In Vitro Antimicrobial Activity of “Last-Resort” Antibiotics Against Unusual Nonfermenting Gram-Negative Bacilli Clinical Isolates

Abstract

In this prospective multicentric study, we assessed the in vitro antimicrobial activity of carbapenems (imipenem, meropenem, and doripenem), tigecycline, and colistin against 166 unusual nonfermenting Gram-negative bacilli (NF-GNB) clinical isolates collected from nine French hospitals during a 6-month period (from December 1, 2008, to May 31, 2009). All NF-GNB isolates were included, except those phenotypically identified as Pseudomonas aeruginosa or Acinetobacter baumannii. Minimal inhibitory concentrations (MICs) of antimicrobial agents were determined by using the E-test technique. The following microorganisms were identified: Stenotrophomonas maltophilia (n=72), Pseudomonas spp. (n=30), Achromobacter xylosoxidans (n=25), Acinetobacter spp. (n=18), Burkholderia cepacia complex (n=9), Alcaligenes faecalis (n=7), and Delftia spp. (n=5). All isolates of Acinetobacter spp., A. faecalis, and Delftia spp. were susceptible to the three carbapenems. Imipenem exhibited the lowest MICs against Pseudomonas spp., and meropenem, as compared with imipenem and doripenem, displayed an interesting antimicrobial activity against A. xylosoxidans and B. cepacia complex isolates. Conversely, no carbapenem exhibited any activity against S. maltophilia. Except for S. maltophilia isolates, tigecycline and colistin exhibited higher MICs than carbapenems, but covered most of the microorganisms tested in this study. To our knowledge, no prior study has compared antimicrobial activity of these five antibiotics, often considered as “last-resort” treatment options for resistant Gram-negative infections, against unusual NF-GNB clinical isolates. Further studies should be carried out to assess the potential clinical use of these antibiotics for the treatment of infections due to these microorganisms.

Introduction

Nonfermenting Gram-negative bacilli (NF-GNB) are ubiquitous bacteria widespread in the environment and are involved in various nosocomial infections. They are also responsible for recurrent infections in cystic fibrosis (CF) patients. These microorganisms are particularly worrying because of the increase of severely ill and immunocompromised patients in hospitals, in conjunction with their growing resistance to multiple antimicrobial agents.⁶

Pathogenicity and epidemiology features of Pseudomonas aeruginosa and Acinetobacter baumannii have been largely described in nosocomial infections,⁶ but little information is available about other NF-GNB, often considered as of a minor clinical significance.⁸ Nevertheless, their involvement in human infections is likely underestimated, partly due to their phenotypic misidentification.¹³

Hence, most studies dealing with antimicrobial susceptibility of NF-GNB have focused on P. aeruginosa and A. baumannii species, particularly on multidrug-resistant clinical isolates.⁴ To date, only a few studies explored susceptibility of unusual NF-GNB to antibiotics,^3,9,21 especially those often considered as “last-resort” agents for resistant Gram-negative infections, that is, carbapenems (including the newest doripenem), tigecycline, or polymyxins.^16,27,30

The aim of this prospective multicentric study was to compare the in vitro antimicrobial activity of carbapenems (imipenem, meropenem, and doripenem), tigecycline, and polymyxin E (also named colistin) against unusual NF-GNB clinical isolates collected in France.

Materials and Methods

Different NF-GNB recovered from clinical samples were prospectively collected from nine French hospitals during a 6-month period (from December 1, 2008, to May 31, 2009). All NF-GNB isolates were included, except those phenotypically identified as P. aeruginosa or A. baumannii. All the strains were identified by 16S rRNA gene sequencing as previously described.¹³ Note that the different species of Pseudomonas (other than P. aeruginosa), Acinetobacter (other than A. baumannii), and Delftia were pooled as Pseudomonas spp., Acinetobacter spp., and Delftia spp. groups, respectively.

