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In vitro activity of new fluoroquinolones and linezolid against non-tuberculous mycobacteria
International Journal of Antimicrobial Agents 21 (2003) 585 /588 www.ischemo.org
In vitro activity of new fluoroquinolones and linezolid against nontuberculous mycobacteria J.C. Rodriguez Dı´az *, M. Lo´pez, M. Ruiz, G. Royo Hospital General Universitario de Elche, Universidad Miguel Herna´ndez, Elche, Alicante, Spain Received 17 May 2002; accepted 16 December 2002
Abstract The objective of the study was to determine the activity of new fluoroquinolones and linezolid against 108 clinical isolates of different species of non-tuberculous mycobacteria isolated in Spain. Gatifloxacin and moxifloxacin were found to be more effective than levofloxacin. Mycobacterium kansasii was more susceptible to the fluoroquinolones tested than M. avium complex and M. fortuitum was more susceptible than M. chelonae . Linezolid was more active against M. kansasii than against M. avium complex. A better understanding of the relationship between the in vitro activity of these compounds and their usefulness in the treatment of these infections is needed. The new fluoroquinolones exhibit good activity against M. kansasii and M. fortuitum and linezolid is active against M. kansasii . # 2003 Elsevier Science B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Fluoroquinolones; Linezolid; Non-tuberculous mycobacteria
1. Introduction Infections due to non-tuberculous mycobacteria have acquired great clinical importance in recent years as they may affect both immunocompromised and immunocompetent patients [1 /9]. The clinical importance of isolating these mycobacteria depends on the patient’s characteristics, the site of the infection, the type of sample and the species isolated [10]. Treatment may be specific for the species isolated. These treatments are limited due to the small number of drugs available, especially those administered orally, and so it is very important to find new antibiotics that are useful in these infections [10]. Furthermore, in clinical practice, correct identification of these genera is slow and tedious and many clinical laboratories do not have the means to perform susceptibility tests with the result that in many cases empirical treatment has to be given.
* Corresponding author. Present address: Seccio´n de Microbiologı´a, Hospital General Universitario de Elche, Camı´ de L’Almazara, no. 11, 03203 Elche, Alicante, Spain; fax: /34-966-679-108. E-mail address: [email protected] (J.C. Rodriguez Dı´az).
We present data on the susceptibility to new fluoroquinolones and linezolid, of nontuberculous mycobacteria isolated in our health district.
2. Material and methods 2.1. Strains studied One hundred and eight clinical isolates of 16 species were studied. The organisms tested included 31 Mycobacterium avium , 23 M. gordonae , 16 M. fortuitum , 11 M. intracellulare , eight M. kansasii , four each of M. chelonae and M. nonchromogenicum , two each of M. aurum and M. terrae, one each of M. malmoense , M. celatum , M. poriferae , M. fluorescens, M. flavescens , M. xenopi and M. scrofulaceum . These isolates were obtained from the same number of patients in the Elche health district, which has 250 000 inhabitants and is situated in the southeast of Spain, on the Mediterranean coast. Between 1 January 1995 and 1 March 2002, we processed 19 723 clinical samples. The most numerous samples were: 13 377 respiratory (67.82%), 3473 urine (17.60%), 944 pleural fluid (4.78%), 732 biopsies, punctures and exudates (3.71%),
0924-8579/03/$30 # 2003 Elsevier Science B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/S0924-8579(03)00048-7
586
J.C. Rodriguez Dı´az et al. / International Journal of Antimicrobial Agents 21 (2003) 585 /588
440 CSF (2.23%), 276 faeces (1.39%) and 220 blood samples (1.11%). The clinical isolates obtained were maintained at /70 8C. In the studies on slow growing mycobacteria, we used Mycobacterium tuberculosis Mt 14323 [11] as the control strain and in the studies on fast growing mycobacteria, Staphylococcus aureus ATCC 29213. 2.2. Isolation and identification techniques
Table 1 Susceptibility of the clinical isolates of slow growing species studied (mg/l) Species
Moxifloxacin Gatifloxacin Levofloxacin Linezolid
M. avium MIC50/90
2/4
8/16
16/16
Modal MIC
4
8
16
/32// 32 /32
M. gordonae MIC50/90 Modal MIC
0.25/0/5 0.25
0.5/1 0.25
0.5/1 1
2/4 1
All clinical samples were cultivated in Lo¨wenstein medium and Coletsos medium and were incubated at 37 8C for 60 days. In addition, from 1996, all samples except sputum and urine were inoculated in ESP II (Difco). Isolates were identified by means of standard techniques. Identification of all strains was confirmed at the Carlos III Institute (Majadahonda, Madrid).
