BODMER, T.

GUERRERO, C.

MARCHESI, F.

ROBERTS, G. D.

STOCKMAN, L.

TELENTI, A.

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Journal of Antimicrobial Chemotherapy (1994) 33, 661-674CorrespondenceAntibacterial activity of local anaestheticagentsJ Antimicroh Chemother 1994; 33: 661Sir,In their excellent overview on antibacterialactivity of non-antibiotic drugs Cederlund &Mardh (1993) did not mention local anaes-thetics such as tetracaine, dibucaine, procaineand lidocaine, which have potent antimicrobialactivity as detected by effects on cell growthrates and viability of Escherichia coli andCandida albicans in vitro. The effects of lido-caine on bacterial growth in a chemicallydefined medium has been compared with thatof antibacterial agents such as ampicillin,chloramphenicol, puromycin and cationicsurface-active agents (Abanzukwe, FazleyBazaz & Salt, 1991). Although local anaes-thetics are 1000 fold less toxic to both pro-caryotic and eucaryotic cells than activequaternary ammonium disinfectants, theirantibacterial activity could be of concern forthe yield of broncho-alveolar lavage (BAL)fluid bacteriology. Bronchoscopists instill10-15 mL 2% lidocaine into the bronchial treebefore performing BAL. If allowed to mix with50 mL BAL fluid, 200-300 mg of lidocainewould attain a concentration of4000-6000 mg/L, enough to induce membranedamage in bacterial organisms and possiblycompromise their growth in culture.Fortunately removal of the local anaestheticpermits bacterial cell recovery and growth.There is, however, no study comparing thebacteriological yield of BAL with and withoutthe use of local anaesthetics.FRANCO de'CLARIIntensive Care Unit,Ospedate Civico,6900 LuganoVia Tesserete 46.SwitzerlandReferencesCederlund,. H. & Mardh, P.-A. (1993). Antibacterialactivities of non-antibiotic drugs. Journal ofAntimicrobial Chemotherapy 32, 355-65.Abanzukwe, T. C , Fazley Bazas, B. S. & Salt, W. G.(1991). Further studies of the antimicrobialactivity of beta-adrenergic blocking agents andlocal anaesthetics. In Program and Abstracts ofthe Seventeenth International Congress ofChemotherapy, Berlin. Abstract 726.Evaluation of the rpoB gene in rifampicin-susceptible and -resistant Mycobacterium aviumand Mycobacterium intraceltulareJ Antimicrob Chemolher 1994; 33: 661-663Sir,Mycobacteria of the Mycobacterium aviumcomplex (MAC) are significant human andanimal pathogens. Treatment options havebeen limited because of resistance to mostantimicrobial agents. Such intrinsic resistancehas been attributed to the characteristics of thecell wall structure of MAC, but the precisemolecular bases have not been elucidated. Incontrast, significant progress has been made inthe understanding, at molecular level, of resist-ance to the main antituberculous drugs inMycobacterium tuberculosis: resistance toisoniazid has been mapped to the gene for thecatalase-peroxidase (Zhang et al., 1992),rifampicin to the RNA polymerase subunit ji(Telenti et al., 1993a), fluoroquinolones to thegyrase A (TakifT, H., Salazar, L., Guerrero, C ,Philipp, W., Huang, W. M., Kreiswirth, B. etal., unpublished observations), streptomycin tothe ribosomal S12 protein and 16S rRNA gene(Douglass & Steyn, 1993; Nair et al., 1993).Recent work has identified mutations in a genepossibly involved in the mycolic acid metabolicpathway as an additional mechanism forisoniazid resistance in M. tuberculosis(Banerjee et al., 1993).We have evaluated the rpoB of severalstrains of M. avium and Mycobacterium intra-cellulare that