Minimal inhibitory concentrations (MICs) of doripenem, meropenem, imipenem, tigecycline, and colistin were determined by using the E-test technique (AB Biodisk, Solna, Sweden) according to the manufacturer's instructions. The EUCAST susceptibility breakpoints (www.eucast.org/) were used for carbapenems: species-specific breakpoints for Pseudomonas spp. (≤4, ≤2, and ≤1 mg/L for imipenem, meropenem, and doripenem, respectively) and Acinetobacter spp. (≤2, ≤2, and ≤1 mg/L for imipenem, meropenem, and doripenem, respectively), and nonspecies related breakpoints for other species (≤2, ≤2, and ≤1 mg/L for imipenem, meropenem, and doripenem, respectively). For colistin, strains exhibiting an MIC ≤2 mg/L were considered as susceptible,¹¹ except for Pseudomonas spp. (≤4 mg/L) (www.eucast.org/). For tigecycline, a susceptibility breakpoint of ≤2 mg/L was used for comparison purposes.^2,7

Results and Discussion

During the study, 166 NF-GNB were collected from 166 clinical samples, mainly respiratory tract samples (n=71) including 19 samples from CF patients, followed by blood (n=28), urine (n=26), gastrointestinal tract (n=20), skin and soft tissue (n=11), ear-nose-throat (n=5), bone and joint (n=4), and genital tract (n=1) samples.

The following bacterial species were identified: Stenotrophomonas maltophilia (n=72), Pseudomonas spp. (n=30), Achromobacter xylosoxidans (n=25), Acinetobacter spp. (n=18), Burkholderia cepacia complex (n=9), Alcaligenes faecalis (n=7), and Delftia spp. (n=5) (Tables 1 and 2).

Table 1.

Distribution of Minimal Inhibitory Concentration Values (mg/L) of Carbapenems, Tigecycline, and Colistin for the 166 Nonfermenting Gram-Negative Bacilli Clinical Isolates

	≤0.01	0.03	0.06	0.125	0.25	0.5	1	2	4	8	16	≥32
Stenotrophomonas maltophilia (72)
Imipenem	0	0	0	0	0	0	0	0	0	0	0	72
Meropenem	0	0	0	0	0	0	0	0	0	0	0	72
Doripenem	0	0	0	0	0	0	0	0	0	0	0	72
Tigecycline	0	0	0	0	2	8	24	14	15	6	2	1
Colistin	1	2	4	10	10	3	4	11	10	1	0	16
Pseudomonas spp. (30)^a
Imipenem	1	0	0	3	4	7	8	1	2	2	0	2
Meropenem	5	2	2	1	0	0	2	3	2	5	0	8
Doripenem	4	1	3	2	1	1	5	1	1	7	1	3
Tigecycline	0	1	1	1	2	4	2	1	6	0	2	9
Colistin	0	0	2	1	1	7	3	8	5	1	1	0
Achromobacter xylosoxidans (25)
Imipenem	0	0	0	0	0	0	11	7	2	0	1	4
Meropenem	0	0	1	3	8	4	1	2	1	1	0	4
Doripenem	0	0	0	2	1	6	4	6	0	2	0	4
Tigecycline	0	0	0	0	0	0	5	6	8	2	4	0
Colistin	0	0	0	0	0	1	1	5	6	4	2	6
Acinetobacter spp. (18)^b
Imipenem	0	3	9	6	0	0	0	0	0	0	0	0
Meropenem	0	2	4	9	3	0	0	0	0	0	0	0
Doripenem	1	2	8	4	3	0	0	0	0	0	0	0
Tigecycline	0	0	2	3	5	5	2	1	0	0	0	0
Colistin	2	4	2	3	2	3	1	0	0	1	0	0
Burkholderia cepacia complex (9)
Imipenem	0	0	0	0	0	0	0	0	0	1	1	7
Meropenem	0	0	0	0	0	0	2	3	0	3	0	1
Doripenem	0	0	0	0	0	0	0	2	2	1	1	3
Tigecycline	0	0	0	0	0	0	1	3	1	0	0	4
Colistin	0	0	0	0	0	0	0	0	0	0	0	9
Alcaligenes faecalis (7)
Imipenem	0	0	0	0	4	3	0	0	0	0	0	0
Meropenem	2	5	0	0	0	0	0	0	0	0	0	0
Doripenem	0	0	6	1	0	0	0	0	0	0	0	0
Tigecycline	0	0	0	0	0	0	1	3	3	0	0	0
Colistin	0	0	0	0	0	1	0	3	2	0	0	1
Delftia spp. (5)
Imipenem	0	0	1	3	1	0	0	0	0	0	0	0
Meropenem	0	0	3	1	1	0	0	0	0	0	0	0
Doripenem	0	0	2	2	1	0	0	0	0	0	0	0
Tigecycline	0	0	0	2	2	1	0	0	0	0	0	0
Colistin	0	0	0	0	0	0	0	0	0	0	0	5

