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Antimicrobial Agents and Chemotherapy, March 2007, p. 1102-1104, Vol. 51, No. 3
0066-4804/07/$08.00+0     doi:10.1128/AAC.01102-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

In Vitro Activities of Tigecycline and Eight Other Antimicrobials against Different Nocardia Species Identified by Molecular Methods{triangledown}

Emilia Cercenado,* Mercedes Marín, Mónica Sánchez-Martínez, Oscar Cuevas, José Martínez-Alarcón, and Emilio Bouza

Servicio de Microbiología Clínica y Enfermedades Infecciosas, Hospital General Universitario Gregorio Marañón, Universidad Complutense, Madrid, Spain

Received 31 August 2006/ Returned for modification 21 November 2006/ Accepted 17 December 2006


   ABSTRACT
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The in vitro activities of tigecycline and other antimicrobials against 51 isolates of Nocardia spp. were evaluated. MIC90s and MIC ranges were as follows: tigecycline, 4 and ≤0.06 to 8 mg/liter, respectively; minocycline, 2 and ≤0.06 to 2 mg/liter, respectively; linezolid, 1 and ≤0.06 to 2 mg/liter, respectively; moxifloxacin, 2 and ≤0.06 to >64 mg/liter, respectively; ertapenem, 32 and ≤0.06->64 mg/liter, respectively; imipenem, 2 and ≤0.06 to >64 mg/liter, respectively; meropenem, 8 and ≤0.06 to >64 mg/liter, respectively; amikacin, 1 and ≤0.06 to 32 mg/liter, respectively; and trimethoprim-sulfamethoxazole, 1/19 and ≤0.5/9.5 to >2/38 mg/liter, respectively.


   TEXT
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Nocardia species cause serious infections, especially in immunocompromised patients (4, 6, 16). Trimethoprim-sulfamethoxazole has traditionally been the agent of choice for the treatment of nocardiosis, with alternative drugs including minocycline, amikacin, and imipenem (4, 16). Resistance to the previous drugs, toxicity, intolerance, and therapeutic failures justify the search for alternative antimicrobial agents. The activity of tigecycline (15) against Nocardia spp. has never been evaluated; and there is very little information regarding the activities of linezolid, moxifloxacin, and ertapenem (1, 2, 5, 10, 20). In the study described here, we compared the in vitro activities of tigecycline, linezolid, ertapenem, and moxifloxacin with those of minocycline, imipenem, meropenem, amikacin, and trimethoprim-sulfamethoxazole against 51 nonduplicate clinical isolates of different Nocardia species.

(This study was presented in part at the 44th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 2004 [E. Cercenado, M. Marín, J. Martínez-Alarcón, and E. Bouza, Abstr. 44th Intersci. Conf. Antimicrob. Agents Chemother., abstr. E-2062, 2006].).

The isolates were obtained from 1995 to 2006 in our laboratory from respiratory samples (n = 41), cutaneous abscesses (n = 5), brain abscesses (n = 2), blood (n = 2), and cerebrospinal fluid (n = 1). Molecular identification was performed by PCR-restriction fragment length polymorphism (RFLP) analysis of the hsp65 gene (hsp65 PCR-RFLP) with PCR primers TB11 and TB12 and restriction analysis with BstEII, MspI, HinfI, and BsaHI, as described previously (17, 18). Identification was confirmed by sequencing the first 500 bp of the 16S rRNA gene (7, 12, 14). A 5'-end 16S rRNA gene-specific PCR was performed with universal primers E8F and E533F. The amplicons obtained were sequenced by the BigDye Terminator method and were detected with an ABI Prism 3100 automatic DNA sequencer (Applied Byosystems, Inc.). The sequences of well-characterized Nocardia isolates (14) deposited in GenBank were used as references for phylogenetic tree construction with ClustalX 1.8 software. Only identifications with 100% similarity were considered. The strains were stored at –70°C in skim milk until they were tested for their susceptibilities.

The solutions of the antimicrobials and the cation-adjusted Mueller-Hinton broth used were made up fresh on the day of testing (3). MICs were determined by the microdilution method, according to the guidelines of the CLSI (former NCCLS) guidelines (13), with twofold dilutions from 64 to 0.06 mg/liter of all antimicrobials except trimethoprim-sulfamethoxazole; trimethoprim-sulfamethoxazole was tested at concentrations ranging from 0.5/9.5 to 2/38 mg/liter. Bacterial suspensions were prepared by using fresh colonies grown on blood agar plates. Readings were performed after 72 h of incubation at 37°C in ambient air. The control strains used were Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213, and Enterococcus faecalis ATCC 29212.

A comparison of the identifications obtained by the hsp65 PCR-RFLP and by the 16S rRNA sequencing methods is shown in Table 1. Isolates identified as Nocardia asteroides VI by PCR-RFLP were heterogeneous, since identification by 16S rRNA sequencing differentiated three different species within this group and three isolates for which no GenBank sequence was found. This variability was also found between isolates identified by hsp65 PCR-RFLP as Nocardia nova and N. asteroides I. All Nocardia farcinica and Nocardia otitidiscaviarum isolates gave identical identifications by both methods.