M. intracellulare MIC50/90 1/2 Modal MIC 2
4/4 4
8/8 8
32//32 32
M. kansasii MIC50/90 Modal MIC
1/4 1
2/16 2
4/ /32 4
2.3. Antibiotics tested
M. nonchromogenicum MIC50/90 1/32
2/32
4/32
16/32
M. aurum MIC50/90
0.06/0.06
0.06/0.125
1/1
0.06/ 0.125
M. terrae MIC50/90
0.25/16
0.25/32
0.5/16
/32// 32
0.25
0.25
1
16
1 0.125 0.06
1 0.06 0.06
2 0.125 0.125
2 0.5 1
0.06 0.06 1
0.06 0.06 1
0.125 0.25 4
1 2 32
The following quinolones were tested: levofloxacin (Hoechst), moxifloxacin (Bayer), gatifloxacin (Bristol Myers Squibb) and linezolid (Pharmacia). 2.4. Determination of in vitro activity 2.4.1. Slow growing mycobacteria Organisms were cultured in Middlebrook 7H11 medium (Difco) supplemented with OADC (Difco) and incubated at 37 8C in a CO2 incubator for 21 days. The following concentrations of antibiotics were tested: 0.5, 1, 2, 4, 8, 16, and 32 mg/l. The inoculum was made from a recent culture of the strain in Lo¨wenstein / Jensen medium. A suspension with a turbidity of 1 on the MacFarland scale was made in distilled water, was then diluted 1:100 in sterile distilled water and 5 ml put on each plate holding the antibiotic and in each control. On reading and interpreting the antibiograms we considered the strain to be susceptible to the antibiotic if less than 1% of the colonies which grew on the control plate were isolated on the plate with antibiotic [11]. 2.4.2. Fast growing mycobacteria The same procedure was followed but two different media were used Middlebrook 7H11 medium (Difco) supplemented with OADC (Difco) and Mueller Hinton agar (Difco); plates were read at 3 and 5 days.
M. malmoense M. celatum M. poriferae M. fluorescens M. flavescens M. xenopi M. scrofulaceum
0.125/2 0.125
the two fluoroquinolones with a C-8 methoxy group (gatifloxacin and moxifloxacin), were more active, as reported previously [12]. Of the three species most frequently involved in clinically important conditions, M. kansasii was the most susceptible, followed by M. intracellulare, while M. avium showed the highest level of resistance. All compounds showed good activity against M. gordonae , an organism not often associated with clinical conditions. These compounds exhibited excellent activity against the limited number of isolates studied of M. malmoense , M. celatum , M. scrofulaceum and M xenopi . Linezolid had little activity against the M. aviumintracellulare complex, but was more active against M. kansasii , M. gordonae , M. xenopi and M. celatum .
3. Results 3.1. Slow growing mycobacteria:
3.2. Fast growing mycobacteria
The susceptibility data are shown in Table 1. There were differences in fluoroquinolone susceptibility between the different species studied. However, in general
The susceptibility data, together with the different media and incubation times, are shown in Table 2. There was a clear difference in the activity of the various
J.C. Rodriguez Dı´az et al. / International Journal of Antimicrobial Agents 21 (2003) 585 /588 Table 2 Susceptibility of fast growing mycobacteria (mg/l) Species
Moxifloxacin Gatifloxacin Levofloxacin Linezolid
M. chelonae MIC50aMIC90a MIC50bMIC90b MIC50cMIC90c MIC50dMIC90d
2/16 4/16 8/16 8/16
1/16 4/16 4/8 4/16
3/32 4/32 1/31 4/32
8/ /32 32//32 8/16 16/16
M. fortuitum MIC50aMIC90a 0.25/0.5
0.25/0.5
0.5/1
MIC50bMIC90b 0.5/0.5
0.25/0.5
0.5/1
MIC50cMIC90c 0.25/0.5 MIC50dMIC90d 0.5/0.5
0.25/0.25 0.25/0.25
0.5/4 4/1
/32// 32 /32// 32 16//32 /32// 32
a b c d
Middlebrook 7H11 medium and incubation for 3 days. Middlebrook 7H11 medium and incubation for 5 days. Mueller Hinton medium and incubation for 3 days. Mueller Hinton medium and incubation for 5 days.