displayed variable levels ofsusceptibility to rifampicin to clarify its role inresistance. For this purpose, the rpoB regionwhere mutations have been identified inM. tuberculosis and Mycobacterium leprae(Honore & Cole, 1993; Telenti et al.,1993a)—the Rif locus—was amplified inM. avium serovar 1, and M. intracellulareserovar 15 reference strains by using primerspreviously described (Telenti et al., 1993a).PCR fragments were sequenced directly orafter cloning into a plasmid vector. In addi-tion, the Rif locus was evaluated in 29 humanMAC isolates displaying a rifampicin-suscept-ible phenotype (MIC < 1 mg/L, n = 4),moderate susceptibility (MIC 1-4 mg/L, n =6), or resistance (MIC £ 4 mg/L, n = 19), bysequencing or by PCR single strand conforma-tion polymorphism (Telenti et al., 19936).661Tabfe. Alignment of the rpoB Rif locus of rifampicin susceptible M. tuberculosis H37rv with sequences from M. avium and M. intracellulare reference strains and 15clinical isolates (grouped as 'types' according to sequence homology) exhibiting susceptible, intermediate resistant, and rifampicin resistant phenotypes. With theexception of the M. intracellulare isolate 'D', all isolates had full amino acid homology with M. tuberculosis H37rv. Shown in bold are the mutations described inrifampicin-resistant M. tuberculosisM. tuberculosis AAC ATC CGG CCG GTG GTC GCC GCG ATC AAG GAG TTC TTC GGC ACC AGC CAG CTG AGC CAA TCC ATG GAC CAG494 Asn He Arg Pro Val Val Ala Ala He Lys Glu Phe Phc Gly Thr Ser Gin U u Ser Gin Phe Met Asp GinM. avium AAC ATC CGT CCC GTC GTG GCG GCG ATC AAG GAG TTC TTC GGC ACC AGC CAG CTG TCC CAG TTC ATG GAC CAGtype A (n = 3)type B (n - 4) Atype C ( n - I ) C CM. intracellulare . . . . G .G .. . . C . C AGtype A (n •= I) G . . G .. . . C . . C AGtype B (n = 3) G .. G C .. C Gtype C(n = 2) G . . G C . C GtypeD(/r=l) G G ..G C ..C AGSerM. tuberculosis AAC AAC CCG CTG TCG GGG TTG ACC CAC AAG CGC CGA CTG TCG GCG CTG GGG CCC GGC GGT CTG TCA CGT GAG518 Asn Asn Pro Leu Ser Gly Leu Thr Hb Lys Arg Arg Leu Ser Ala Leo Gly Pro Gly Gly Leu Ser Arg GluM. avium AAC AAC CCG CTG TCG GGG CTC ACC CAC AAG CGC CGC CTG TCG GCG CTG GGC CCG GGT GGT CTG TCC CGG GAGtype A (n = 3)type B (/. = 4)type C (rt= I) T . . G T . . .M. inlracellulare C .T ..G C C ..C T ...typeA(n=l) C ..T .G C C ,.C T ...typeB(n = 3) C . T ,.G C C . C T ...typeC(n = 2) C .. ..G T ..T . C C T . .type D ( n = l ) G T .. T G ..C T ...no•g3.Correspondence 663In M. avium and M. intracellulare referencestrains, the rpoB region homologous to the Riflocus of M. tuberculosis exhibited differencesat nucleotide level but a full amino acid iden-tity with rifampicin-susceptible M. tuberculosis(Table). Sequence information from clinicalisolates demonstrated significant sequenceheterogeneity, in particular among strainsidentified as M. intracellulare. However, withonly one exception, all retained the amino acidsequence corresponding to a rifampicin-susceptible M. tuberculosis (Table). One isolatepresented an Asn -> Ser in codon 494, amutation not reported previously in rifam-picin-resistant M. tuberculosis (Telenti et al.,1993a), or M. leprae (Honore & Cole, 1992).This isolate did not display high levelresistance—it had no growth at 4 mg/L ofrifampicin; thus, the contribution of themutation to the resistant phenotype wasdeemed doubtful.In summary, our data confirm at themolecular level that the most frequent mechan-ism of resistance to nfampicin among clinicalisolates of M. avium and M. intracellulare doesnot involve alterations