Includes P. putida (n=8), Pseudomonas spp. (n=8), P. stutzeri (n=4), P. oryzihabitans (n=4), P. fluorescens (n=3), P. monteilii (n=1), P. mosselii (n=1), and P. luteola (n=1).

Includes A. junii (n=5), A. lwoffii (n=5), A. johnsonii (n=3), A. ursingii (n=2), A. schindleri (n=1), A. radioresistens (n=1), and Acinetobacter spp. (n=1).

MIC, minimal inhibitory concentration.

Table 2.

In Vitro Susceptibility to Carbapenems, Tigecycline, and Colistin of the 166 Nonfermenting Gram-Negative Bacilli Clinical Isolates

Organism (no. of isolates)	MIC₅₀ (mg/L)	MIC₉₀ (mg/L)	Range (mg/L)	Susceptibility breakpoints (mg/L) ^a	% Susceptible
Stenotrophomonas maltophilia (72)
Imipenem	≥32	≥32	≥32	≤2	0
Meropenem	≥32	≥32	≥32	≤2	0
Doripenem	≥32	≥32	≥32	≤1	0
Tigecycline	2	8	0.25 to ≥32	≤2	66.7
Colistin	2	≥32	≤0.01 to ≥32	≤2	62.5
Pseudomonas spp. (30)^b
Imipenem	0.5	8	≤0.01 to ≥32	≤4	86.7
Meropenem	2	≥32	≤0.01 to ≥32	≤2	50
Doripenem	1	16	≤0.01 to ≥32	≤1	56.7
Tigecycline	4	≥32	0.03 to ≥32	≤2	41.4
Colistin	2	4	0.06 to 16	≤4	93.1
Achromobacter xylosoxidans (25)
Imipenem	2	≥32	1 to ≥32	≤2	72
Meropenem	0.5	≥32	0.06 to ≥32	≤2	76
Doripenem	1	≥32	0.125 to ≥32	≤1	52
Tigecycline	4	16	1 to 16	≤2	44
Colistin	4	≥32	0.5 to ≥32	≤2	28
Acinetobacter spp. (18)^c
Imipenem	0.06	0.125	0.03 to 0.125	≤2	100
Meropenem	0.125	0.25	0.03 to 0.25	≤2	100
Doripenem	0.06	0.25	≤0.01 to 0.25	≤1	100
Tigecycline	0.25	1	0.06 to 2	≤2	100
Colistin	0.125	1	≤0.01 to 8	≤2	94.4
Burkholderia cepacia complex (9)
Imipenem	—	—	8 to ≥32	≤2	0
Meropenem	—	—	1 to ≥32	≤2	55.6
Doripenem	—	—	2 to ≥32	≤1	0
Tigecycline	—	—	1 to ≥32	≤2	44.4
Colistin	—	—	≥32	≤2	0
Alcaligenes faecalis (7)
Imipenem	—	—	0.25 to 0.5	≤2	100
Meropenem	—	—	≤0.01 to 0.03	≤2	100
Doripenem	—	—	0.06 to 0.125	≤1	100
Tigecycline	—	—	1 to 4	≤2	57.1
Colistin	—	—	0.5 to ≥32	≤2	57.1
Delftia spp. (5)
Imipenem	—	—	0.06 to 0.25	≤2	100
Meropenem	—	—	0.06 to 0.25	≤2	100
Doripenem	—	—	0.06 to 0.25	≤1	100
Tigecycline	—	—	0.125 to 0.5	≤2	100
Colistin	—	—	≥32	≤2	0

