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The combination of doripenem and colistin is bactericidal and synergistic against 2

colistin-resistant, carbapenemase-producing Klebsiella pneumoniae 3

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Meredith G. Jernigan1, Ellen G. Press2, M. Hong Nguyen2, Cornelius J. Clancy2,3, 5

Ryan K. Shields2 6

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1 Department of Pharmacy and Therapeutics, University of Pittsburgh 8

2 Department of Medicine, University of Pittsburgh; 9

3 VA Pittsburgh Healthcare System, Pittsburgh, Pennsylvania 10

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Keywords: Klebsiella pneumoniae, doripenem, colistin, synergy 12

Running Title: Doripenem and colistin are synergistic against KPCs 13

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Copyright © 2012, American Society for Microbiology. All Rights Reserved.Antimicrob. Agents Chemother. doi:10.1128/AAC.06364-11 AAC Accepts, published online ahead of print on 19 March 2012

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Abstract 20

We tested two-drug combinations of doripenem, colistin, gentamicin, and doxycycline 21

against 12 carbapenemase-producing Klebsiella pneumoniae (KPC) isolates by time-22

kill. Doripenem and colistin reduced the starting inocula by 2-logs for each isolate 23

(range: 2.02-6.01 log10) and was bactericidal or synergistic against 75 and 50%, 24

respectively. Among colistin- and pandrug-resistant isolates, synergy was identified in 25

60 and 67%, respectively. All other combinations were inferior. We are currently 26

evaluating doripenem and colistin as frontline therapy for KPC infection. 27

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Carbapenemase producing Klebsiella pneumoniae (KPC) are emerging causes 40

of morbidity and mortality at institutions worldwide (4) (12, 14-16). KPC isolates are 41

defined by resistance to the carbapenems and are generally resistant to most other 42

antimicrobial classes. Salvage agents that may retain activity against KPC isolates 43

include colistin, gentamicin, doxycycline and tigecycline (9-10, 18). Resistance to these 44

agents is increasing (3, 7), however, and treatment failures are well-recognized with 45

each (9). Given the dearth of effective antimicrobial agents and poor clinical outcomes 46

among patients with KPC infections, combination therapy has been utilized. 47

Unfortunately, in vitro synergy studies have yielded conflicting results and left clinicians 48

with no clear direction in selecting combination regimens. At our center, we have 49

utilized the combination of doripenem and colistin to treat patients with KPC infections, 50

a decision that was extrapolated from our encouraging preliminary experience in 51

treating extensively drug-resistant (XDR)-Acinetobacter baumannii infections (17). The 52

primary objective of this study was to determine if this combination is active against 53

KPC isolates recovered from our patients. In particular, we focused on colistin-resistant 54

isolates determined in vitro by broth dilution. We also sought to test other two-drug 55

combinations that include commonly employed salvage agents. 56

Toward these ends, we determined the in vitro activity of doripenem, colistin, 57

gentamicin and doxycycline, alone and in combination, against 12 KPC isolates 58

collected over a 3-year period from unique patients at our center. We included 59

doxycycline rather than tigecycline because it is more commonly used at our center, 60

particularly as part of treatment of KPC urinary tract infections. 61

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We confirmed the presence of KPC for each isolate by modified-Hodge test, and 62

determined minimum inhibitory concentrations (MICs) by standard broth microdilution 63

methods (1). One hundred percent (12/12), 83% (10/12), 83% (10/12) and 58% (7/12) 64

of isolates were resistant to doripenem, colistin, gentamicin and doxycycline, 65

respectively; 50% (6/12) were resistant to all agents. The corresponding MIC90’s were 66

>128, 16, 64 and 16 µg/mL, respectively. Using time-kill assays (2), we found that 67

colistin was bactericidal (>3 log10 decrease from the starting inoculum) against 33% 68

(4/12), including both colistin-susceptible strains. Doripenem and gentamicin were 69

bactericidal against one isolate each, whereas doxycycline was not bactericidal against 70

any isolate (Table 1). 71

We next assessed the activity of two drug combinations by time-kills using 50mL 72

flasks containing 9.9mL of cation-adjusted Mueller-hinton broth, 0.1mL of organism 73

