Genetic analysis of the {beta}-lactamases of Mycobacterium tuberculosis and Mycobacterium smegmatis and susceptibility to {beta}-lactam antibiotics

Anthony R. Flores1, Linda M. Parsons2 and Martin S. Pavelka,, Jr1

1 University of Rochester School of Medicine and Dentistry and Department of Microbiology and Immunology, Rochester, NY 14642, USA
2 The Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA

Correspondence
Martin S. Pavelka, Jr
Martin_Pavelka{at}urmc.rochester.edu


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Mycobacteria produce {beta}-lactamases and are intrinsically resistant to {beta}-lactam antibiotics. In addition to the {beta}-lactamases, cell envelope permeability and variations in certain peptidoglycan biosynthetic enzymes are believed to contribute to {beta}-lactam resistance in these organisms. To allow the study of these additional mechanisms, mutants of the major {beta}-lactamases, BlaC and BlaS, were generated in the pathogenic Mycobacterium tuberculosis strain H37Rv and the model organism Mycobacterium smegmatis strain PM274. The mutants M. tuberculosis PM638 ({Delta}blaC1) and M. smegmatis PM759 ({Delta}blaS1) showed an increase in susceptibility to {beta}-lactam antibiotics, as determined by disc diffusion and minimal inhibitory concentration (MIC) assays. The susceptibility of the mutants, as assayed by disc diffusion tests, to penicillin-type {beta}-lactam antibiotics was affected most, compared to the cephalosporin-type {beta}-lactam antibiotics. The M. tuberculosis mutant had no detectable {beta}-lactamase activity, while the M. smegmatis mutant had a residual type 1 {beta}-lactamase activity. We identified a gene, blaE, encoding a putative cephalosporinase in M. smegmatis. A double {beta}-lactamase mutant of M. smegmatis, PM976 ({Delta}blaS1{Delta}blaE : : res), had no detectable {beta}-lactamase activity, but its susceptibility to {beta}-lactam antibiotics was not significantly different from that of the {Delta}blaS1 parental strain, PM759. The mutants generated in this study will help determine the contribution of other {beta}-lactam resistance mechanisms in addition to serving as tools to study the biology of peptidoglycan biosynthesis in these organisms.


Abbreviations: ATM, aztreonam; CLA, clavulanic acid; LOT, cephalothin; PBP, penicillin-binding protein; PEN, benzylpenicillin

The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AY332268 and AY442183.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The mycobacteria consist of pathogens responsible for significant morbidity and mortality throughout the world, such as Mycobacterium leprae and Mycobacterium tuberculosis, as well as opportunistic pathogens, such as Mycobacterium avium, Mycobacterium fortuitum and Mycobacterium smegmatis. It is estimated that one-third of the world population, approximately 2 billion people, are latently infected with M. tuberculosis, and that more than 2 million people die each year from tuberculosis (Dye et al., 1999). Interest in the treatment of tuberculosis has grown in recent years due to the increased prevalence of M. tuberculosis in the immunocompromised (e.g. AIDS patients) and the emergence and spread of multi-drug-resistant strains.

The {beta}-lactam class of antibiotics has not been used in the treatment of M. tuberculosis or other mycobacterial infections as mycobacteria are resistant to these antibiotics and produce {beta}-lactamases (Kasik, 1979; Kwon et al., 1995). However, the effectiveness of {beta}-lactam/{beta}-lactamase inhibitor combinations has been shown in vitro for M. tuberculosis (Chambers et al., 1995; Cynamon & Palmer, 1983; Segura et al., 1998; Sorg & Cynamon, 1987), M. avium (Casal et al., 1987), M. fortuitum (Utrup et al., 1995; Wong et al., 1988) and M. smegmatis (Yabu et al., 1985). Clinical evidence suggests that {beta}-lactam antibiotics in combination with {beta}-lactamase inhibitors may be useful in the treatment of M. tuberculosis infection (Chambers et al., 1998; Nadler et al., 1991).

The major {beta}-lactamase, BlaA, of the avirulent M. tuberculosis strain H37Ra, has been described in both biochemical and molecular terms and is identical to BlaC, found in the virulent M. tuberculosis strain H37Rv (Hackbarth et al., 1997; Voladri et al., 1998). This M. tuberculosis {beta}-lactamase bears significant homology to molecular class A {beta}-lactamase enzymes; functionally, it appears to be a penicillinase or type 2b {beta}-lactamase (Ambler, 1980; Ambler et al., 1991; Bush et al., 1995). A recombinant form of the M. tuberculosis H37Rv BlaC enzyme has been described biochemically (Voladri et al., 1998). However, direct genetic studies of BlaC in M. tuberculosis H37Rv are lacking. In addition, a minor {beta}-lactamase having predominantly cephalosporinase activity has been described in M. tuberculosis H37Ra (Voladri et al., 1998), but its role in the resistance of M. tuberculosis to {beta}-lactam antibiotics is not understood and no gene has been identified.

The major {beta}-lactamase in M. smegmatis mc2155 has been biochemically described and is similar to BlaF, the well-studied molecular class A {beta}-lactamase from M. fortuitum (Kaneda & Yabu, 1983; Quinting et al., 1997). A recent report identified a gene, designated blaA, encoding the major {beta}-lactamase in M. smegmatis (Li et al., 2004), which is the same gene we previously designated blaS and describe in this work (A.R. Flores & M. S. Pavelka, Abstr. 43rd Intersci. Conf. Antimicrob. Agents Chemother. Abstr. 674, 2003). Biochemical studies have revealed the presence of a cephalosporinase in M. smegmatis SN2, which, from inhibitor and substrate profiles, appears to be a functional group 2e {beta}-lactamase (Basu et al., 1997). However, an N-terminal sequence from the purified {beta}-lactamase bears significant homology to class C or functional group 1 enzymes (Basu et al., 1997).

Resistance to {beta}-lactam antibiotics in mycobacteria is generally believed to result from the following mechanisms, singly or in combination: (1) enzymic inactivation by {beta}-lactamases, (2) exclusion of the drug from the site of action by an impermeable cell envelope, and (3) the susceptibility of the target penicillin-binding proteins (PBPs) to inhibition. Drug export pumps may contribute to resistance, but the influence of these pumps appears to be limited (Li et al., 2004). The presence of {beta}-lactamases in these organisms complicates the study of the other {beta}-lactam resistance mechanisms and also interferes with the use of {beta}-lactam antibiotics in the study of peptidoglycan biosynthesis. Here, we have used a genetic approach to study the contribution of the {beta}-lactamases to {beta}-lactam antibiotic resistance in M. tuberculosis H37Rv and M. smegmatis mc2155. We identified the major {beta}-lactamase gene, blaS, and the minor {beta}-lactamase, blaE, in the genome of M. smegmatis. The {beta}-lactamase of M. tuberculosis (blaC), and those of M. smegmatis (blaS and blaE), were deleted by allelic exchange. The resulting mutants were significantly more susceptible to {beta}-lactam antibiotics and had reduced or non-detectable {beta}-lactamase activities; however, the susceptibility of the mutants to penicillin-type {beta}-lactam antibiotics was affected most, compared to the cephalosporin-type {beta}-lactam antibiotics. These mutants will serve as tools for the study of other {beta}-lactam resistance mechanisms and of the interaction of {beta}-lactam antibiotics with the peptidoglycan biosynthesis machinery of M. tuberculosis and M. smegmatis.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and growth conditions.
The strains used in this study are shown in Table 1. M. tuberculosis H37Rv is a virulent laboratory strain. M. smegmatis PM274 is a lysine auxotroph of the common laboratory strain M. smegmatis mc2155 (Consaul et al., 2003). Escherichia coli cultures were grown in Luria–Bertani (LB) broth (Difco; BD Biosciences) or on LB agar. Mycobacterial cultures were grown in Middlebrook 7H9 broth (Difco; BD Biosciences) or on Middlebrook 7H11 medium (Difco; BD Biosciences). All Middlebrook media were supplemented with 0·05 % Tween 80, 0·2 % glycerol (v/v) and ADS (0·5 % BSA, fraction V, 0·2 % glucose and 0·85 % NaCl). Sucrose was added to media at a concentration of 2 %. L-Lysine was added to all M. smegmatis cultures at 40 µg ml–1. When necessary, ampicillin (50 µg ml–1; Sigma-Aldrich Chemical) and hygromycin (50 µg ml–1; Roche Applied Science) were added to media. M. smegmatis plates were incubated for 3–5 days, while M. tuberculosis plates were incubated for 3–4 weeks at 37 °C. Inoculation and growth conditions for allelic exchange in M. smegmatis and M. tuberculosis were as previously described (Pavelka & Jacobs, 1999).


