Activity of Polymyxin B and the Novel Polymyxin Analogue CB-182,804 Against Contemporary Gram-Negative Pathogens in New York City

Abstract

Multidrug-resistant Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa have become common in many regions, often requiring therapy with colistin or polymyxin B. An increase in resistance to these agents would render many infections untreatable. We tested the activity of polymyxin B and the novel polymyxin analogue CB-182,804 against over 5,000 recent Gram-negative clinical isolates from New York City, a region with a high prevalence of multiresistant strains. Over 96% of Escherichia coli, K. pneumoniae, A. baumannii, and P. aeruginosa were susceptible to polymyxin B; only 76% of Enterobacter spp. was susceptible. The MICs of CB-182,804 were generally two-fold higher than polymyxin B and cross-resistance was observed. The addition of rifampin resulted in synergistic inhibition and bactericidal activity in time kill studies, and restored activity against all polymyxin-resistant strains. The synergistic effect of the combination with rifampin was most pronounced against A. baumannii strains, and was slightly greater with CB-182,804 than with polymyxin B against K. pneumoniae and Enterobacter spp. Despite considerable usage of polymyxin B and colistin in this region, polymyxin B retains excellent activity against most Gram-negative isolates. CB-182,804 shows similar activity, particularly when combined with rifampin. The clinical utility of CB-182,804 remains to be determined.

Introduction

Gram-negative bacterial pathogens have relentlessly acquired antibiotic resistance mechanisms over the last three decades. Carbapenem resistance has been caused by carbapenemases of the OXA-type in A. baumannii, of the VIM- and IMP-types in P. aeruginosa, and of the KPC-type in K. pneumoniae and other Enterobacteriaceae. Due to resistance to most or all other antibiotic classes, the polymyxins have become the standard treatment for infections due to MDR strains.⁹ The use of polymyxins is limited by nephrotoxicity and the emergence of resistance. The development of a polymyxin with less nephrotoxicity and strategies to reduce the emergence of polymyxin resistance would clearly be beneficial. Resistance to these agents has been well-documented in clinical isolates,⁹ and the clinical utility of this class has been predicted to be shortlived.¹⁰ Polymyxin B is widely used in Brazil^7,12 and most hospitals in New York City (personal communications), two regions with widespread multidrug-resistant A. baumannii and K. pneumoniae. Given the high prevalence of MDR Gram-negative pathogens and regular usage of the polymyxins at New York City hospitals, this region would appear to be at risk for the emergence and spread of polymyxin resistance. In this report, we evaluate the in vitro activity of polymyxin B against a large collection of recent clinical isolates from New York City hospitals. We also evaluate the in vitro activity of the investigational compound CB-182,804, a novel polymyxin analogue.

Materials and Methods

Bacterial isolates and susceptibility testing

During a 3-month period in 2009, clinical isolates of E. coli, K. pneumoniae, Enterobacter spp., A. baumannii, and P. aeruginosa were collected at 16 hospitals in Brooklyn, NY and Staten Island, NY. All unique-patient isolates of the targeted species were obtained from the hospital Microbiology laboratories. MICs of a panel of antibiotics were performed by agar-dilution according to CLSI methodology.³ The following antibiotics were tested: ceftazidime, ciprofloxacin, meropenem, amikacin, polymyxin B, and CB-182,804 (Cubist Pharmaceuticals, Lexington, MA). MICs of polymyxin B and CB-182,804 were also tested in the presence of rifampin (1 μg/ml), an antibiotic that has shown synergy with polymyxins in several reports.⁹ Susceptibility breakpoints followed CLSI guidelines; for Enterobacteriaceae, isolates with an MIC ≤2 μg/ml were considered susceptible to polymyxin B. ATCC strains P. aeruginosa 27853 and E. coli 25922 were used as controls.

Time-kill studies

A total of 25 isolates were selected for time-kill studies, including five isolates of each Gram-negative species. All Enterobacter spp. selected were Enterobacter cloacae. For each species, two of five were selected because of resistance to polymyxin B, and the remaining three polymyxin-susceptible isolates were selected because of resistance to other antibiotic classes. Multiplex PCR was performed to detect the presence of bla_KPC, bla_IMP, bla_VIM, bla_OXA23-like, bla_OXA24-like, and bla_OXA58-like using previously described primers and conditions.^1,14 Time-kill studies were performed in fresh cation-supplemented Mueller-Hinton broth as previously described.² The following concentrations of polymyxin B and CB-182,804 were tested: 0.125, 0.25, 0.5, 1, 2, 4, and 8 μg/ml. Each antibiotic was tested alone and in the presence of rifampin 1 μg/ml. The concentrations tested were chosen to reflect clinically relevant levels. Antibiotic carryover was eliminated by diluting the final cultures 200-fold in pour plates. Bactericidal activity was defined as a decrease of ≥3 log cfu/mL in 24 h. Synergy was defined as a ≥100-fold increase in killing at 24 h by a combination compared with the most active single agent, assuming no significant inhibition by at least one agent.