(starting inoculums of 1 x 106 CFU/mL) and fixed drug concentrations alone and in 74

combination (doripenem: 8 µg/mL, colistin: 1 µg/mL, gentamicin: 2 µg/mL, and 75

doxycycline: 2 µg/mL). Flasks were incubated with agitation at 37°C and viable bacterial 76

colonies were enumerated at 0, 4, 8, 12, and 24 hours as previously reported (17). 77

Antimicrobial concentrations were representative of achievable serum levels. Synergy 78

and antagonism were defined as ≥2 log10 greater and lesser kill in combination versus 79

the most active single agent, respectively (21). The combination of doripenem and 80

colistin was bactericidal against 75% (9/12) of isolates (Table 2), and synergistic against 81

50% (6/12; Table 3). Log-kills ranged from 2.02 to 6.01 log10 CFU/mL (median: 3.95 82

log10 CFU/mL; Table 2), and this was the only combination that reduced the starting 83

inocula of all strains. Moreover, doripenem and colistin was synergistic against 60% 84

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(6/10) and 67% (4/6) of isolates that were resistant to colistin (MICs: 4 to 64 µg/mL) or 85

resistant to all agents, respectively. 86

Other combinations were less active (Table 3). Colistin and gentamicin was 87

bactericidal and synergistic against 50% (6/12) and 25% (3/12) of isolates, respectively. 88

The next most active combination, gentamicin plus doxycycline, was bactericidal and 89

synergistic against 25% (3/12) and 42% (5/12) of isolates, respectively. Synergy was 90

achieved against isolates that were doxycycline susceptible; bactericidal activity or 91

synergy was not identified among doxycycline-resistant strains. Doripenem and 92

gentamicin was also bactericidal against 25% (3/12), but synergy was evident against 93

only 8% (1/12). The least active combinations were colistin plus doxycycline and 94

doripenem plus doxycycline, which were bactericidal against 17% (2/12) and 8% (1/12) 95

of isolates, respectively. Doripenem and doxycycline was synergistic against 25% 96

(3/12) of isolates, whereas colistin plus doxycycline was synergistic against 8% (1/12). 97

Antagonism was identified with all combinations except doripenem and colistin (Table 98

3). The combination of colistin and doxycycline was antagonistic in 25% (3/12). 99

Doripenem plus colistin or gentamicin were associated with the lowest and 100

highest median AUCs, respectively (Table 4). Indeed, the median AUC for colistin and 101

doripenem was lower than the other combinations (p=0.004), including the individual 102

combinations of gentamicin plus doxycycline (p=0.04) and doripenem plus doxycycline 103

(p<0.0001; Figure 1, GraphPad Prism Software (La Jolla, CA)). 104

Taken together, our data demonstrate that doripenem plus colistin was the most 105

active two-drug combination against KPC isolates. Doripenem and colistin attained the 106

highest levels of bactericidal activity and synergy, and was the only combination that did 107

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not show any antagonism. Moreover, the combination was effective at clinically-108

achievable concentrations of both drugs (5, 8) and it was particularly active against 109

colistin- and pan-drug resistant isolates. In this regard, our findings are similar to those 110

we reported against XDR-Acinetobacter baumannii isolates from our center (17). 111

Furthermore, our results extend our understanding of in vitro synergy against KPC 112

isolates, which have been studied in colistin-susceptible strains (11, 20, 22) or by 113

checkerboard broth microdilution (6) previously. 114

At present, the treatment options for patients with KPC infections are limited, as 115

evident by crude mortality rates that are reported to range from 42 to 69% (13-14, 19, 116

24). Mortality rates are higher among patients infected with colistin-resistant isolates 117

(24). Investigators have reported that combination therapy may improve outcomes of 118

KPC infections compared to monotherapy (9, 23), but optimal regimens are not defined 119

and none have been shown to be effective against colistin resistant-isolates. Of note, 120

we reported promising preliminary data in a pilot clinical study using doripenem plus 121

colistin to treat XDR-A. baumannii infections (17). Based on these data and the in vitro 122

findings of the current study, we are currently using the combination as the 123

recommended front-line regimen against KPC infections. 124

The other combinations tested in this study demonstrated inferior bactericidal 125

activity and less synergy. Furthermore, the second most potent combination in vitro 126

(colistin plus gentamicin), is limited in clinical settings by the potential for additive 127

toxicity. Our data suggest that doxycycline and gentamicin may be useful against 128

isolates that retain susceptibility to the former agent, but at our center such isolates are 129

in the minority. In the end, this combination was synergistic against only 25% (3/12) of 130