quinolones depending on the species involved, although, as in the case of the other mycobacteria studied, the fluorquinolones with a C-8 methoxy group (moxifloxacin and gatifloxacin) were more effective. M. fortuitum was much more susceptible to all fluoroquinolones than M. chelonae but linezolid was more active against M. chelonae than M. fortuitum . The concentration of antibiotics necessary to inhibit growth of the strains was slightly increased as the incubation time was increased. The MIC also varied slightly with the medium.
4. Discussion The treatment of serious infections with nontuberculous mycobacteria is a problem and the new fluorquinolones have allowed important advances in therapy, especially in the treatment of infections with certain species [13]. Levofloxacin gave improved results over existing quinolones, and had greater in vitro activity than ofloxacin [14,15]. However, our data confirm that quinolones with the 8-methoxy group (gatifloxacin and moxifloxacin) exhibit even better in vitro activity [12]. It has been reported that the new quinolones are not very active against strains of the M. avium-intracellulare complex [16] and this concurs with our findings. However, they are more active against M. kansasii , one of the most important pathogens [17]. Our data confirm the excellent activity of these compounds against M. fortuitum , the great majority of our isolates being inhibited by /2 mg/l of quinolones, as previously reported [17,18]. On the other hand, our findings also confirmed the lower activity against M. chelonae [14].
587
These data were confirmed on studying the characteristics of the gene encoding for quinolone resistance, the quinolone resistance-determining regions (QRDR) in the A and B subunits of DNA gyrase. Peptide sequences of the QRDR of GyrB were identical in all species. Peptide sequences of the QRDR of GyrA were identical in all the species, except for the amino acid at position 83, which was an alanine in the two less susceptible groups and a serine in the most susceptible one, suggesting that this amino acid is involved in the differences in quinolone susceptibility observed within the Mycobacterium genus [19]. In addition to this mechanism, others that affect the permeability of the membrane and the efflux pump are also involved [19]. Few studies exist on the activity of linezolid against the Mycobacterium genus [20]. Wallace et al. report a MIC90 of 16 mg/l for linezolid against M. fortuitum and M. chelonae [21], but our strains of M. fortuitum and M. chelonae were more resistant. Linezolid was active against our strains of M. kansasii. Variations in susceptibility arising when different media are used have been described previously [22] and emphasise the need for careful evaluation of standardised studies. There are also problems of correlation between in vitro susceptibility data and the therapeutic activity of the drugs in the treatment of infections [23]. One of the most important parameters to be established is the cut-off point that relates the in vitro activity of these compounds to their clinical efficacy, which has not yet been established for these bacteria. It has been suggested that for ciprofloxacin and ofloxacin the cutoff point may be 2 mg/l [25] and this was supported by a recent multicentre study [26]. However, there are no clear data concerning levofloxacin, gatifloxacin and moxifloxacin. It has recently been suggested that mycobacteria with an MIC ]/32 mg/l linezolid could be considered resistant to this drug [21] if the serum concentrations reached after administration of the recommended dose are considered. Despite these questions, the fluoroquinolones appear to be drugs with therapeutic possibilities against M. fortuitum and M. kansasii . Linezolid also has good activity against M. kansasii . However, the limited standardization of in vitro studies on these bacteria and lack of data that correlate the findings with the clinical efficacy of the drugs, suggest that it is necessary to analyze these studies in depth to evaluate the true utility of these new drugs in the treatment of these infections.
