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
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AY332268 and AY442183.
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INTRODUCTION |
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The -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
-lactamases (Kasik, 1979
; Kwon et al., 1995
). However, the effectiveness of
-lactam/
-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
-lactam antibiotics in combination with
-lactamase inhibitors may be useful in the treatment of M. tuberculosis infection (Chambers et al., 1998
; Nadler et al., 1991
).
The major -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
-lactamase bears significant homology to molecular class A
-lactamase enzymes; functionally, it appears to be a penicillinase or type 2b
-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
-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
-lactam antibiotics is not understood and no gene has been identified.
The major -lactamase in M. smegmatis mc2155 has been biochemically described and is similar to BlaF, the well-studied molecular class A
-lactamase from M. fortuitum (Kaneda & Yabu, 1983
; Quinting et al., 1997
). A recent report identified a gene, designated blaA, encoding the major
-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
-lactamase (Basu et al., 1997
). However, an N-terminal sequence from the purified
-lactamase bears significant homology to class C or functional group 1 enzymes (Basu et al., 1997
).
Resistance to -lactam antibiotics in mycobacteria is generally believed to result from the following mechanisms, singly or in combination: (1) enzymic inactivation by
-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
-lactamases in these organisms complicates the study of the other
-lactam resistance mechanisms and also interferes with the use of
-lactam antibiotics in the study of peptidoglycan biosynthesis. Here, we have used a genetic approach to study the contribution of the
-lactamases to
-lactam antibiotic resistance in M. tuberculosis H37Rv and M. smegmatis mc2155. We identified the major
-lactamase gene, blaS, and the minor
-lactamase, blaE, in the genome of M. smegmatis. The
-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
-lactam antibiotics and had reduced or non-detectable
-lactamase activities; however, the susceptibility of the mutants to penicillin-type
-lactam antibiotics was affected most, compared to the cephalosporin-type
-lactam antibiotics. These mutants will serve as tools for the study of other
-lactam resistance mechanisms and of the interaction of
-lactam antibiotics with the peptidoglycan biosynthesis machinery of M. tuberculosis and M. smegmatis.
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METHODS |
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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
).
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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
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 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 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
blaE1 of pMP307 to generate pMP330. Finally, a 6·3 kb fragment containing
blaE1 : : res-aph-res was excised from pMP330 using PvuII and inserted into the EcoRV site of pYUB657 to generate pMP332.
Resolution of blaE1 : : res-aph-res using
-resolvase.
The res-aph-res cassette was resolved in strain PM939 (blaS1
blaE1 : : res-aph-res) using the
-resolvase supplied in trans on a mycobacterial shuttle vector (pGH542; a gift from Graham Hatfull, University of Pittsburgh) to yield strain PM976 (
blaS1
blaE1 : : res). The resulting allele,
blaE1 : : res, contains an out-of-frame insertion in the
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 -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 0·40·6) 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 23 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 0·40·6), 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 (OD6001·0) 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
-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 -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 -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.
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RESULTS |
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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
-lactam antibiotics, with the exception of cefoxitin and imipenem. As expected, PM274 was susceptible to amoxicillin in the presence of the
-lactamase inhibitor clavulanic acid. For the M. smegmatis
blaS1 mutant PM759, increased susceptibility to
-lactam antibiotics was evident from the appearance of zones of growth inhibition. This phenotype was observed for the mutant with all
-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
-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-
-lactam antibiotics isoniazid and rifampicin (data not shown).
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Complementation of both the mutant strains PM638 and PM759 with their wild-type -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
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 -lactamase knockout, PM976. Disc diffusion tests showed little difference between the single
-lactamase knockout, PM759, and the double
-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
-lactams were observed, with the exception of piperacillin. Similarly, most of the cephalosporin-based
-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
).
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Finally, to demonstrate that this residual -lactamase activity in PM759 was due to the minor
-lactamase, BlaE, we performed nitrocefin assays on whole-cell lysates of the double mutant PM976. As shown in Table 8
, the
-lactamase activity of PM976 was reduced compared to the major
-lactamase mutant, PM759, to a level similar to that seen for the M. tuberculosis mutant PM638.
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DISCUSSION |
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In this study, we showed that the M. tuberculosis H37Rv 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
-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
-lactamase preparations and consequently may have been more sensitive than our study, which used whole-cell lysates. In addition, the aforementioned study harvested
-lactamase activities from the supernatants of 34-week-old cultures. If a minor
-lactamase is produced at a much slower rate and is subsequently secreted or released into the medium due to cell lysis, a 34-week-old culture could yield larger amounts of the minor
-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
-lactamase activity at this stage is cell-associated.
Alternatively, it has been proposed that this minor cephalosporinase of M. tuberculosis is not a -lactamase per se, but is D,D-carboxypeptidase, capable of hydrolysing
-lactam antibiotics (Voladri et al., 1998
).
BlaS, the major -lactamase of M. smegmatis described here, shows a high degree of homology to the molecular class A
-lactamase enzymes (Ambler, 1980
; Ambler et al., 1991
). This same classification has been previously suggested based on N-terminal sequencing of a purified
-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
-lactamase classification. Previous biochemical studies suggested that the M. smegmatis major
-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 -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 -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
-lactamase detection in the lysates. Alternatively, the reduced
-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
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
-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
-lactamase enzyme. In contrast, an environmental organism such as M. smegmatis would presumably rely on resistance mechanisms such as
-lactamases to ensure its survival and is under selective pressure to maintain a higher level of
-lactamase activity.
Disc diffusion tests showed an overall increase in susceptibility, relative to the wild-type of both mutants, to most -lactam antibiotics. Specifically, the greatest increase was observed for the penicillin-based
-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
-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 -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 -lactamases and the low permeability of the mycobacterial cell wall are believed to act synergistically to produce a
-lactam resistance phenotype (Jarlier & Nikaido, 1994
). The roles of
-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
-lactam antibiotics (Jarlier & Nikaido, 1990
). The low permeability of M. chelonae acts synergistically with its
-lactamase production to produce an organism with extreme resistance to
-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
-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 -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
-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
-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
-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 -lactamases contribute significantly to the resistance of M. tuberculosis and M. smegmatis to
-lactam antibiotics. Our biochemical evidence indicates that there is only one
-lactamase in M. tuberculosis and two in M. smegmatis. However, the antibiotic susceptibility data suggest that there may be additional, difficult to detect,
-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
-lactamases in both strains. We did not detect any additional
-lactamase activity in the mutants PM638 (
blaC1) and PM976 (
blaS1
blaE1 : : res). There is a possibility that additional
-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 -lactamases but is the result of effects that clavulanic acid can have on cell wall biosynthesis. It has been previously shown that
-lactamase-negative pneumococci grown with subinhibitory concentrations of clavulanic acid are more susceptible to
-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.
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ACKNOWLEDGEMENTS |
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Received 14 September 2004;
revised 1 November 2004;
accepted 3 November 2004.
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