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isolates and may be unsuitable for the treatment of severe or life-threatening KPC 131

infections. At present, we reserve doxycycline-containing regimens for the treatment of 132

uncomplicated cystitis at our center. The remaining combinations were even less 133

effective in vitro, and we do not employ them in the clinic. Finally, it is important to note 134

that the number of strains evaluated here are limited and future studies are needed. 135

Towards this, the exact mechanisms underlying the synergistic effects between 136

doripenem and colistin have not been identified and merit further investigation. It is 137

possible that colistin permeabilizes the outer cell membrane of KPC isolates allowing 138

high concentrations of doripenem to access penicillin-binding proteins in the 139

cytoplasmic membrane where the drug acts. 140

In conclusion, it is important to stress that these results were obtained against 141

KPC isolates from a single center. As such, the results may not be applicable to 142

isolates from other centers or geographic locations. It is advisable for centers to test 143

isolates recovered from patients at their institution. Having introduced doripenem and 144

colistin as the recommended therapy for KPC infections at our center, we are in the 145

process of accumulating our clinical experience and assessing the impact of the 146

regimen on preventing the emergence of colistin resistance. 147

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Acknowledgements. The project described was supported by Award Number KL2 152

RR024154 from the National Center for Research Resources to R.K.S. The content is solely 153

the responsibility of the authors and does not necessarily represent the official views of the 154

National Center for Research Resources or the National Institutes of Health. 155

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Table 1. Minimum inhibitory concentrations for KPC isolates

Strain

MIC (mcg/mL) Log Δ from the Starting Inoculum (log10 CFU/mL) †

Doripenem Colistin Gentamicin Doxycycline Doripenem Colistin Gentamicin Doxycycline

1* 64 8 16 8 3.67 -3.76 3.78 -1.40

18 16 64 32 4 -4.96 -2.86 3.75 2.73

82 16 8 8 4 3.64 3.64 3.22 3.95

124* 16 16 64 16 2.99 -1.89 -2.35 2.87

133* 128 16 64 16 3.83 -3.60 3.62 3.70

136 128 4 >64 4 3.53 -2.05 2.78 3.24

141 >128 0.5 1 4 4.44 -3.19 -2.74 3.90

145* >128 16 64 16 2.66 -1.11 -0.03 2.12

167 32 1 16 8 3.75 -4.74 4.43 3.49

180* 32 8 32 16 4.06 -0.47 3.53 3.69

182* 32 8 64 16 2.66 -0.47 3.93 3.98

183 64 16 1 2 4.39 -0.79 -3.70 3.52

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* Indicates isolates resistant to all agents tested.

† Log Δ = Final inoculum – Starting inoculum (log10 CFU/mL)

Negative numbers represent a decrease from the starting inoculum and positive numbers represent an increase. Fixed concentrations of doripenem (8 µg/mL), colistin (1 µg/mL), gentamicin (2 µg/mL), and doxycycline (2 µg/mL) were used for all isolates.

Note. The following susceptibility breakpoints were used: doripenem and colistin (≤ 2µg/mL) and gentamicin and doxycycline (≤ 4µg/mL).

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Table 2. Log change (log10 cfu/mL) from the starting inoculum and most active single agent after 24 hours of incubation.

Combination Isolate

1 18 82 124 133 136 141 145 167 180 182 183 Log Δ (log10 CFU/mL)*

Colistin + Doripenem -6.01 -4.12 -2.09 -5.99 -3.57 -3.95 -3.94 -4.65 -5.76 -2.02 -2.73 -3.90

-2.25 0.84 -5.73 -4.10 0.03 -1.90 -0.75 -3.54 -1.02 -1.55 -2.26 -3.11

Colistin + Gentamicin -4.56 -3.07 -3.20 -5.87 -2.07 -2.22 -5.60 1.70 -2.05 -0.60 -0.56 -5.00

-0.80 -0.21 -6.84 -3.52 1.53 -0.17 -2.41 1.73 2.69 -0.13 -0.09 -1.30

Colistin + Doxycycline -1.21 -0.38 0.29 -0.57 -1.06 -0.85 -3.37 -0.24 -5.48 0.02 -0.60 -1.15

2.55 2.48 -3.35 1.35 2.54 1.20 -0.18 0.87 -0.74 0.49 -0.13 -0.36

Doripenem + Gentamicin 3.69 -5.95 -2.40 3.85 4.11 0.99 -4.43 2.63 3.88 3.94 3.40 -5.04