References [1] Bouza E, Albadalejo J, Cercenado E, Ruiz Serrano MJ, Vicente T, Ortega A. Impact of the human immunodeficiency virus
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J.C. Rodriguez Dı´az et al. / International Journal of Antimicrobial Agents 21 (2003) 585 /588 epidemic on detection of Mycobacterium isolates in a general hospital. J Clin Microbiol 2000;35:1013 /5. Ginsburg CM. Nontuberculous mycobacterial infections. Pediatr Infect Dis J 1992;11:875 /7. Debrunner M, Salfinger M, Brandli O, von Graevenitz A. Epidemiology and clinical significance of non-tuberculous mycobacteria in patients negative for human immunodeficiency virus in Switzerland. Clin Infect Dis 1992;15:330 /45. Hoyt L, Oleske J, Holland B, Connor E. Non-tuberculous mycobacteria in children with acquired immunodeficiency syndrome. Pediatr Infect Dis J 1992;11:354 /60. Kilby JM, Gilligan PH, Yankaskas JR, Highsmith WE, Jr, Edwards LJ, Knowles MR. Non-tuberculous mycobacteria in adult patients with cystic fibrosis. Chest 1992;102:70 /5. Wolinsky E. Mycobacterial diseases other than tuberculosis. Clin Infect Dis 1992;15:1 /10. Patel R, Roberts GD, Keating MR, Paya CV. Infections due to non-tuberculous mycobacteria in kidney, heart, and liver transplant recipients. Clin Infect Dis 1994;19:263 /73. Choudhri S, Manfreda J, Wolfe J, Parker S, Long R. Clinical significance of non-tuberculous mycobacteria isolates in a Canadian tertiary care center. Clin Infect Dis 1995;21:128 /33. Skogberg K, Ruutu P, Tukiainen P, Valtonen VV. Non-tuberculous mycobacterial infection in HIV-negative patients receiving immunosuppressive therapy. Eur J Clin Microbiol Infect Dis 1995;14:755 /63. Wallace RJ, Jr, Meier A, Brown BA, et al. American Thoracic Society statement: diagnosis and treatment of disease caused by non tuberculous mycobacteria. Am Respir Crit Care Med 1997;156:S1 /S25. Rodrı´guez JC, Ruiz M, Climent A, Royo G. In vitro activity of four fluoroquinolones against Mycobacterium tuberculosis . Int J Antimicrobial Agents 2001;17:229 /31. Renau TE, Gage JW, Dever JA, et al. Structure-activity relationships of quinolone agents against mycobacteria: effect of structural modifications at the 8 position. Antimicrob Agents Chemother 1996;40(10):2363 /8. Jacobs MR. Activity of quinolones against mycobacteria. Drugs 1999;58(Suppl.):219 /22. Khardori N, Nguyen H, Rosenbaum B, Rolston K, Bodey GP. In vitro susceptibilities of rapidly growing mycobacteria to newer antimicrobial agents. Antimicrob Agents Chemother 1994;38(1):134 /7.
[15] Rastogi N, Goh KS, Bryskier A, Devallois A. Spectrum of activity of levofloxacin against non-tuberculous mycobacteria and its activity against the Mycobacterium avium complex in combination with ethambutol, rifampin, roxithromycin, amikacin, and clofazimine. Antimicrob Agents Chemother 1996;40(11):2483 /7. [16] Fung Tomc J, Minassian B, Kolek B, Washo T, Huczko E, Bonner DJ. In vitro antibacterial spectrum of a new broadspectrum 8-methoxy fluoroquinolone, gatifloxacin. Antimicrob Chemother 2000;45(4):437 /46. [17] Guillemin I, Jarlier V, Cambau E. Correlation between quinolone susceptibility patterns and sequences in the A and B subunits of DNA gyrase in mycobacteria. Antimicrob Agents Chemother 1998;42(8):2084 /8. [18] Fernandez Roblas R, Esteban J, Cabria F, Lopez JC, Jimenez MS, Soriano F. In vitro susceptibilities of rapidly growing mycobacteria to telithromycin (HMR 3647) and seven other antimicrobials. Antimicrob Agents Chemother 2000;44(1):181 /2. [19] Guillemin I, Sougakoff W, Cambau E, Revel Viravau V, Moreau N, Jarlier V. Purification and inhibition by quinolones of DNA gyrases from Mycobacterium avium , Mycobacterium smegmatis and Mycobacterium fortuitum bv. peregrinum. Microbiology 1999;145:2527 /32. [20] Watt B. In-vitro sensitivities and treatment of less common mycobacteria. J Antimicrob Chemother 1997;39(5):567 /74. [21] Wallace RJ, Jr, Brown-Elliot BA, Ward SC, Crist CJ, Mann LB, Wilson RW. Activities of linezolid against rapidly growing mycobacteria. Antimicrob Agents Chemother 2001;45:764 /7. [22] Hernandez AM, Arias A, Felipe A, Alvarez R, Sierra A. Determination of the susceptibility in vitro of 54 isolates of Mycobacterium fortuitum against three fluoroquinolones using two methods. J Chemother 1995;7(2):109 /13. [23] Heginbothom ML. The relationship between the in vitro drug susceptibility of opportunist mycobacteria and their in vivo response to treatment. Int J Tuberc Lung Dis 2001;5(6):539 /45. [25] Chen CH, Shih JF, Lindholm Levy PJ, Heifets LB. Minimal inhibitory concentration of rifabutin, ciprofloxacin and ofloxacin against Mycobacterium tuberculosis isolated before treatment of patients in Taiwan. Am Rev Resp Dis 1989;140:987 /9. [26] Tomioka H, Sato K, Kajitani H, Akaki T, Shishido S. Comparative antimicrobial activities of the newly synthesized quinolone WQ-3034, levofloxacin, sparfloxacin, and ciprofloxacin against Mycobacterium tuberculosis and Mycobacterium avium complex. Antimicrob Agents Chemother 2000;44:283 /6.