The study was approved by the Institutional Review Board at SUNY Downstate Medical Center.

Results

Susceptibility results

Over 5,400 unique patient isolates were collected during the 3-month period (Table 1). A high prevalence of isolates were multidrug-resistant, with 29% of K. pneumoniae and 7% of Enterobacter spp. possessing bla_KPC, and approximately 85% of A. baumannii and 25% of P. aeruginosa exhibiting carbapenem resistance. bla_OXA-23 or bla_OXA-72 was detected in 19% of A. baumannii isolates. No other carbapenemases were detected in A. baumannii isolates and none were detected in P. aeruginosa isolates. Overall, polymyxin B retained excellent activity against E. coli, K. pneumoniae, A. baumannii, and P. aeruginosa, with no increase in resistance compared with a prior surveillance conducted in 2006.⁸ However, polymyxin B resistance among Enterobacter spp. increased from 6% to 24% (p<0.0001). The presence of rifampin (1 μg/ml) resulted in lower polymyxin B MICs for all species, with all isolates susceptible to the combination (polymyxin B MIC ≤2 μg/ml). The effect of rifampin was most pronounced with polymyxin B-resistant strains, with polymyxin B MICs often decreasing from >16 μg/ml to ≤0.125 μg/ml. In addition, the effect of rifampin was much greater with A. baumannii (polymyxin B MIC₅₀ ≥eight-fold lower) than with the other species (MIC₅₀ two-fold lower).

Table 1.

Susceptibility Results for over 5,000 Clinical Isolates at New York City Hospitals

	MIC₅₀ (μg/ml)	MIC₉₀ (μg/ml)	Range	Susceptible (%)	Concentration inhibiting ≥95% isolates
Escherichia coli (n=3,049)
Ceftazidime	≤0.25	2	≤0.25–>32	93
Meropenem	≤0.12	≤0.12	≤0.12–2	99.9
Ciprofloxacin	≤0.12	>4	≤0.12–>4	67
Amikacin	2	4	≤0.5–>64	93
Polymyxin B	1	1	≤0.12–>8	99.8	2 μg/ml
CB-182,804	2	4	≤0.12–>8		4 μg/ml
Klebsiella pneumoniae (n=1,155)
Ceftazidime	2	>32	≤0.25–>32	52
Meropenem	≤0.12	8	≤0.12–>16	85
Ciprofloxacin	2	>4	≤0.12–>4	49
Amikacin	1	32	≤0.5–>64	70
Polymyxin B	1	2	0.25–>16	96	2 μg/ml
Polymyxin B + Rifampin^a	0.5	1	≤0.12–2	100	1 μg/ml
CB-182,804	2	4	≤0.25–>8		4 μg/ml
CB-182,804 + Rifampin	0.25	0.25	≤0.12–>4		0.5 μg/ml
Enterobacter spp (n=199)
Ceftazidime	0.5	>32	≤0.25–>32	68
Meropenem	≤0.12	0.5	≤0.12–16	99
Ciprofloxacin	≤0.12	>4	≤0.12–>4	84
Amikacin	1	4	≤0.5–64	97
Polymyxin B	1	>8	≤0.12–>16	76	>16 μg/ml
Polymyxin B + Rifampin	0.5	0.5	≤0.12–1	100	1 μg/ml
CB-182,804	2	>8	≤0.25–>8		>8 μg/ml
CB-182,804 + Rifampin	≤0.12	0.5	≤0.12–>4		0.5 μg/ml
Acinetobacter baumannii (n=407)
Ceftazidime	>32	>32	≤0.25–>32	13
Meropenem	>16	>16	≤0.12–>16	15
Ciprofloxacin	>4	>4	≤0.12–>4	11
Amikacin	32	>64	≤0.5–>64	38
Polymyxin B	1	2	≤0.25–>16	97	2 μg/ml
Polymyxin B + Rifampin	≤0.12	0.25	≤0.12–1	100	0.5 μg/ml
CB-182,804	2	4	1–>16		4 μg/ml
CB-182,804 + Rifampin	≤0.12	0.5	≤0.12–>4		1 μg/ml
Pseudomonas aeruginosa (n-679)
Ceftazidime	4	>32	≤0.25–>32	75
Meropenem	2	8	≤0.12–>16	74
Ciprofloxacin	0.5	>4	≤0.12–>4	54
Amikacin	8	16	≤0.5–>64	92
Polymyxin B	1	2	0.25–4	99.5	2 μg/ml
Polymyxin B + Rifampin	0.5	1	≤0.12–2	100	1 μg/ml
CB-182,804	2	8	≤0.25–16		8 μg/ml
CB-182,804 + Rifampin	1	2	≤0.12–>4		2 μg/ml

Rifampin concentration 1 μg/ml.