0.02 -0.99 -5.62 6.20 0.49 -1.79 -1.69 2.66 0.13 0.41 0.74 -1.34

Doripenem + Doxycycline 4.47 -0.88 3.90 -3.03 4.13 3.21 3.95 2.70 4.49 -0.43 4.06 -1.99

5.87 4.08 0.26 -5.90 0.89 -0.03 0.05 0.58 1.00 -4.12 1.40 -5.51

Gentamicin +Doxycycline -1.61 -0.54 -5.98 4.01 2.93 -1.38 -5.65 2.74 4.51 3.42 3.73 -5.73

-0.21 -3.27 -9.20 6.36 -0.69 -4.16 -2.91 2.77 1.02 -0.11 -0.20 -2.03

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* Log Δ is defined as:

Non-shaded rows: Final inoculum – Starting inoculum (log10 CFU/mL). Negative numbers represent a decrease from the starting inoculum and positive numbers represent an increase.

Shaded rows: Final inoculum of the most active single drug – Final inoculum of the two-drug combination (log10 CFU/mL). Negative numbers represent greater killing with two-drugs versus one.

Fixed concentrations of doripenem (8 µg/mL), colistin (1 µg/mL), gentamicin (2 µg/mL), and doxycycline (2 µg/mL) were used for all isolates.

Note. Bold numbers indicate synergy.

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Table 3. Interactions between two drugs tested in combination

Backbone Agent

Agents Tested in Combination

Synergy n (%)

Indifference n (%)

Antagonism n (%)

Colistin

Doripenem 6 (50) 6 (50) 0

Gentamicin 3 (25) 8 (67) 1 (8)

Doxycycline 1 (8) 8 (67) 3 (25)

Doripenem Gentamicin 1 (8) 9 (75) 2 (17)

Doxycycline 3 (25) 7 (58) 2 (17)

Gentamicin Doxycycline 5 (42) 5 (42) 2 (17)

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Table 4. Area under the curve for two-drug combinations after 24 hours of incubation

NA = Not applicable; area under the curve was not calculated for antagonistic combinations.

Isolate Colistin plus Doripenem plus Gentamicin

plus Doripenem Gentamicin Doxycycline Gentamicin Doxycycline Doxycycline

1 62.34 88.02 NA 209.5 NA 106.6

18 69.6 103.9 NA 81.7 NA 134.2

82 74.8 57.4 141.0 62.8 150.6 52.9

124 47.8 37.46 132.5 NA 128.2 NA

133 87.9 110.2 NA 176.5 168.5 206.0

136 84.8 121.4 138.5 202.9 213.6 143.4

141 59.4 29.1 99.9 22.4 156.7 50.8

145 81.5 139.0 157.4 NA 195.3 NA

167 33.9 NA 51.0 207.6 207.4 220.1

180 118.3 132.4 146.7 155.4 138.7 203.7

182 112.3 137.0 135.5 183.8 164.8 168.2

183 82.1 10.0 132.1 15.9 107.5 26.4 Median (Range)

78.2 (33.9 – 118.3)

103.9 (10.0 – 139.0)

135.5 (51.0 – 157.4)

165.9 (15.9 – 209.5)

160.8 (107.5 – 213.6)

138.8 (26.4 – 220.1)

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Figure 1. Representative area under the curve (AUC) for isolates alone and in combination for the following drug combinations: a) colistin and doripenem; 

b) gentamicin and doxycycline; and c) doripenem and doxycyclineb) gentamicin and doxycycline; and c) doripenem and doxycycline

a) Representative AUC for colistin (light grey), doripenem (dark grey),or both (striped)

10

5

log

(CFU

/mL) c) Representative AUC for doripenem (light grey), doxycycline (dark grey),

or both (striped)10

U/m

L)

0 5 10 15 200

Time of Incubation (Hours)

0 5 10 15 200

5

log

(CFU

b) Representative AUC for gentamicin (light grey), doxycycline (dark grey),or both (striped)

10

/mL)

Time of Incubation (Hours)

0 5 10 15 200

5

log

(CFU

/

Figure 1 Legend. Representative AUC graphs of three synergistic combinations.  Shading with t i t th AUC f t d0 5 10 15 20

Time of Incubation (Hours)stripes represents the AUC for two‐drug combinations.

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