In vitro activity of new fluoroquinolones and linezolid against nontuberculous mycobacteria J.C. Rodriguez Dı´az *, M. Lo´pez, M. Ruiz, G. Royo Hospital General Universitario de Elche, Universidad Miguel Herna´ndez, Elche, Alicante, Spain Received 17 May 2002; accepted 16 December 2002
Abstract The objective of the study was to determine the activity of new fluoroquinolones and linezolid against 108 clinical isolates of different species of non-tuberculous mycobacteria isolated in Spain. Gatifloxacin and moxifloxacin were found to be more effective than levofloxacin. Mycobacterium kansasii was more susceptible to the fluoroquinolones tested than M. avium complex and M. fortuitum was more susceptible than M. chelonae . Linezolid was more active against M. kansasii than against M. avium complex. A better understanding of the relationship between the in vitro activity of these compounds and their usefulness in the treatment of these infections is needed. The new fluoroquinolones exhibit good activity against M. kansasii and M. fortuitum and linezolid is active against M. kansasii . # 2003 Elsevier Science B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Fluoroquinolones; Linezolid; Non-tuberculous mycobacteria
1. Introduction Infections due to non-tuberculous mycobacteria have acquired great clinical importance in recent years as they may affect both immunocompromised and immunocompetent patients [1 /9]. The clinical importance of isolating these mycobacteria depends on the patient’s characteristics, the site of the infection, the type of sample and the species isolated [10]. Treatment may be specific for the species isolated. These treatments are limited due to the small number of drugs available, especially those administered orally, and so it is very important to find new antibiotics that are useful in these infections [10]. Furthermore, in clinical practice, correct identification of these genera is slow and tedious and many clinical laboratories do not have the means to perform susceptibility tests with the result that in many cases empirical treatment has to be given.
* Corresponding author. Present address: Seccio´n de Microbiologı´a, Hospital General Universitario de Elche, Camı´ de L’Almazara, no. 11, 03203 Elche, Alicante, Spain; fax: /34-966-679-108. E-mail address: [email protected] (J.C. Rodriguez Dı´az).
We present data on the susceptibility to new fluoroquinolones and linezolid, of nontuberculous mycobacteria isolated in our health district.
2. Material and methods 2.1. Strains studied One hundred and eight clinical isolates of 16 species were studied. The organisms tested included 31 Mycobacterium avium , 23 M. gordonae , 16 M. fortuitum , 11 M. intracellulare , eight M. kansasii , four each of M. chelonae and M. nonchromogenicum , two each of M. aurum and M. terrae, one each of M. malmoense , M. celatum , M. poriferae , M. fluorescens, M. flavescens , M. xenopi and M. scrofulaceum . These isolates were obtained from the same number of patients in the Elche health district, which has 250 000 inhabitants and is situated in the southeast of Spain, on the Mediterranean coast. Between 1 January 1995 and 1 March 2002, we processed 19 723 clinical samples. The most numerous samples were: 13 377 respiratory (67.82%), 3473 urine (17.60%), 944 pleural fluid (4.78%), 732 biopsies, punctures and exudates (3.71%),
0924-8579/03/$30 # 2003 Elsevier Science B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/S0924-8579(03)00048-7
586
J.C. Rodriguez Dı´az et al. / International Journal of Antimicrobial Agents 21 (2003) 585 /588
440 CSF (2.23%), 276 faeces (1.39%) and 220 blood samples (1.11%). The clinical isolates obtained were maintained at /70 8C. In the studies on slow growing mycobacteria, we used Mycobacterium tuberculosis Mt 14323 [11] as the control strain and in the studies on fast growing mycobacteria, Staphylococcus aureus ATCC 29213. 2.2. Isolation and identification techniques
Table 1 Susceptibility of the clinical isolates of slow growing species studied (mg/l) Species
Moxifloxacin Gatifloxacin Levofloxacin Linezolid
M. avium MIC50/90
2/4
8/16
16/16
Modal MIC
4
8
16
/32// 32 /32
M. gordonae MIC50/90 Modal MIC
0.25/0/5 0.25
0.5/1 0.25
0.5/1 1
2/4 1
All clinical samples were cultivated in Lo¨wenstein medium and Coletsos medium and were incubated at 37 8C for 60 days. In addition, from 1996, all samples except sputum and urine were inoculated in ESP II (Difco). Isolates were identified by means of standard techniques. Identification of all strains was confirmed at the Carlos III Institute (Majadahonda, Madrid).