View this table:
[in this window]
[in a new window]
 
Table 1. Strains used in this study

 
Antibiotics.
The antibiotics used in minimal inhibitory concentration (MIC) determinations, amoxicillin, ampicillin, carbenicillin, cefoxitin, ceftriaxone and cephalothin, were obtained from Sigma-Aldrich, while clavulanic acid was kindly provided by GlaxoSmithKline. Sensi-Discs (BBL; BD Biosciences) used in this study were ampicillin (10 µg), amoxicillin/clavulanic acid (20 µg/10 µg), carbenicillin (100 µg), cefazolin (30 µg), cefixime (5 µg), cefoperazone (75 µg), cefoxitin (30 µg), ceftazidime (30 µg), ceftriaxone (30 µg), cephalothin (30 µg), imipenem (10 µg), isoniazid (5 µg), mezlocillin (75 µg), oxacillin (10 µg), piperacillin (100 µg) and rifampicin (5 µg). In addition, the antibiotics amoxicillin, ampicillin and cefoxitin were tested at a higher concentration by spotting of the appropriate amount of antibiotic on a sterile paper disc (BBL; BD Biosciences) from a stock antibiotic solution.

DNA manipulation.
Basic DNA methods were essentially as described by Maniatis et al. (1982). Plasmids used in this study are listed in Table 2. Plasmids were constructed in E. coli DH10B and were prepared by an alkaline lysis protocol or by Qiagen columns, if used for recombination. DNA fragments were isolated using agarose gel electrophoresis and absorption to a silica matrix (GeneClean; Bio 101), or by QIAquick spin columns (Qiagen). Genomic DNA was prepared as described previously for M. tuberculosis (Pavelka & Jacobs, 1999) and M. smegmatis (Jacobs et al., 1991). Southern blotting was done as described previously (Pavelka & Jacobs, 1996). Oligonucleotides for PCR were generated by Invitrogen Life Technologies. All restriction and DNA modifying enzymes were from Fermentas or New England Biolabs. Electroporation of M. smegmatis and M. tuberculosis was as previously described (Pavelka & Jacobs, 1999).


View this table:
[in this window]
[in a new window]
 
Table 2. Plasmids used in this study

 
PCR and cloning of M. smegmatis and M. tuberculosis {beta}-lactamases.
The oligonucleotide primers Pv152 (5'-CGTGGTGCTCGAGGAAATCGC-3') and Pv153 (5'-AGCCCGAGTACTCGCGGATG-3') were used to amplify the blaS coding region, including 1 kb of flanking DNA on each side of the gene, from M. smegmatis mc2155 genomic DNA (http://www.tigr.org/tdb/mdb/mdbinprogress.html) using Pfu polymerase (Stratagene). PCR was performed in a Perkin-Elmer GeneAmp 2400 temperature cycler (Applied Biosystems) with the following programme: 94 °C for 45 s, 1 cycle; 94 °C for 45 s and 60 °C for 4 min, 30 cycles; 72 °C for 10 min. The resulting 2·8 kb PCR fragment was digested with XmaI and XhoI and ligated into pKSI+ digested with the same enzymes to generate pMP222.

Similarly, blaE and adjacent sequence was amplified from M. smegmatis mc2155 genomic DNA (http://www.tigr.org/tdb/mdb/mdbinprogress.html) using the oligonucleotide pair Pv187 (5'-CCGAAGGACATCTCGAGTCGTTGCGGTTCG-3') and Pv188 (5'-GCGGACCTCTCGAGAGCACGCTTGTCATCG-3') and Pfu polymerase. The 6·7 kb product was subsequently digested with the restriction endonucleases XhoI and NotI and inserted into the same sites of pKSI+ to generate pMP295.

The M. tuberculosis H37Rv blaC gene was cloned from cosmid MTCY49 of the M. tuberculosis H37Rv genome sequencing project (Cole et al., 1998). We excised a 6·5 kb fragment containing blaC from MTCY49 using the restriction endonuclease NotI and inserted it into the NotI site of pKSI+, to generate pMP159.

Construction of suicide plasmids.
Construction of suicide plasmids was done essentially as described (Pavelka & Jacobs, 1999). For blaC and with pMP159 as a template, the oligonucleotide primer pair Pv135 (5'-GTCACGGAGCTAGCCATTGCCATCGCTACCAGCAGTTC-3') and Pv136 (5'-CGGGCACTGCTAGCGATTGGATGGCGCGCAACACCACC-3') was used for inverse XL-PCR with rTth DNA polymerase (Applied Biosystems) in a Perkin-Elmer GeneAmp 2400 temperature cycler with the following parameters: 94 °C for 5 min, 1 cycle; 94 °C 1 min and 60 °C 10 min, 16 cycles; 94 °C 1 min and 60 °C 10 min with time increasing by 15 s for each cycle, 12 cycles; 72 °C for 30 min. The PCR product was purified, digested with NheI, and self-ligated to generate pMP179. The result was an in-frame 615 bp deletion in the ORF of blaC marked with an NheI restriction site. A NotI digest of pMP179 liberated the 5·9 kb fragment containing {Delta}blaC1, which was subsequently treated with Klenow polymerase and ligated into the EcoRV site of pYUB657 to generate the suicide plasmid pMP180.

The plasmid pMP222, containing wild-type blaS, was used as template in an inverse PCR reaction (Pfu polymerase; Stratagene) with primers Pv176 (5'-GGCGACTACGTATCCACCAACGATGTG-3') and Pv177 (5'-GGGATCTACGTAGACACGATCGTCCAGC-3') in a Perkin-Elmer GeneAmp 2400 temperature cycler with the following parameters: 94 °C for 45 s, 1 cycle; 94 °C for 45 s and 63 °C for 7 min, 30 cycles; 72 °C for 10 min. The PCR product was purified, digested with SnaBI and self-ligated to generate pMP225. The result was an in-frame, 426 bp deletion in the open reading frame of the M. smegmatis blaS gene marked by a SnaBI restriction site The 2·0 kb {Delta}blaS1 fragment was excised from pMP225 using XhoI and XbaI, treated with Klenow polymerase, and ligated into EcoRV-digested pYUB657 to yield the suicide plasmid pMP252.

