistin combinations. If our findings are validated at other centers and in larger populations, MICs and genotypes of CR-Kp strains should be considered by clinicians as they make treatment decisions, along with the type of infection being treated, underlying diseases, and immune status. There are limited numbers of new antimicrobials in development with activity against carbapenem-resistant pathogens (15). Phenotypic assays and molecular markers that predict antimicrobial responsiveness among CR-Kp strains may allow clinicians to reserve new agents for cases in which they are most needed.

ACKNOWLEDGMENTS

This project was supported by funding provided to the XDR Pathogen Laboratory by the University of Pittsburgh Medical Center and by the National Center for Advanced Translational Sciences of the National Institutes of Health (NIH) under award no. KL2 RR024154 given to R.K.S.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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