EUCAST interpretative criteria (www.eucast.org/) were used for carbapenems: species-specific breakpoints for Pseudomonas spp. and Acinetobacter spp. and nonspecies related breakpoints for other species. A susceptibility breakpoint of ≤2 mg/L was used for tigecycline for comparison purposes.^2,7 A susceptibility breakpoint of ≤2 mg/L was used for colistin, except for Pseudomonas spp. (≤4 mg/L).¹⁰

Includes P. putida (n=8), Pseudomonas spp. (n=8), P. stutzeri (n=4), P. oryzihabitans (n=4), P. fluorescens (n=3), P. monteilii (n=1), P. mosselii (n=1), and P. luteola (n=1).

Includes A. junii (n=5), A. lwoffii (n=5), A. johnsonii (n=3), A. ursingii (n=2), A. schindleri (n=1), A. radioresistens (n=1), and Acinetobacter spp. (n=1).

In our study, all isolates of S. maltophilia exhibited high-level resistance to all carbapenems (MIC₅₀ ≥32 mg/L) (Tables 1 and 2). Few studies explored susceptibility of S. maltophilia to newer carbapenems. In a global survey carried out between 2003 and 2007, Castanheira et al. reported that 0.7% and 1.3% of isolates were inhibited with 2 mg/L of doripenem and meropenem, respectively.³ A Canadian study conducted in intensive care units in 2005 and 2006 showed that 1.9% of isolates were susceptible to meropenem (according to the CLSI non-Enterobacteriaceae breakpoints).²⁹

Concerning Acinetobacter spp., all carbapenems displayed low MIC₅₀ values: 0.06 mg/L for imipenem and doripenem and 0.125 mg/L for meropenem (Tables 1 and 2). Surprisingly, most studies showed higher MIC₅₀ to carbapenems.^3,14,24–26 Castanheira et al. showed MIC₅₀ of 1 mg/L for imipenem, and 2 mg/L for meropenem and doripenem.³ Similar results were found both in European and Chinese studies, with MIC₅₀ of 1 mg/L both for imipenem and doripenem.^24,25 Interestingly, a French study found MIC₅₀ of 0.5 mg/L both for imipenem and meropenem.¹⁴ Moreover, variability among worldwide areas was heterogeneous, carbapenem-resistant Acinetobacter strains being more prevalent in the Middle East, Latin America, and Asia than in Europe and North America.²⁶

For Pseudomonas spp., imipenem displayed the best activity (MIC₅₀=0.5 mg/L; 86.7% susceptible) as compared with that of meropenem (MIC₅₀=2 mg/L; 50% susceptible) and doripenem (MIC₅₀=1 mg/L; 56.7% susceptible) (Tables 1 and 2). Such reports concerning Pseudomonas other than P. aeruginosa are scarce, and similar results have been found for Pseudomonas fluorescens/Pseudomonas putida concerning imipenem and meropenem susceptibility.²¹ Conversely, meropenem and doripenem are known to be more potent than imipenem against P. aeruginosa,^{5,14,15,24,25,30} and these results point out a probable difference in intrinsic susceptibility to carbapenems between P. aeruginosa and related species.