An in-frame deletion allele of blaE was generated using inverse PCR with pMP295 as a template. Pfu polymerase and the primer pair Pv199 (5'-CGGCTATTACTACGTAGGCCCGATGG-3') and Pv200 (5'-CGGTGAATGTCTTGCTTACGTAGCCGA-3') generated an in-frame deletion of 591 bp in the ORF of blaE. Initial attempts to generate an unmarked deletion of blaE were unsuccessful; therefore, the {Delta}blaE1 allele was marked using a resolvable kanamycin resistance cassette to ensure a definitive phenotype upon exchange with the wild-type blaE. The cassette was excised from pYUB638 (Pavelka & Jacobs, 1999) using MluI and inserted into a SnaBI site of {Delta}blaE1 of pMP307 to generate pMP330. Finally, a 6·3 kb fragment containing {Delta}blaE1 : : res-aph-res was excised from pMP330 using PvuII and inserted into the EcoRV site of pYUB657 to generate pMP332.

Resolution of {Delta}blaE1 : : res-aph-res using {gamma}{delta}-resolvase.
The res-aph-res cassette was resolved in strain PM939 ({Delta}blaS1 {Delta}blaE1 : : res-aph-res) using the {gamma}{delta}-resolvase supplied in trans on a mycobacterial shuttle vector (pGH542; a gift from Graham Hatfull, University of Pittsburgh) to yield strain PM976 ({Delta}blaS1 {Delta}blaE1 : : res). The resulting allele, {Delta}blaE1 : : res, contains an out-of-frame insertion in the {Delta}blaE1-coding region.

Antimicrobial susceptibility testing.
Zones of inhibition measured by disc diffusion (Sensi-Disc) and MICs were used to determine changes in antibiotic susceptibility in the {beta}-lactamase mutants. The procedure used for disc diffusion in M. tuberculosis and M. smegmatis was as follows. M. tuberculosis cultures were grown to approximately mid to late exponential phase in 10 ml Middlebrook 7H9. Hygromycin was added for PM669 and PM670. Cells were pelleted, washed once in fresh medium, and resuspended in 10 ml fresh medium. Then, 150 µl of washed culture was spread on Middlebrook 7H11 and the antibiotic Sensi-Disc was placed in the centre. Plates were incubated for 2 weeks and zones of inhibition measured to the nearest 5 mm. M. smegmatis cultures were grown to mid-exponential phase (OD600 0·4–0·6) in 10 ml Middlebrook 7H9. Hygromycin was added to PM791 and PM876 cultures. Cells were washed once in fresh medium, and resuspended in an equal volume. A pour-plate technique was used by adding 200 µl washed cells to 3·5 ml molten top agar (0·6 % agarose, 0·2 % glycerol, v/v) and pouring onto Middlebrook 7H11 plates. Sensi-Discs were placed in the centre and the plates were incubated for 2–3 days. Zones of inhibition were measured to the nearest 1 mm.

M. tuberculosis MICs were determined at The Wadsworth Center using the radiometric (BACTEC) method. The source of the inoculum was freshly grown M. tuberculosis from a primary 7H12 liquid medium (BACTEC TB vial) with a GI (growth index) reading between 900 and 999. After vortexing and passing the suspension through a syringe to break up clumps of bacteria, 0·1 ml of the suspension was used to inoculate vials containing various concentrations of drugs and a control vial (C-0) without any drug. A 1 : 100 dilution of the suspension was used to inoculate a second control vial (C-100). When the growth in the C-100 vial, inoculated with 1 % of the inoculum in the drug-containing vials, reached a GI of 30, it was used to compare increases in daily readings of the drug-containing vials.

M. smegmatis MICs were determined using a broth macrodilution method (Jorgensen et al., 1999). Briefly, cultures were harvested at mid-exponential phase (OD600 0·4–0·6), washed once in fresh media, and diluted 100-fold. The diluted culture was used to inoculate 4 ml media containing serially diluted antibiotic (approx. 105 c.f.u. per tube). The tubes were incubated for 3 days on a rotary drum at 37 °C. The MIC was determined to be the lowest concentration at which no growth was observed after 3 days incubation.

Whole-cell lysates.
M. smegmatis lysates were obtained by French press (Aminco). Cells from saturated cultures (OD600>=1·0) were pelleted, washed twice in cold buffer (1x PBS, pH 7·0), and subsequently resuspended in 3 ml buffer with DNase (100 U; Roche Applied Science), RNase A (100 µg; Sigma-Aldrich) and protease inhibitor [3 mM 4-(2-aminoethyl)benzenesulfonylfluoride (AEBSF); Calbiochem] added. Cells were broken in a French pressure cell [14 000 p.s.i. (96·6 MPa); four applications] and cell debris removed by centrifugation (12 000 g; 30 min). Sterile M. tuberculosis lysates were obtained using a FastPrep instrument (Qbiogene) and FastPROTEIN Blue lysing matrix. Control experiments using M. smegmatis cells showed that lysate preparation using the FastPrep machine was as efficient as using the French pressure cell. Cells were pelleted from 50 ml saturated cultures, washed twice in cold buffer (1x PBS, pH 7·0) and resuspended in 5 ml buffer. DNase, RNase A and protease inhibitor were added as for M. smegmatis, above. Resuspended cells were distributed to lysing matrix tubes (1 ml each) and subjected to two disruptions at a speed setting of 6·0 for 30 s. The cells were chilled on ice for 5 min between disruptions. Lysate and lysing matrix were transferred to a 15 ml conical tube and the debris was pelleted (4700 g, 10 min). The resulting lysate was filtered twice through a 0·2 µm syringe filter. Control experiments using M. smegmatis lysates showed that filtration of lysates does not affect the detection of {beta}-lactamase activity. Protein concentration of whole-cell lysates was determined using the Bradford method (Bio-Rad).

Nitrocefin assays.
The chromogenic cephalosporin nitrocefin (Oxoid) was used to assay {beta}-lactamase activity in whole-cell lysates (O'Callaghan et al., 1972). We empirically determined the amount of total lysate protein to add to achieve a linear response. Assays were performed at 22 °C with 100 µM nitrocefin in 1x PBS, pH 7·0. Hydrolysis was monitored at 486 nm using a Beckman DU530 spectrophotometer (Beckman Instruments) and absorbance recorded every 30 s for 15 min. The amount of nitrocefin hydrolysed per unit time was determined using Beer's law and the molar extinction coefficient of nitrocefin, at 486 nm, of 20 500. Finally, a slope was calculated to estimate the amount of nitrocefin hydrolysed per min. The rate of nitrocefin hydrolysis for each strain was expressed as micrograms of nitrocefin hydrolysed per minute per milligram total lysate protein.

For inhibition and competition assays with M. smegmatis lysates, the {beta}-lactam antibiotics aztreonam (ATM; ICN Pharmaceuticals), clavulanic acid (CLA), benzylpenicillin (PEN; ICN Pharmaceuticals) or cephalothin (LOT; Sigma-Aldrich) were added prior to each assay and nitrocefin hydrolysis was determined as described above.