The activity of the novel polymyxin analogue CB-182,804 was slightly less than polymyxin B, with MICs that were generally two-fold higher than those of polymyxin B (Table 1). Isolates resistant to polymyxin B required higher concentrations of CB-182,804 as well. Rifampin enhanced the activity of CB-182,804 similar to polymyxin B against A. baumannii and P. aeruginosa (MIC₅₀ ≥16-fold and two-fold lower, respectively). However, the effect of rifampin was much more pronounced with CB-182,804 against K. pneumoniae and Enterobacter spp.; MIC₅₀ ≥eight-fold lower with CB-182,804 versus two-fold lower with polymyxin B.

Time-kill study results

Time-kill studies were performed on 25 isolates (Table 2). Two of five isolates of each species were resistant to polymyxin B. The remaining polymyxin B-susceptible isolates were all multidrug-resistant including 10 of 15 that were carbapenem-resistant. bla_KPC was present in 2 of 3 polymyxin B-susceptible E. coli and K. pneumoniae and 1 of 3 E. cloacae. bla_OXA24-like was present in 2 of 3 polymyxin B-susceptible A. baumannii isolates. No other carbapenemases were detected. Rifampin MICs ranged from 4–128 μg/ml in the 25 isolates, with most having an MIC of 32 μg/ml. Both antibiotics exhibited concentration-dependent killing with polymyxin B bactericidal at 1× MIC and CB-182,804 bactericidal at 1–2× MIC for the polymyxin B-susceptible strains. The combination of each antibiotic with rifampin was synergistic against 22 of 25 isolates (including all polymyxin B-resistant strains) and bactericidal against all strains. In general, CB-182,804 was bactericidal at a two-fold lower concentration than polymyxin B when combined with rifampin.

Table 2.

Combined Results of Time-Kill Studies Against Strains of E. coli, K. pneumoniae, Enterobacter cloacae, A. baumannii, and P. aeruginosa

	Change in log CFU/ml (mean±sd)
	Polymyxin B susceptible (n=15)		Polymyxin B resistant (n=10)
Antibiotic (μg/ml)^a	at 4 h	at 24 h	at 4 h	at 24 h
Polymyxin B (0.125)	+0.1±2.1	+2.3±2.1	+2.1±0.4	+3.3±0.4
Polymyxin B (0.25)	−1.9±2.2	0±4.0	+1.3±1.6	+2.9±0.4
Polymyxin B (0.5)	−3.4±1.2	−2.8±3.9	+0.3±3.7	+2.9±0.5
Polymyxin B (1)	−4.2±0.6	−4.7±1.6	+0.3±1.9	+2.6±0.9
Polymyxin B (2)	−4.3±0.5	−5.0±0.4	−0.5±2.7	+1.8±2.6
Polymyxin B (4)	−4.3±0.5	−5.1±0.4	−1.4±3.0	+0.8±3.2
Polymyxin B (8)	−4.3±0.5	−5.0±0.4	−1.3±3.0	+0.7±3.2
Polymyxin B (0.125)^a	−1.6±2.2	+0.3±3.8	−0.2±3.0	0±3.8
Polymyxin B (0.25)^a	−3.3±1.8	−3.1±3.7	−2.4±2.3	+0.5±3.7
Polymyxin B (0.5)^a	−3.8±1.1	−4.1±2.3	−2.6±2.0	−2.7±3.6
Polymyxin B (1)^a	−4.1±0.8	−4.9±0.8	−3.5±1.4	−3.7±2.7
Polymyxin B (2)^a	−3.8±1.5	−4.9±0.7	−4.2±0.9	−5.2±0.3
Polymyxin B (4)^a	−4.1±1.1	−5.1±0.4	−4.2±0.8	−5.2±0.3
Polymyxin B (8)^a	−4.3±0.5	−5.1±0.4	−4.1±1.3	−4.9±0.9
CB-182,804 (0.125)	+1.3±1.5	+2.6±2.3	+2.3±0.9	+2.9±0.4
CB-182,804 (0.25)	−0.7±2.4	+1.3±3.1	+1.9±1.3	+3.0±0.3
CB-182,804 (0.5)	−2.5±2.0	−0.1±3.8	+1.8±1.6	+3.0±0.3
CB-182,804 (1)	−3.6±1.8	−3.4±3.4	+1.6±1.4	+2.9±0.4
CB-182,804 (2)	−4.0±1.0	−4.0±2.6	+0.9±1.7	+2.7±0.3
CB-182,804 (4)	−4.1±0.6	−4.3±2.0	+0.1±2.5	+1.6±2.0
CB-182,804 (8)	−4.3±0.5	−5.0±0.4	−1.0±2.4	+1.7±2.5
CB-182,804 (0.125)^a	−3.1±1.9	−2.6±3.8	−1.2±2.3	−0.4±3.9
CB-182,804 (0.25)^a	−3.6±1.3	−4.4±2.2	−2.4±2.2	−2.3±2.7
CB-182,804 (0.5)^a	−3.8±1.2	−4.9±0.8	−2.7±2.0	−3.2±3.0
CB-182,804 (1)^a	−3.9±1.1	−4.8±1.0	−3.4±1.4	−4.1±2.0
CB-182,804 (2)^a	−3.9±1.2	−4.9±0.8	−3.8±1.2	−4.8±1.0
CB-182,804 (4)^a	−3.8±1.2	−4.8±0.9	−3.9±1.0	−4.4±2.4
CB-182,804 (8)^a	−4.1±0.9	−5.1±0.4	−4.1±0.9	−5.0±0.6
Rifampin (1)	+1.9±1.2	+3.1±0.5	+1.9±1.1	+2.8±0.7
Control	+2.1±0.6	+3.2±0.4	+2.2±0.9	+3.1±0.3