Regarding A. xylosoxidans, higher MICs to carbapenems were shown (Tables 1 and 2), and the best antimicrobial activity was observed for meropenem (MIC₅₀=0.5 mg/L; 76% susceptible), as previously described.⁹

Although all isolates of the B. cepacia complex were resistant to imipenem and doripenem, meropenem remained active against 55.6% of isolates (Tables 1 and 2). All isolates of A. faecalis and Delftia spp. were susceptible to carbapenems (Tables 1 and 2), with MICs much lower than those observed in previous studies.^9,21

Tigecycline exhibited a moderate antimicrobial activity against S. maltophilia isolates (MIC₅₀=2 mg/L) (Tables 1 and 2), while previous studies reported similar results with MIC₅₀ ranges from 0.5 to 2 mg/L.^2,7,28 For Acinetobacter spp., we observed an MIC₅₀ of 0.25 mg/L (Tables 1 and 2), close to values described in previous worldwide studies.^12,17 In our study, we observed an MIC₅₀ of 4 mg/L for Pseudomonas spp. consistent with the low antimicrobial activity of this drug against P. aeruginosa.^20,28

More than 50% of B. cepacia group complex isolates were categorized resistant to tigecycline (Tables 1 and 2), consistent with a previous European collection (MIC₅₀=4 mg/L),¹⁸ whereas two recent studies showed a better antimicrobial activity with MIC₅₀<1 mg/L.^2,5 The five isolates of Delftia spp. were susceptible to tigecycline, whereas it showed a moderate activity against A. xylosoxidans (MIC₅₀=4 mg/L; 44% susceptible) and A. faecalis (57.1% susceptible) (Tables 1 and 2). Most previous reports dealt with tetracycline antimicrobial activity, and little is known about antimicrobial activity of tigecycline on these uncommon microorganisms.^1,2,21

All isolates of B. cepacia complex and Delftia spp. were highly resistant to colistin (Tables 1 and 2). This antibiotic displayed a good activity against S. maltophilia (MIC₅₀=2 mg/L; 62.5% susceptible) (Tables 1 and 2), whereas previous studies reported MIC₅₀ of polymyxins in a range of 1–8 mg/L.^7,19,21,28 Colistin exhibited a good antimicrobial activity against Pseudomonas spp. (MIC₅₀=2 mg/L; 93.1% susceptible) (Tables 1 and 2) consistent with results observed with polymyxin B,²¹ and a moderate activity against A. xylosoxidans isolates (MIC₅₀=4 mg/L; 28% susceptible) (Tables 1 and 2), as previously described.¹ In our study, colistin exhibited an interesting antimicrobial activity against Acinetobacter spp. (MIC₅₀=0.125 mg/L; 94.4% susceptible) (Tables 1 and 2), whereas previous studies described higher MICs (from 0.5 to 1 mg/L).^10,15 This difference could be due to the fact that we assessed the susceptibility on a very large range of concentrations, and that we included only species other than A. baumannii whereas most studies pooled the results of all Acinetobacter species.

To our knowledge, this is the first study comparing the activity of these five “last-resort” antibiotics against such a collection of unusual NF-GNB clinical isolates. All isolates of Acinetobacter spp., A. faecalis, and Delftia spp. were susceptible to the three carbapenems. Noteworthy, imipenem exhibited the lowest MICs against Pseudomonas spp., and meropenem, as compared with imipenem and doripenem, displayed an interesting antimicrobial activity against A. xylosoxidans and B. cepacia complex isolates. Conversely, not one carbapenem exhibited any activity against S. maltophilia. Overall, the interpretation of the susceptibility of these microorganisms must be considered very prudently. Indeed, variability is described in MIC determination between different methods for tigecycline and colistin.^19,23 Moreover, susceptibility breakpoints are not yet established for NF-GNB concerning tigecycline, and could lead to discrepant susceptibility interpretations for carbapenems according to FDA, EUCAST, or CLSI recommendations.^15,22

Footnotes

Acknowledgments

The authors are grateful to Michel Auzou (Caen) and Brigitte Couzon (Versailles) for excellent technical assistance.

Disclosure Statement

This study was supported by the GMC Group Study. E-test strips were graciously provided by AstraZeneca, MSD, Janssen-Cilag, Pfizer, and Sanofi-Aventis (for meropenem, imipenem, doripenem, tigecycline, and colistin, respectively).

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