Nucleotide sequence accession numbers.
The DNA sequence of a 2482 bp PCR product containing the blaS ORF was submitted to GenBank and given the accession number AY332268. The DNA sequence of a 2133 bp fragment containing the blaE ORF was submitted to GenBank and given the accession number AY442183.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Identification of the M. smegmatis major and minor {beta}-lactamases
We used the mycobacterial {beta}-lactamase gene blaF from M. fortuitum (GenBank accession no. AAA19882 to search the unfinished M. smegmatis mc2155 genome (The Institute for Genomic Research, TIGR; http://www.tigr.org/tdb/mdb/mdbinprogress.html), and retrieved a gene (we term blaS) whose product was 70 % homologous to BlaF. An alignment of this M. smegmatis {beta}-lactamase, BlaS, the M. fortuitum BlaF, and the BlaC {beta}-lactamase from M. tuberculosis H37Rv (GenBank accession no. NP_216584) is shown in Fig. 1.



View larger version (55K):
[in this window]
[in a new window]
 
Fig. 1. Multiple alignment of M. smegmatis BlaS, M. fortuitum BlaF and M. tuberculosis H37Rv BlaC {beta}-lactamases using CLUSTAL W version 1.8 (http://clustalw.genome.ad.jp/). The M. smegmatis BlaS protein is 70 % and 37 % identical to the M. fortuitum BlaF protein and the M. tuberculosis BlaC protein, respectively (BLASTP 2.2.4; http://www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html). Structural motifs indicative of class A {beta}-lactamases (Ambler, 1980; Ambler et al., 1991) are boxed, while the regions deleted in the blaC and blaS mutants are underlined.

 
Another putative {beta}-lactamase gene, that we designate blaE, was identified in the M. smegmatis mc2155 unfinished genome using the previously reported N-terminal sequence of a purified cephalosporinase from M. smegmatis SN2 (Basu et al., 1997). Fig. 2 shows an alignment of the M. smegmatis BlaE protein and known class C {beta}-lactamases.



View larger version (83K):
[in this window]
[in a new window]
 
Fig. 2. Multiple alignment using CLUSTAL W version 1.8 (http://clustalw.genome.ad.jp/) of the known class C {beta}-lactamases Enterobacter cloacae P99 (GenBank accession no. P05364), Escherichia coli K-12 AmpC (GenBank accession no. NP_418574) and Pseudomonas aeruginosa PAO1 AmpC (GenBank accession no. NP_252799) with the putative {beta}-lactamase from M. smegmatis mc2155, BlaE (GenBank accession no. AY442183). Motifs indicative of class C {beta}-lactamases are boxed. The N-terminal sequence of the purified M. smegmatis SN2 cephalosporinase (Basu et al., 1997) used to identify the putative M. smegmatis mc2155 {beta}-lactamase, BlaE, is shown in italics with the homologous region in BlaE in bold. The sequence deleted in the {Delta}blaE1 mutant PM976 is underlined.

 
Construction of deletion mutants
Mutants with deletions of the {beta}-lactamase genes were constructed by a previously described methodology using a mycobacterial suicide vector containing the counter-selectable marker sacB (Pavelka & Jacobs, 1999). Southern hybridizations (data not shown) verified the gene replacements in the mutants used in this study, PM638 (M. tuberculosis {Delta}blaC1), PM759 (M. smegmatis {Delta}blaS1) and PM976 (M. smegmatis {Delta}blaS1{Delta}blaE1 : : res).

Antimicrobial susceptibility testing
Susceptibility testing for both M. smegmatis and M. tuberculosis included disc diffusion using Sensi-Discs. This method was chosen for the initial screening of the allelic exchange mutants because of ease of use and also because more antibiotics are readily available as Sensi-Discs than are available in powder form. This disc diffusion method has been used in the past for susceptibility determinations in fast-growing (Cynamon & Patapow, 1981; Wallace et al., 1979) and slow-growing (Jarboe et al., 1998) mycobacteria.

Results from disc diffusion experiments for the M. smegmatis blaS deletion mutant are shown in Table 3. The parental strain, PM274, was not susceptible to {beta}-lactam antibiotics, with the exception of cefoxitin and imipenem. As expected, PM274 was susceptible to amoxicillin in the presence of the {beta}-lactamase inhibitor clavulanic acid. For the M. smegmatis {Delta}blaS1 mutant PM759, increased susceptibility to {beta}-lactam antibiotics was evident from the appearance of zones of growth inhibition. This phenotype was observed for the mutant with all {beta}-lactam antibiotics except oxacillin, ceftriaxone and cefixime. Note that there was no increase in susceptibility to cefoxitin and imipenem and that the presence of the {beta}-lactamase inhibitor clavulanic acid did not affect the susceptibility of the mutant to amoxicillin. We also observed no change in susceptibility between the parental strain and the mutant for the non-{beta}-lactam antibiotics isoniazid and rifampicin (data not shown).


View this table:
[in this window]
[in a new window]
 
Table 3. Susceptibility determined by disc diffusion for M. smegmatis strains

Zone diameters reported are the mean of triplicate determinations with variation <2 mm. The genotypes of the respective strains are as follows: PM274, blaS+; PM759, {Delta}blaS1; PM791, PM759 attB : : pMP283; PM876, PM759 attB : : pMV361.hyg.

 
Similarly, wild-type M. tuberculosis H37Rv was not susceptible to the {beta}-lactam antibiotics tested, except for ceftriaxone, imipenem, and the amoxicillin/clavulanic acid combination (Table 4). The blaC knockout, PM638, showed susceptibility to all {beta}-lactam antibiotics except oxacillin and cefixime. However, there was no increase in the susceptibility of the mutant to ceftriaxone and the susceptibility to imipenem was inconsistent. There was an increase in zone size for amoxicillin/clavulanic acid for PM638, but this was close to the variation for this method, and no such increase was seen for the mutant bearing the vector, control strain PM670 (Table 4). There was no increase in the susceptibility of the mutant to the non-{beta}-lactam antibiotics isoniazid and rifampicin (data not shown).


View this table:
[in this window]
[in a new window]
 
Table 4. Susceptibility determined by disc diffusion for M. tuberculosis strains

Zone diameters reported are the mean of triplicate determinations with variation <10 mm. The genotypes of the respective strains are as follows: H37Rv, blaC+; PM638, {Delta}blaC1; PM669, PM638 attB : : pMP199; PM670, PM638 attB : : pMV361.hyg.

 
We determined MICs to further define the extent of susceptibility of PM759 and PM638. As shown in Table 5, the M. smegmatis mutant PM759 showed little or no change in MIC values for cefoxitin or ceftriaxone. However, the mutant showed a 16- to 64-fold increase in susceptibility to amoxicillin, ampicillin, carbenicillin and oxacillin. The presence of clavulanic acid resulted in an eight to 16-fold increase in the MIC value for amoxicillin for the wild-type strain and an four- to eightfold increase for the mutant strain.


View this table:
[in this window]
[in a new window]
 
Table 5. Susceptibility determined by MIC for M. smegmatis and M. tuberculosis

The genotypes of M. smegmatis strains are as follows: PM274, blaS+; PM759, {Delta}blaS1; PM791, PM759 attB : : pMP283; PM876, PM759 attB : : pMV361.hyg. The genotypes of M. tuberculosis strains are as follows: H37Rv, blaC+; PM638, {Delta}blaC1; PM669, PM638 attB : : pMP199; PM670, PM638 attB : : pMV361.hyg. Values are reported in µg ml–1. All MICs were performed in duplicate on at least two independent cultures. Values are reported as a range, or as a single number in cases of non-variant results. ND, Not determined.