of the RNA polymerasesubunit /?. Thus, alternative mechanisms ofresistance are responsible for the intrinsicresistance to rifampicin in MAC. Thefrequency with which these isolates exhibitresistance to multiple structurally unrelatedantimicrobial agents, and the existence ofintermediate and high-level resistance pheno-types are most consistent with changes in druguptake or with efflux mechanisms. A signifi-cant permeability barrier to rifampicin, thatcould be reduced with Tween, has beendescribed in a type strain of M. intracellulareand Mycobacterium smegmatis shown topossess a rifampicin-susceptible RNA poly-merase (Hui, Gordon & Kajioka, 1977). Thegenetic determinants of permeability, and thepossibility for additional mechanisms of resist-ance to antimicrobial agents in MAC,including the acquisition of exogenous geneticelements encoding for drug resistance, havenot yet been established. This information willbe important in the development of moreactive drugs against MAC.C. GUERRERO"L. STOCKMAN*F. MARCHES!"T. BODMER"G. D. ROBERTS*A. TELENTI""Institute for Medical Microbiology,University of Berne,3010 Bern, Switzerland;*Section of Clinical Microbiology,Mayo Clinic,Rochester MN 55905, USAReferencesBanerjee A., Dubnau, E., Quemard, A. & Jacobs,W. R. (1993). Cloning of a novel gene conferringresistance to isoniazid in mycobacteria: a possibletarget for isoniazid. In Abstracts of theNinety-Third General Meeting of the AmericanSociety for Microbiology, Atlanta, 1993. AbstractU-15, p. 171. American Society for Microbiology,Washington. DC.Douglass, J. & Steyn, LM. (1993). A ribosomalgene mutation in streptomycin-resistantMycobacterium tuberculosis isolates. Journal ofInfectious Diseases 167, 1505-6.Honore, N. & Cole, S. T. (1993). The molecularbasis of nfampicin resistance in Mycobacteriumleprae. Antimicrobial Agents and Chemotherapy37,414-8.Hui, J., Gordon, N & Kajioka, R. (1977).Permeability barrier to rifampin in mycobacteria.Antimicrobial Agents and Chemotherapy 11,773-9.Nair, J., Rouse, D., Bai, G. & Morris, S. (1993)Genetic analyses of streptomycin resistance insingle and multiple drug resistant strains ofMycobacterium tuberculosis. In Abstracts of theNinety-Third General Meeting of the AmericanSociety of Microbiology, Atlanta, 1993. AbstractU-14, p. 171. American Society for Microbiology,Washington, DC.Telenti, A., Imboden, P., Marchesi, F., Lowne D.,Cole, S., Colston, M.J. el al. (1993a). Detection ofrifampicin-resistance mutations in Mycobacteriumtuberculosis. Lancet 341, 647-50.Telenti, A., Imboden, P., Marchesi, F., Schmidheini,T. & Bodmer, T. (1993b). Direct, automateddetection of rifampicin-resistant M. tuberculosisby PCR and single strand conformationpolymorphism. Antimicrobial Agents andChemotherapy. 37, 2054-8.Zhang, Y., Heym B., Allen, B., Young, D. & Cole,S. (1992). The catalase-peroxidase gene andisoniazide resistance of Mycobacteriumtuberculosis. Nature 358, 591-3.Outer membrane protein profiles ofXanthomonas maltophilia isolates displayingtemperature-dependent susceptibility togenUmicinJ Antimicrob Chemother 1994; 33: 663-666Sir,Xanthomonas maltophilia is increasinglyisolated from hospitalized patients, parti-cularly immunocompromised individualsreceiving broad-spectrum antibiotics.Treatment is difficult because of its inherentresistance to many antibiotics. Wheat,

Evaluation of the rpoB gene in rifampicin-susceptible and -resistant Mycobacterium avium and Mycobacterium intracellulare

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