M. intracellulare MIC50/90 1/2 Modal MIC 2
4/4 4
8/8 8
32//32 32
M. kansasii MIC50/90 Modal MIC
1/4 1
2/16 2
4/ /32 4
2.3. Antibiotics tested
M. nonchromogenicum MIC50/90 1/32
2/32
4/32
16/32
M. aurum MIC50/90
0.06/0.06
0.06/0.125
1/1
0.06/ 0.125
M. terrae MIC50/90
0.25/16
0.25/32
0.5/16
/32// 32
0.25
0.25
1
16
1 0.125 0.06
1 0.06 0.06
2 0.125 0.125
2 0.5 1
0.06 0.06 1
0.06 0.06 1
0.125 0.25 4
1 2 32
The following quinolones were tested: levofloxacin (Hoechst), moxifloxacin (Bayer), gatifloxacin (Bristol Myers Squibb) and linezolid (Pharmacia). 2.4. Determination of in vitro activity 2.4.1. Slow growing mycobacteria Organisms were cultured in Middlebrook 7H11 medium (Difco) supplemented with OADC (Difco) and incubated at 37 8C in a CO2 incubator for 21 days. The following concentrations of antibiotics were tested: 0.5, 1, 2, 4, 8, 16, and 32 mg/l. The inoculum was made from a recent culture of the strain in Lo¨wenstein / Jensen medium. A suspension with a turbidity of 1 on the MacFarland scale was made in distilled water, was then diluted 1:100 in sterile distilled water and 5 ml put on each plate holding the antibiotic and in each control. On reading and interpreting the antibiograms we considered the strain to be susceptible to the antibiotic if less than 1% of the colonies which grew on the control plate were isolated on the plate with antibiotic [11]. 2.4.2. Fast growing mycobacteria The same procedure was followed but two different media were used Middlebrook 7H11 medium (Difco) supplemented with OADC (Difco) and Mueller Hinton agar (Difco); plates were read at 3 and 5 days.
M. malmoense M. celatum M. poriferae M. fluorescens M. flavescens M. xenopi M. scrofulaceum
0.125/2 0.125
the two fluoroquinolones with a C-8 methoxy group (gatifloxacin and moxifloxacin), were more active, as reported previously [12]. Of the three species most frequently involved in clinically important conditions, M. kansasii was the most susceptible, followed by M. intracellulare, while M. avium showed the highest level of resistance. All compounds showed good activity against M. gordonae , an organism not often associated with clinical conditions. These compounds exhibited excellent activity against the limited number of isolates studied of M. malmoense , M. celatum , M. scrofulaceum and M xenopi . Linezolid had little activity against the M. aviumintracellulare complex, but was more active against M. kansasii , M. gordonae , M. xenopi and M. celatum .