 
Wild-type M. tuberculosis H37Rv showed a relatively high level of resistance to all {beta}-lactams tested using MIC determination. The inclusion of clavulanic acid reduced the MIC for amoxicillin by at least 16-fold for the wild-type H37Rv and by eightfold for the {Delta}blaC1 mutant PM638 The mutant showed a 16-fold increase in susceptibility to amoxicillin and carbenicillin, a 32-fold increase in sensitivity to cefoxitin, and an eightfold increase in sensitivity to ceftriaxone. This differed from the M. smegmatis mutant, which did not show an increase in susceptibility to either cefoxitin or ceftriaxone. No changes in the MICs were observed in the M. tuberculosis mutant compared to wild-type when the standard anti-tubercular drugs streptomycin, isoniazid, pyrazinamide, ethambutol and rifampicin were tested (data not shown).

Complementation of both the mutant strains PM638 and PM759 with their wild-type {beta}-lactamase genes restored the parental resistance pattern in both disc diffusion tests (Tables 3 and 4) and MIC determinations (Table 5). The blaC gene of M. tuberculosis was incapable of complementing the M. smegmatis {Delta}blaS1 mutant, PM759, in single copy but it restored the parental phenotype in multi-copy (disc diffusion data not shown).

Susceptibility studies were also performed on the M. smegmatis double {beta}-lactamase knockout, PM976. Disc diffusion tests showed little difference between the single {beta}-lactamase knockout, PM759, and the double {beta}-lactamase knockout, PM976 (Table 6). The presence of clavulanic acid did not change the susceptibility of the double mutant to amoxicillin (Table 6). Because the sequence comparisons suggested that BlaE might be a cephalosporinase, a wider variety of cephalosporins was included in the disc diffusion studies. As seen in Table 6, no significant differences in susceptibility to penicillin-based {beta}-lactams were observed, with the exception of piperacillin. Similarly, most of the cephalosporin-based {beta}-lactam antibiotics showed no major differences. However, cephalothin, cefazolin and ceftriaxone showed very small but consistent zones of inhibition with PM976, whereas no zone was observed for PM759. The MICs were determined to be nearly identical for the two strains, PM759 and PM976 (Table 7).


View this table:
[in this window]
[in a new window]
 
Table 6. Susceptibility determined by disc diffusion for the M. smegmatis double mutant

The genotypes of respective strains are as follows: PM759, {Delta}blaS1; PM976, {Delta}blaS1 {Delta}blaE1 : : res. Zone diameters reported are one of triplicate determinations with variation <2 mm.

 

View this table:
[in this window]
[in a new window]
 
Table 7. Susceptibility as determined by MIC for the M. smegmatis double mutant

The genotypes of respective strains are as follows: PM759, {Delta}blaS1; PM976, {Delta}blaS1 {Delta}blaE1 : : res. MIC values are reported in µg ml–1. All MICs were performed in duplicate on at least two independent cultures. Values are reported as a range, or a single number in cases of non-variant results. The ratio of amoxicillin : clavulanic acid (Amox/clav) was maintained at 2 : 1 for all dilutions.

 
Nitrocefin assays
We performed nitrocefin hydrolysis assays to compare the {beta}-lactamase activity in whole-cell lysates of the wild-type and mutant strains. We chose to use whole-cell lysates since the majority of {beta}-lactamase activity appears to be cell-associated in late-exponential-phase cultures of mycobacteria (data not shown; Fattorini et al., 1991; Zhang et al., 1992). The {beta}-lactamase activity in M. smegmatis PM274 and in the single {beta}-lactamase knockout, PM759, lysate is shown in Table 8. A significant decrease in activity was observed in PM759, compared to PM274, with a residual {beta}-lactamase activity present in PM759. The M. tuberculosis mutant PM638 also showed significantly reduced {beta}-lactamase activity compared to wild-type, but, in contrast to the M. smegmatis mutant, had no detectable residual {beta}-lactamase activity (Table 8). The amount of nitrocefin hydrolysis in the PM638 lysates was essentially the same as that of a negative buffer control. We also noted that the {beta}-lactamase activity in the wild-type strain of M. tuberculosis was 10-fold lower than that in wild-type M. smegmatis.


View this table:
[in this window]
[in a new window]
 
Table 8. Nitrocefin assays on whole-cell lysates of M. smegmatis and M. tuberculosis wild-type and {beta}-lactamase knockout strains

Nitrocefin activity is expressed as µg nitrocefin hydrolysed min–1 (mg total protein)–1±SD. Each assay was performed in triplicate.

 
A group 2e cephalosporinase has been reported in M. smegmatis SN2 (Basu et al., 1997), suggesting that a similar {beta}-lactamase may be present in M. smegmatis mc2155 that might account for the phenotype of the {Delta}blaS1 mutant. We attempted to partially characterize the residual {beta}-lactamase in PM759 through competition assays using benzylpenicillin and the cephalosporin cephalothin. As shown in Fig. 3, the residual {beta}-lactamase activity in PM759 was able to hydrolyse both antibiotics. We found that 10 µM of either benzylpenicillin or cephalothin decreased the nitrocefin hydrolysing activity by 71 % and 59 %, respectively (P<0·001). Even at the lowest concentration of competitor (1 µM), we found a significant decrease in nitrocefin hydrolysis, 24 % and 43 % for benzylpenicillin and cephalothin, respectively (P<0·002), suggesting that the activity has an affinity for both classes of {beta}-lactams.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 3. Nitrocefin competition assays with M. smegmatis PM759 whole-cell lysates. All assays were performed in triplicate and on lysates from two cultures. Each column represents the mean of three assays from one lysate and error bars represent the standard deviation. CLA, clavulanic acid; ATM, aztreonam; LOT, cephalothin; PEN, penicillin.

 
Cephalosporinases are differentiated through their susceptibility to clavulanic acid and aztreonam (Bush et al., 1995). The functional type 1 cephalosporinases are more susceptible to inhibition by aztreonam than by clavulanic acid. Conversely, cephalosporinases of type 2 can be inhibited by low levels of clavulanic acid. Since previous reports suggest the presence of a type 2e cephalosporinase in M. smegmatis (Basu et al., 1997), we examined the nitrocefin hydrolysing ability in PM759 in the presence of either clavulanic acid or aztreonam. As shown in Fig. 3, the addition of aztreonam at 1 µM resulted in a 55 % decrease in nitrocefin hydrolysing ability while the addition of 10 µM aztreonam reduced {beta}-lactamase activity to less than 4 % of the level seen in the control reaction (P<0·001). In contrast, the addition of 100 µM clavulanic acid to the lysate resulted in only a 15 % decrease in {beta}-lactamase activity. While statistically significant (P<0·05), the effect was much less pronounced than that seen with aztreonam, suggesting that the residual activity in PM759 is a type 1 {beta}-lactamase.

Finally, to demonstrate that this residual {beta}-lactamase activity in PM759 was due to the minor {beta}-lactamase, BlaE, we performed nitrocefin assays on whole-cell lysates of the double mutant PM976. As shown in Table 8, the {beta}-lactamase activity of PM976 was reduced compared to the major {beta}-lactamase mutant, PM759, to a level similar to that seen for the M. tuberculosis mutant PM638.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The goal of this study was to investigate the contribution of the major {beta}-lactamases, BlaC and BlaS, in M. tuberculosis and M. smegmatis to {beta}-lactam antibiotic resistance and to examine any additional {beta}-lactamases in these organisms. We have demonstrated that deletion of the major {beta}-lactamases of M. smegmatis and M. tuberculosis resulted in an increase in susceptibility to {beta}-lactam antibiotics. The M. smegmatis {beta}-lactamase-deficient mutant retained a residual {beta}-lactamase activity, and we determined that this activity is the result of an additional {beta}-lactamase, BlaE. However, the contribution of this minor {beta}-lactamase to the resistance of M. smegmatis appears to be minimal.