Plus Rifampin, 1 μg/ml.

Discussion

For many patients infected with MDR Gram-negative pathogens, the polymyxins remain the last available options for therapy. However, resistance develops frequently in vitro, occurs with clinical isolates, and can emerge during therapy. The data presented suggest that in New York City, a region affected by endemic MDR A. baumannii and K. pneumoniae for many years, resistance to polymyxin B has remained low with the exception of Enterobacter spp. The reason that polymyxin resistance has not become more common in this setting is unclear. While this phenomenon may be related to factors intrinsic to the polymyxins, one of the possible explanations may be the routine use of combination therapy by many clinicians. In one review involving a limited number of patients, most polymyxin B treated patients received combination therapy.⁶ Combination therapy with rifampin and other agents is used for most patients at many New York City hospitals (personal communications). Numerous studies have demonstrated in vitro synergy between the polymyxins and other antibiotics against MDR Gram-negative strains.⁹ Also, the in vitro phenomenon of regrowth frequently occurs when testing polymyxin B and colistin, often resulting in resistance. Combining polymyxins with other antibiotics can prevent the occurrence of regrowth.⁵ Combination therapy with rifampin was associated with improved outcomes in animal models;⁹ however, adequate controlled studies in humans are lacking.⁴ Whether the use of antibiotic combinations improves clinical outcomes or reduces the spread of polymyxin resistance requires further study.

Polymyxin B resistance markedly increased among Enterobacter spp. compared with a similar surveillance conducted in 2006. The reason why E. cloacae and Enterobacter aerogenes differ from the other Gram-negative bacteria is unknown. Previous studies have noted the occurrence of colistin hetero-resistance among E. cloacae strains.¹¹ Additional testing of the polymyxin B-resistant Enterobacter spp. in this study revealed that hetero-resistance was common (data not shown). Whether this phenomenon is related to the emergence of polymyxin resistance among Enterobacter spp. remains to be determined.

This study confirms the routine presence of synergistic activity when polymyxin B is combined with rifampin, especially against polymyxin B-resistant strains of each species. Currently, there are no simplified reliable methods of polymyxin B or colistin susceptibility testing approved for use in the United States.⁹ Therefore, polymyxin-resistant bacteria are often not identified at many hospitals. Aside from the possibility that combination therapy might reduce the emergence of resistance to polymyxins, combination therapy would provide reliable activity against both polymyxin-susceptible and resistant strains in this region.

The spread of MDR Gram-negative bacteria has created a need for the development of new antimicrobial agents. CB-182,804, a novel polymyxin analogue, was previously demonstrated to have similar in vitro activity to colistin.¹³ This larger study demonstrated that the overall activity of CB-182,804 was similar to polymyxin B. In general, CB-182,804 MICs were two-fold higher than polymyxin B against each of the tested species, and uniform cross-resistance was observed. The presence of cross-resistance is not surprising given the structural similarities of the two compounds. Of interest, the synergy between rifampin and CB-182,804 was somewhat more pronounced than with polymyxin B for K. pneumoniae and Enterobacter spp. Whether this would allow the use of lower doses and the overall clinical utility of CB-182,804 remain to be determined.

In summary, polymyxin B retains excellent activity against MDR Gram-negative bacteria from New York City despite considerable usage at regional hospitals. The combination of polymyxin B with rifampin is synergistic and bactericidal even against polymyxin-resistant strains. The novel polymyxin CB-182,804 has activity similar to polymyxin B particularly when combined with rifampin and deserves further study.

Footnotes

Acknowledgment

This work was supported by Cubist Pharmaceuticals, Lexington, MA. The sponsor had no decision-making role in the design, execution, analysis, or reporting of the research.

Disclosure Statement

No competing financial interests exist.

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