3. Results 3.1. Slow growing mycobacteria:
3.2. Fast growing mycobacteria
The susceptibility data are shown in Table 1. There were differences in fluoroquinolone susceptibility between the different species studied. However, in general
The susceptibility data, together with the different media and incubation times, are shown in Table 2. There was a clear difference in the activity of the various
J.C. Rodriguez Dı´az et al. / International Journal of Antimicrobial Agents 21 (2003) 585 /588 Table 2 Susceptibility of fast growing mycobacteria (mg/l) Species
Moxifloxacin Gatifloxacin Levofloxacin Linezolid
M. chelonae MIC50aMIC90a MIC50bMIC90b MIC50cMIC90c MIC50dMIC90d
2/16 4/16 8/16 8/16
1/16 4/16 4/8 4/16
3/32 4/32 1/31 4/32
8/ /32 32//32 8/16 16/16
M. fortuitum MIC50aMIC90a 0.25/0.5
0.25/0.5
0.5/1
MIC50bMIC90b 0.5/0.5
0.25/0.5
0.5/1
MIC50cMIC90c 0.25/0.5 MIC50dMIC90d 0.5/0.5
0.25/0.25 0.25/0.25
0.5/4 4/1
/32// 32 /32// 32 16//32 /32// 32
a b c d
Middlebrook 7H11 medium and incubation for 3 days. Middlebrook 7H11 medium and incubation for 5 days. Mueller Hinton medium and incubation for 3 days. Mueller Hinton medium and incubation for 5 days.
quinolones depending on the species involved, although, as in the case of the other mycobacteria studied, the fluorquinolones with a C-8 methoxy group (moxifloxacin and gatifloxacin) were more effective. M. fortuitum was much more susceptible to all fluoroquinolones than M. chelonae but linezolid was more active against M. chelonae than M. fortuitum . The concentration of antibiotics necessary to inhibit growth of the strains was slightly increased as the incubation time was increased. The MIC also varied slightly with the medium.
4. Discussion The treatment of serious infections with nontuberculous mycobacteria is a problem and the new fluorquinolones have allowed important advances in therapy, especially in the treatment of infections with certain species [13]. Levofloxacin gave improved results over existing quinolones, and had greater in vitro activity than ofloxacin [14,15]. However, our data confirm that quinolones with the 8-methoxy group (gatifloxacin and moxifloxacin) exhibit even better in vitro activity [12]. It has been reported that the new quinolones are not very active against strains of the M. avium-intracellulare complex [16] and this concurs with our findings. However, they are more active against M. kansasii , one of the most important pathogens [17]. Our data confirm the excellent activity of these compounds against M. fortuitum , the great majority of our isolates being inhibited by /2 mg/l of quinolones, as previously reported [17,18]. On the other hand, our findings also confirmed the lower activity against M. chelonae [14].
587
These data were confirmed on studying the characteristics of the gene encoding for quinolone resistance, the quinolone resistance-determining regions (QRDR) in the A and B subunits of DNA gyrase. Peptide sequences of the QRDR of GyrB were identical in all species. Peptide sequences of the QRDR of GyrA were identical in all the species, except for the amino acid at position 83, which was an alanine in the two less susceptible groups and a serine in the most susceptible one, suggesting that this amino acid is involved in the differences in quinolone susceptibility observed within the Mycobacterium genus [19]. In addition to this mechanism, others that affect the permeability of the membrane and the efflux pump are also involved [19]. Few studies exist on the activity of linezolid against the Mycobacterium genus [20]. Wallace et al. report a MIC90 of 16 mg/l for linezolid against M. fortuitum and M. chelonae [21], but our strains of M. fortuitum and M. chelonae were more resistant. Linezolid was active against our strains of M. kansasii. Variations in susceptibility arising when different media are used have been described previously [22] and emphasise the need for careful evaluation of standardised studies. There are also problems of correlation between in vitro susceptibility data and the therapeutic activity of the drugs in the treatment of infections [23]. One of the most important parameters to be established is the cut-off point that relates the in vitro activity of these compounds to their clinical efficacy, which has not yet been established for these bacteria. It has been suggested that for ciprofloxacin and ofloxacin the cutoff point may be 2 mg/l [25] and this was supported by a recent multicentre study [26]. However, there are no clear data concerning levofloxacin, gatifloxacin and moxifloxacin. It has recently been suggested that mycobacteria with an MIC ]/32 mg/l linezolid could be considered resistant to this drug [21] if the serum concentrations reached after administration of the recommended dose are considered. Despite these questions, the fluoroquinolones appear to be drugs with therapeutic possibilities against M. fortuitum and M. kansasii . Linezolid also has good activity against M. kansasii . However, the limited standardization of in vitro studies on these bacteria and lack of data that correlate the findings with the clinical efficacy of the drugs, suggest that it is necessary to analyze these studies in depth to evaluate the true utility of these new drugs in the treatment of these infections.
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