In this study, we showed that the M. tuberculosis H37Rv {Delta}blaC1 mutant was devoid of any detectable nitrocefin hydrolysing activity. This is in contrast to the results of a previous study suggesting that M. tuberculosis H37Ra produces an additional minor {beta}-lactamase with predominant cephalosporinase activity (Voladri et al., 1998). This discrepancy may be due to strain differences, as we have used the strain H37Rv for our work and Voladri et al. (1998) used the strain H37Ra. Alternatively, the previous study used purified {beta}-lactamase preparations and consequently may have been more sensitive than our study, which used whole-cell lysates. In addition, the aforementioned study harvested {beta}-lactamase activities from the supernatants of 3–4-week-old cultures. If a minor {beta}-lactamase is produced at a much slower rate and is subsequently secreted or released into the medium due to cell lysis, a 3–4-week-old culture could yield larger amounts of the minor {beta}-lactamase. Our study used whole-cell lysates of younger, late-exponential-phase cultures; the studies in M. fortuitum (Fattorini et al., 1991), M. tuberculosis (Zhang et al., 1992) and our own data (not shown) indicate that the majority of {beta}-lactamase activity at this stage is cell-associated.

Alternatively, it has been proposed that this minor cephalosporinase of M. tuberculosis is not a {beta}-lactamase per se, but is D,D-carboxypeptidase, capable of hydrolysing {beta}-lactam antibiotics (Voladri et al., 1998).

BlaS, the major {beta}-lactamase of M. smegmatis described here, shows a high degree of homology to the molecular class A {beta}-lactamase enzymes (Ambler, 1980; Ambler et al., 1991). This same classification has been previously suggested based on N-terminal sequencing of a purified {beta}-lactamase from M. smegmatis mc2155 (Quinting et al., 1997). The analysis of the coding region for the enzyme reported in this study supports its molecular class A {beta}-lactamase classification. Previous biochemical studies suggested that the M. smegmatis major {beta}-lactamase hydrolyses penicillins and cephalosporins in an equally efficient manner. These biochemical data support the class A designation indicated by protein homology.

This study presents the first description of a minor {beta}-lactamase gene in M. smegmatis. BlaE was identified based on an N-terminal sequence reported from a purified cephalosporinase in M. smegmatis SN2 (Basu et al., 1997). However, in that same work, biochemical studies suggested a group 2e functional classification for the enzyme. Our study shows that the protein sequence, activity and inhibitor profile are consistent with the classification of the BlaE enzyme as a group 1 cephalosporinase. However, substrate and inhibitor profiles using purified enzyme are necessary to confirm this classification.

We found a higher {beta}-lactamase activity in extracts of wild-type M. smegmatis than in extracts of wild-type M. tuberculosis. This might be due to differences in lysate preparation, as we had to pass the M. tuberculosis lysates though a 0·2 µm filter for safety purposes. However, control experiments (not shown) indicated that filtration does not reduce {beta}-lactamase detection in the lysates. Alternatively, the reduced {beta}-lactamase activity of M. tuberculosis could be the result either of accumulated changes within the coding region of blaC or of the weakness of the blaC promoter compared to the blaS promoter. Our complementation studies suggest that the former is more likely, since the wild-type M. tuberculosis blaC gene, when expressed from the strong heterologous groEL promoter, is able to restore a wild-type phenotype in the M. smegmatis {Delta}blaS1 mutant when it is in multi-copy, but not when it is in single copy (data not shown). Furthermore, the amino acid identity between the BlaC and BlaS proteins is only 37 %, making these proteins as similar to each other as they are to {beta}-lactamases of non-mycobacterial species. We hypothesize that the blaC gene of M. tuberculosis has accumulated mildly deleterious mutations over time that have decreased the activity of the BlaC enzyme. Such mutations would likely be tolerated, as there is no selective pressure on an obligate human pathogen such as M. tuberculosis to maintain a functioning {beta}-lactamase enzyme. In contrast, an environmental organism such as M. smegmatis would presumably rely on resistance mechanisms such as {beta}-lactamases to ensure its survival and is under selective pressure to maintain a higher level of {beta}-lactamase activity.

Disc diffusion tests showed an overall increase in susceptibility, relative to the wild-type of both mutants, to most {beta}-lactam antibiotics. Specifically, the greatest increase was observed for the penicillin-based {beta}-lactam antibiotics. This was expected for M. tuberculosis, as initial biochemical descriptions of BlaC indicated that it possessed a predominant penicillinase activity. We observed a similar susceptibility profile in the BlaS mutant of M. smegmatis. However, little or no change in susceptibility was observed for oxacillin, ceftriaxone or cefixime (depending upon the species). Essentially no differences were observed for the M. smegmatis double {beta}-lactamase mutant PM976.

MIC determination confirmed the differences in the susceptibility patterns observed between wild-type and mutant strains in the disc diffusion test in both M. smegmatis and M. tuberculosis. However, some discrepancies were readily apparent with oxacillin, ceftriaxone, cefoxitin and the comparison of amoxicillin and amoxicillin/clavulanic acid. The differences observed could be due to subtle differences between growth on liquid versus solid media, differences in inocula size between MIC and disc diffusion tests, or a combination of these factors.

The M. tuberculosis knockout, PM638, appeared to be more susceptible to {beta}-lactam antibiotics, as measured by disc diffusion, than was the M. smegmatis mutant PM759. It is difficult to make the same comparison for the MIC values between the two species, due to the use of two different methods for MIC determination. However, the fold change in cephalosporin MICs of the M. tuberculosis mutant compared to wild-type was greater than that observed between the wild-type and mutant M. smegmatis strains. Differences in cell envelope permeability may be responsible for these observations.

Production of {beta}-lactamases and the low permeability of the mycobacterial cell wall are believed to act synergistically to produce a {beta}-lactam resistance phenotype (Jarlier & Nikaido, 1994). The roles of {beta}-lactamase production and low permeability have been studied in M. chelonae. While the cell wall of M. chelonae is 10-fold less permeable than that of Pseudomonas aeruginosa and 1000-fold less permeable than that of E. coli, low permeability by itself is insufficient to produce high resistance to {beta}-lactam antibiotics (Jarlier & Nikaido, 1990). The low permeability of M. chelonae acts synergistically with its {beta}-lactamase production to produce an organism with extreme resistance to {beta}-lactam antibiotics (Jarlier et al., 1991). Mycobacterial permeability studies show that fast-growing saprophytic organisms such as M. chelonae (Jarlier & Nikaido, 1990) and M. smegmatis (Trias & Benz, 1994) are less permeable to {beta}-lactam antibiotics than slow-growing obligate pathogens such as M. tuberculosis (Jarlier & Nikaido, 1994). It is reasonable to surmise that higher permeability is responsible for the increase in susceptibility seen with M. tuberculosis.