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  		TABLE 1. Comparison of identifications of different Nocardia species obtained by PCR-RFLP of the hsp65 gene and 5'-end sequencing of the 16S rRNA gene

 
The activities of the different antimicrobials against all isolates and species are shown in Tables 2 and 3. The MIC50 and MIC90 of tigecycline were 1 and 4 mg/liter, respectively. These values were 1 and 2 mg/liter, respectively, for minocycline, and 0.5 and 1 mg/liter, respectively, for linezolid. Imipenem showed the best activity among the carbapenems, with meropenem being 4-fold less active than imipenem and with ertapenem being 16-fold less active than imipenem. One isolate was resistant to amikacin, and four isolates (two isolates each of Nocardia cyriacigeorgica and N. farcinica) were resistant to trimethoprim-sulfamethoxazole. There was species variability in the susceptibility profiles.


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  		TABLE 2. Activities of antimicrobial agents against 51 clinical isolates of Nocardia spp.a

 

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  		TABLE 3. MIC ranges of antimicrobial agents for different species of Nocardia

 
In general, all antimicrobials evaluated except ertapenem were active against different Nocardia species. Among the new agents, tigecycline and moxifloxacin were active against the majority of the isolates, and linezolid presented excellent activity against all isolates. To our knowledge, this is the first study to have evaluated the activities of tigecycline and ertapenem against Nocardia species. Tigecycline was active against imipenem-resistant and trimethoprim-sulfamethoxazole-resistant isolates, and ertapenem should not be assumed to be an alternative to other carbapenenems.

Nocardia species can vary in their antimicrobial susceptibility patterns, and accurate identification is essential if we are to know the epidemiological distribution of species and to predict their antimicrobial susceptibilities. We identified our isolates by PCR-RFLP since most susceptibility studies are based on this type of identification; however, identification based on 16S rRNA gene sequencing demonstrated that isolates identified by PCR-RFLP as belonging to the same species (N. asteroides VI, N. nova, and N. asteroides I) were heterogeneous. Three of the former N. asteroides VI isolates were identified as Nocardia abscessus by 16S rRNA gene sequencing; and all three were susceptible to carbapenems, which is in contrast to the findings of previous reports, which indicated that most N. abscessus isolates (type I drug pattern) are resistant to imipenem (21). This difference probably reflects the existence of underspecific species identification of the former N. abscessus species. Nevertheless, other studies have described N. abscessus isolates that are susceptible to carbapenems (10). Most of the former N. asteroides I isolates (type I drug pattern) were identified as Nocardia beijingensis and were also susceptible to carbapenems. In the case of the former N. nova isolates, three of the isolates matched with the species Nocardia veterana, and the MICs of imipenem and amikacin were very low for all isolates, as described previously (4, 10, 21). Only one isolate of Nocardia brasiliensis was studied, and that isolate was susceptible to imipenem. Although it has been described that this species is resistant to imipenem (10), the existence of susceptible isolates has been documented in other studies (11).

Trimethoprim-sulfamethoxazole had excellent activity, in general, and minocycline was active against all species; however, 47% of the isolates were inhibited by minocycline at 2 mg/liter, a value that falls in the intermediate susceptibility category (13), as described in previous studies (10, 19, 21). Moxifloxacin was active against the majority of the isolates and has the advantage of being available in an oral formulation. However, its variable activity makes it necessary to perform susceptibility studies before its use (20). Amikacin was uniformly active against all isolates but one, which corresponded to the intrinsically amikacin-resistant species Nocardia transvalensis (4). Linezolid presented the lowest MICs of all antimicrobials evaluated, as described previously (4, 5, 10, 11, 16). Its availability as an oral agent gives it the potential to be used as a primary drug for the treatment of nocardiosis, but issues regarding tolerance and the toxicity of linezolid in long-term therapy need to be clarified (8, 9).

These results show that we have many therapeutic options for the treatment of nocardiosis. However, since identification of Nocardia species requires the use of molecular techniques and since different species and isolates vary in their antimicrobial susceptibility patterns, it is necessary to perform in vitro susceptibility testing of all clinically significant isolates.


   ACKNOWLEDGMENTS
 
This is a fully independent study that has not received financial support from any pharmaceutical company. This study was financed by the Red Española de Investigación en Patología Infecciosa (grant REIPI-ISCIII C03/14).


   FOOTNOTES
 
* Corresponding author. Mailing address: Servicio de Microbiología Clínica y Enfermedades Infecciosas, Hospital General Universitario Gregorio Marañón, Dr. Esquerdo 46, 28007 Madrid. Spain. Phone: 34-91-586-8459. Fax: 34-91-5044906. E-mail: ecercenado{at}terra.es. Back

{triangledown} Published ahead of print on 28 December 2006. Back


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Antimicrobial Agents and Chemotherapy, March 2007, p. 1102-1104, Vol. 51, No. 3
0066-4804/07/$08.00+0     doi:10.1128/AAC.01102-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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