An additional key element in the entry of the hydrophilic {beta}-lactam antibiotics is the presence of porins in the mycobacterial cell wall. Substantial information exists regarding the porins and porin genes of M. smegmatis, while less is known regarding the porin(s) of M. tuberculosis (Niederweis, 2003). Hydrophilic compounds, such as the {beta}-lactam antibiotics, are predicted to permeate the mycobacterial cell envelope through porins (Trias & Benz, 1994). Specifically, the porins of M. smegmatis and M. chelonae appear to be cationic or zwitterionic selective. Thus, the rate of permeation by {beta}-lactam antibiotics is most likely dependent upon the overall charge of the molecule. A recent study showed that porins do influence the uptake of antibiotics, particularly {beta}-lactams, in M. tuberculosis (Mailaender et al., 2004). In addition, it has also been noted that the M. smegmatis porin density is significantly less than that observed in Gram-negative bacteria (Engelhardt et al., 2002). Porin selectivity and density may contribute to the differences in susceptibility observed here between M. smegmatis and M. tuberculosis.

Our results suggest that the major {beta}-lactamases contribute significantly to the resistance of M. tuberculosis and M. smegmatis to {beta}-lactam antibiotics. Our biochemical evidence indicates that there is only one {beta}-lactamase in M. tuberculosis and two in M. smegmatis. However, the antibiotic susceptibility data suggest that there may be additional, difficult to detect, {beta}-lactamase enzymes in these organisms. The susceptibility of the mutants (PM638 and PM759) to amoxicillin as assayed by MIC was increased in the presence of clavulanic acid by four- to eightfold in certain cases (Table 5), but not in others (Table 7). In addition, an effect of clavulanic acid on the mutants was not seen for the disc diffusion tests (Table 3 and 4). This could suggest the presence of additional clavulanic acid-sensitive, but low-activity {beta}-lactamases in both strains. We did not detect any additional {beta}-lactamase activity in the mutants PM638 ({Delta}blaC1) and PM976 ({Delta}blaS1 {Delta}blaE1 : : res). There is a possibility that additional {beta}-lactamases in the lysates were lost in the cell-wall fraction if they were somehow tightly associated with the cell wall. Our method to prepare lysates of M. smegmatis included a centrifugation step to pellet debris that would also pellet the cell wall; however, the same centrifugation step was done at a much slower speed, insufficient to pellet the cell wall, for the preparation of the M. tuberculosis lysates.

Another possibility is that the effect of clavulanic acid on the amoxicillin susceptibility of the mutants is not due to inhibition of {beta}-lactamases but is the result of effects that clavulanic acid can have on cell wall biosynthesis. It has been previously shown that {beta}-lactamase-negative pneumococci grown with subinhibitory concentrations of clavulanic acid are more susceptible to {beta}-lactam antibiotics and have alterations in their cell wall indicative of inhibition of a D,D-carboxypeptidase (Severin et al., 1997). We surmise that a similar phenomenon might occur in mycobacteria growing in the presence of clavulanic acid.


   ACKNOWLEDGEMENTS
 
We thank members of the Pavelka laboratory for review of this manuscript. We are particularly thankful for the comments of M. Braunstein during the preparation of this manuscript. We also gratefully acknowledge the assistance of Max Salfinger at the Wadsworth Center, Albany, NY. This work was supported by a grant from the National Institute of Allergy and Infectious Disease of the National Institutes of Health (AI47311) and a Burroughs Wellcome Fund Career Award in the Biomedical Sciences. A. R. F. is a trainee supported by the Molecular Pathogenesis of Bacteria and Viruses NIH training Grant T32 AI07362 and a trainee in the Medical Scientist Training Program funded by the NIH grant T32 GM07356.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Ambler, R. P. (1980). The structure of {beta}-lactamases. Philos Trans R Soc Lond B Biol Sci 289, 321–331.[Medline]

Ambler, R. P., Coulson, A. F., Frere, J. M., Ghuysen, J. M., Joris, B., Forsman, M., Levesque, R. C., Tiraby, G. & Waley, S. G. (1991). A standard numbering scheme for the class A {beta}-lactamases. Biochem J 276, 269–270.[Medline]

Basu, D., Narayankumar, D. V., Van Beeumen, J. & Basu, J. (1997). Characterization of a {beta}-lactamase from Mycobacterium smegmatis SN2. Biochem Mol Biol Int 43, 557–562.[Medline]

Bush, K., Jacoby, G. A. & Medeiros, A. A. (1995). A functional classification scheme for {beta}-lactamases and its correlation with molecular structure. Antimicrob Agents Chemother 39, 1211–1233.[Free Full Text]

Casal, M. J., Rodriguez, F. C., Luna, M. D. & Benavente, M. C. (1987). In vitro susceptibility of Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium avium, Mycobacterium fortuitum, and Mycobacterium chelonae to ticarcillin in combination with clavulanic acid. Antimicrob Agents Chemother 31, 132–133.[Medline]

Chambers, H. F., Moreau, D., Yajko, D. & 7 other authors (1995). Can penicillins and other {beta}-lactam antibiotics be used to treat tuberculosis? Antimicrob Agents Chemother 39, 2620–2624.[Abstract]

Chambers, H. F., Kocagoz, T., Sipit, T., Turner, J. & Hopewell, P. C. (1998). Activity of amoxicillin/clavulanate in patients with tuberculosis. Clin Infect Dis 26, 874–877.[Medline]

Cole, S. T., Brosch, R., Parkhill, J. & 39 other authors (1998). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537–544.[CrossRef][Medline]

Consaul, S. A., Jacobs, W. R., Jr & Pavelka, M. S., Jr (2003). Extragenic suppression of the requirement for diaminopimelate in diaminopimelate auxotrophs of Mycobacterium smegmatis. FEMS Microbiol Lett 225, 131–135.[CrossRef][Medline]

Cynamon, M. H. & Palmer, G. S. (1983). In vitro activity of amoxicillin in combination with clavulanic acid against Mycobacterium tuberculosis. Antimicrob Agents Chemother 24, 429–431.[Medline]

Cynamon, M. H. & Patapow, A. (1981). In vitro susceptibility of Mycobacterium fortuitum to cefoxitin. Antimicrob Agents Chemother 19, 205–207.[Medline]

Dye, C., Scheele, S., Dolin, P., Pathania, V. & Raviglione, M. C. (1999). Consensus statement. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. WHO Global Surveillance and Monitoring Project. JAMA 282, 677–686.[Abstract/Free Full Text]

Engelhardt, H., Heinz, C. & Niederweis, M. (2002). A tetrameric porin limits the cell wall permeability of Mycobacterium smegmatis. J Biol Chem 277, 37567–37572.[Abstract/Free Full Text]

Fattorini, L., Scardaci, G., Jin, S. H., Amicosante, G., Franceschini, N., Oratore, A. & Orefici, G. (1991). {beta}-Lactamase of Mycobacterium fortuitum: kinetics of production and relationship with resistance to {beta}-lactam antibiotics. Antimicrob Agents Chemother 35, 1760–1764.[Medline]

Hackbarth, C. J., Unsal, I. & Chambers, H. F. (1997). Cloning and sequence analysis of a class A {beta}-lactamase from Mycobacterium tuberculosis H37Ra. Antimicrob Agents Chemother 41, 1182–1185.[Abstract]

Jacobs, W. R., Jr, Kalpana, G. V., Cirillo, J. D., Pascopella, L., Snapper, S. B., Udani, R. A., Jones, W., Barletta, R. G. & Bloom, B. R. (1991). Genetic systems for mycobacteria. Methods Enzymol 204, 537–555.[Medline]

Jarboe, E., Stone, B. L., Burman, W. J., Wallace, R. J., Jr, Brown, B. A., Reves, R. R. & Wilson, M. L. (1998). Evaluation of a disk diffusion method for determining susceptibility of Mycobacterium avium complex to clarithromycin. Diagn Microbiol Infect Dis 30, 197–203.[CrossRef][Medline]

Jarlier, V. & Nikaido, H. (1990). Permeability barrier to hydrophilic solutes in Mycobacterium chelonei. J Bacteriol 172, 1418–1423.[Medline]

Jarlier, V. & Nikaido, H. (1994). Mycobacterial cell wall: structure and role in natural resistance to antibiotics. FEMS Microbiol Lett 123, 11–18.[CrossRef][Medline]

Jarlier, V., Gutmann, L. & Nikaido, H. (1991). Interplay of cell wall barrier and {beta}-lactamase activity determines high resistance to {beta}-lactam antibiotics in Mycobacterium chelonae. Antimicrob Agents Chemother 35, 1937–1939.[Medline]

Jorgensen, J. H., Turnidge, J. D. & Washington, J. A. (1999). Antibacterial susceptibility tests: dilution and disk diffusion methods. In Manual of Clinical Microbiology, pp. 1526–1543. Edited by P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover & R. H. Yolken. Washington, DC: American Society for Microbiology.

Kaneda, S. & Yabu, K. (1983). Purification and some properties of {beta}-lactamase from Mycobacterium smegmatis. Microbiol Immunol 27, 191–193.[Medline]

Kasik, J. E. (1979). Mycobacterial {beta}-lactamases. In {beta}-Lactamases, pp. 339–350. Edited by J. M. T. Hamilton-Miller & J. T. Smith. New York: Academic Press.

Kwon, H. H., Tomioka, H. & Saito, H. (1995). Distribution and characterization of {beta}-lactamases of mycobacteria and related organisms. Tuber Lung Dis 76, 141–148.[Medline]

Li, X.-Z., Zhang, L. & Nikaido, H. (2004). Efflux-pump mediated intrinsic drug resistance in Mycobacterium smegmatis. Antimicrob Agents Chemother 48, 2415–2423.[Abstract/Free Full Text]

Mailaender, C., Reiling, N., Engelhardt, H., Bossmann, S., Ehlers, S. & Niederweis, M. (2004). The MspA porin promotes growth and increases antibiotic susceptibility of both Mycobacterium bovis BCG and Mycobacterium tuberculosis. Microbiology 150, 853–864.[CrossRef][Medline]

Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Nadler, J. P., Berger, J., Nord, J. A., Cofsky, R. & Saxena, M. (1991). Amoxicillin-clavulanic acid for treating drug-resistant Mycobacterium tuberculosis. Chest 99, 1025–1026.[Abstract]

Niederweis, M. (2003). Mycobacterial porins – new channel proteins in unique outer membranes. Mol Microbiol 49, 1167–1177.[CrossRef][Medline]

O'Callaghan, C. H., Morris, A., Kirby, S. M. & Shingler, A. H. (1972). Novel method for detection of {beta}-lactamases by using a chromogenic cephalosporin substrate. Antimicrob Agents Chemother 1, 283–288.[Medline]

Pavelka, M. S., Jr & Jacobs, W. R., Jr (1996). Biosynthesis of diaminopimelate, the precursor of lysine and a component of peptidoglycan, is an essential function of Mycobacterium smegmatis. J Bacteriol 178, 6496–6507.[Abstract/Free Full Text]

Pavelka, M. S., Jr & Jacobs, W. R., Jr (1999). Comparison of the construction of unmarked deletion mutations in Mycobacterium smegmatis, Mycobacterium bovis bacillus Calmette-Guerin, and Mycobacterium tuberculosis H37Rv by allelic exchange. J Bacteriol 181, 4780–4789.[Abstract/Free Full Text]

Quinting, B., Galleni, M., Timm, J., Gicquel, B., Amicosante, G. & Frere, J. M. (1997). Purification and properties of the Mycobacterium smegmatis mc(2)155 {beta}-lactamase. FEMS Microbiol Lett 149, 11–15.[CrossRef][Medline]

Segura, C., Salvado, M., Collado, I., Chaves, J. & Coira, A. (1998). Contribution of {beta}-lactamases to {beta}-lactam susceptibilities of susceptible and multidrug-resistant Mycobacterium tuberculosis clinical isolates. Antimicrob Agents Chemother 42, 1524–1526.[Abstract/Free Full Text]

Severin, A., Severina, E. & Tomasz, A. (1997). Abnormal physiological properties and altered cell wall composition in Streptococcus pneumoniae grown in the presence of clavulanic acid. Antimicrob Agents Chemother 41, 504–510.[Abstract]

Sorg, T. B. & Cynamon, M. H. (1987). Comparison of four {beta}-lactamase inhibitors in combination with ampicillin against Mycobacterium tuberculosis. J Antimicrob Chemother 19, 59–64.[Abstract]

Trias, J. & Benz, R. (1994). Permeability of the cell wall of Mycobacterium smegmatis. Mol Microbiol 14, 283–290.[Medline]

Utrup, L. J., Moore, T. D., Actor, P. & Poupard, J. A. (1995). Susceptibilities of nontuberculosis mycobacterial species to amoxicillin-clavulanic acid alone and in combination with antimycobacterial agents. Antimicrob Agents Chemother 39, 1454–1457.[Abstract]

Voladri, R. K., Lakey, D. L., Hennigan, S. H., Menzies, B. E., Edwards, K. M. & Kernodle, D. S. (1998). Recombinant expression and characterization of the major {beta}-lactamase of Mycobacterium tuberculosis. Antimicrob Agents Chemother 42, 1375–1381.[Abstract/Free Full Text]

Wallace, R. J., Jr, Dalovisio, J. R. & Pankey, G. A. (1979). Disk diffusion testing of susceptibility of Mycobacterium fortuitum and Mycobacterium chelonei to antibacterial agents. Antimicrob Agents Chemother 16, 611–614.[Medline]

Wong, C. S., Palmer, G. S. & Cynamon, M. H. (1988). In-vitro susceptibility of Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium kansasii to amoxycillin and ticarcillin in combination with clavulanic acid. J Antimicrob Chemother 22, 863–866.[Abstract]

Yabu, K., Kaneda, S. & Ochiai, T. (1985). Relationship between {beta}-lactamase activity and resistance to {beta}-lactam antibiotics in Mycobacterium smegmatis. Microbiol Immunol 29, 803–809.[Medline]

Zhang, Y., Steingrube, V. A. & Wallace, R. J., Jr (1992). Beta-lactamase inhibitors and the inducibility of the beta-lactamases of Mycobacterium tuberculosis. Am Rev Respir Dis 145, 657–660.[Medline]

Received 14 September 2004; revised 1 November 2004; accepted 3 November 2004.



This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Flores, A. R.
Articles by Pavelka,, M. S.
Articles citing this Article
PubMed
PubMed Citation
Articles by Flores, A. R.
Articles by Pavelka,, M. S., Jr
Agricola
Articles by Flores, A. R.
Articles by Pavelka,, M. S.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
INT J SYST EVOL MICROBIOL MICROBIOLOGY J GEN VIROL
J MED MICROBIOL ALL SGM JOURNALS
Copyright © 2005 Society for General Microbiology.