Abstract
The distribution of β-lactamase activities in a collection of actinomycete strains was surveyed. Six of 127 strains were found to produce β-lactamase. This low frequency was in contrast to the case with Streptomyces species. The producing strains were not related phylogenetically. MICs of benzylpenicillin did not correlate with β-lactamase production.
β-Lactamases are distributed ubiquitously in both gram-positive and gram-negative bacteria (4). β-Lactamases are referred to as such on the basis of only one common property: they carry out the hydrolysis of β-lactam antibiotics to produce antibacterially inactive products. Because of this, β-lactamases are the main cause of β-lactam resistance in many pathogenic bacteria (11). These enzymes, however, are also produced by nonpathogenic bacteria such as Streptomyces species (5, 8). Most of the Streptomyces species produce β-lactamases constitutively (5, 8).
In contrast to some other enzymes involved in the resistance of antibiotics, β-lactamases are not implicated in the biosynthesis of their corresponding antibiotics, although at least with some bacteria such as Streptomyces clavuligerus (10) and Nocardia lactamdurans (2), a β-lactamase gene is a member of the gene cluster for β-lactam biosynthesis. In Streptomyces, the resistance to β-lactams may be due to very low affinity to penicillin-binding proteins (6). Furthermore, β-lactamase activity has not been detected in β-lactam-producing fungi such as Penicillium species and Cephalosporium species. The role of β-lactamases, particularly in nonpathogenic bacteria like Streptomyces, therefore, remains to be clarified, although it has been suggested that β-lactamase induction by β-lactams is linked with cell wall metabolism in gram-negative bacteria (3). In this sense, the roles of the β-lactamases from gram-positive bacteria and those from gram-negative bacteria may be different, although they are in common in hydrolyzing β-lactams. The fact that β-lactamases from gram-positive bacteria and those from gram-negative bacteria are only remotely related phylogenetically (7) supports this suggestion. Thus, it would be intriguing to know the distribution of β-lactamases in actinobacteria other than Streptomyces. This report describes the distribution of β-lactamases and β-lactam resistance in uncommon actinobacteria.
Actinomycete strains.
The actinomycetes used for the screening of β-lactamase activity were 127 strains from 36 genera found in the Japan Collection of Microorganisms (JCM, RIKEN) and are shown in Table 1. Streptomyces cacaoi subsp. cacaoi JCM 4352 was used as a positive β-lactamase-producing control strain, and Streptomyces lavendulae subsp. lavendulae JCM 4985 was used as a non-β-lactamase-producing control strain.
TABLE 1.
List of actinomycete strains used in this study
| Species | JCM accession no. | |
|---|---|---|
| Actinokineospora spp. | ||
| A. diospyrosa | 9921 | |
| A. globicatena | 9922 | |
| Actinomadura | ||
| A. atramentaria | 6250 | |
| A. aurantiaca | 8201 | |
| A. citrea | 3295 | |
| A. coerulea | 3320 | |
| A. cremea subsp. cremea | 3308 | |
| A. cremea subsp. rifamicini | 3309 | |
| A. fulvescens | 6833 | |
| A. hibisca | 9627 | |
| A. kilaniata | 3306 | |
| A. libanotica | 3284 | |
| A. luteofluorescens | 4203 | |
| A. rugatobispora | 3366 | |
| A. umbrina | 6837 | |
| A. verrucosospora | 3147 | |
| A. vinacea | 3325 | |
| A. yumaensis | 3369 | |
| Actinoplanes spp. | ||
| A. brasiliensis | 3196 | |
| A. consettensis | 7624 | |
| A. deccanensis | 3247 | |
| A. derwentensis | 7556 | |
| A. durhamensis | 7625 | |
| A. ferrugineus | 3277 | |
| A. italicus | 3165 | |
| A. minutisporangius | 9458 | |
| A. pallerronii | 7626 | |
| A. philippinensis | 3001 | |
| A. rectilineatus | 3194 | |
| A. utahensis | 3122 | |
| Actinosynnema spp. | ||
| A. mirum | 3225 | |
| A. pretiosum subsp. auranticum | 7343 | |
| A. pretiosum subsp. pretiosum | 7344 | |
| Aeromicrobium spp. | ||
| A. erythreum | 8359 | |
| A. fastidiosum | 8088 | |
| Amycolatopsis spp. | ||
| A. azurea | 3275 | |
| A. fastidiosa | 3276 | |
| A. mediterranei | 4789 | |
| A. methanolica | 8087 | |
| A. orientalis subsp. lurida | 3141 | |
| A. orientalis subsp. orientalis | 4600 | |
| Arthrobacter spp. | ||
| A. globiformis | 1332 | |
| A. polychromogenes | 2523 | |
| Brevibacterium spp. | ||
| B. linens | 1327 | |
| B. linens | 2590 | |
| Catellatospora spp. | ||
| C. citrea subsp. citrea | 7542 | |
| C. ferruginea | 7544 | |
| C. matsumotoense | 9104 | |
| C. tsunoense | 9105 | |
| Catenuloplanes spp. | ||
| C. atrovinosus | 9535 | |
| C. japonicus | 9106 | |
| Cellulomonas fimi | 1341 | |
| Corynebacterium aquaticum | 1368 | |
| Couchioplanes caeruleus subsp. azureus | 3246 | |
| Curtobacterium spp. | ||
| C. citreum | 1345 | |
| C. luteum | 1480 | |
| C. pusillum | 1350 | |
| Dactylosporangium spp. | ||
| D. aurantiacum | 3083 | |
| D. roseum | 3364 | |
| D. thailandense | 3084 | |
| D. vinaceum | 3307 | |
| Geodermatophilus obscurus | 3152 | |
| Glycomyces spp. | ||
| G. harbinensis | 7347 | |
| G. rutgersensis | 6238 | |
| Gordona terrae | 3229 | |
| Herbidospora cretacea | 8553 | |
| Kibdelosporangium spp. | ||
| K. aridum subsp. aridum | 7912 | |
| K. aridum subsp. largum | 9107 | |
| Kineosporia aurantiaca | 3230 | |
| Microbispora spp. | ||
| M. rosea subsp. rosea | 3006 | |
| M. amethystogenes | 3021 | |
| Micromonospora spp. | ||
| M. carbonacea subsp. carbonacea | 3139 | |
| M. chalcea | 3031 | |
| M. halophytica subsp. halophytica | 3125 | |
| M. olivasterospora | 7348 | |
| Microtetraspora spp. | ||
| M. fusca | 3138 | |
| M. glauca | 3300 | |
| M. niveoalba | 3149 | |
| Nocardioides spp. | ||
| N. albus | 3185 | |
| N. luteus | 3358 | |
| N. plantarum | 9626 | |
| N. pyridinolyticus | 10369 | |
| N. simplex | 1363 | |
| Nocardiopsis spp. | ||
| N. alba | 9419 | |
| N. dassonvillei | 7437 | |
| N. prasina | 3336 | |
| Nonomuria spp. | ||
| N. roseola | 3323 | |
| N. spiralis | 3286 | |
| Planobispora spp. | ||
| P. longispora | 3092 | |
| P. rosea | 3166 | |
| Planomonospora spp. | ||
| P. parontospora subsp. antibiotica | 3094 | |
| P. parontospora subsp. parontospora | 3093 | |
| P. venezuelensis | 3167 | |
| Planotetraspora mira | 9131 | |
| Prauserella rogosa | 9736 | |
| Rhodococcus spp. | ||
| R. equi | 1311 | |
| R. erythropolis | 3201 | |
| R. rhodochrous | 3202 | |
| Saccharomonospora spp. | ||
| S. azurea | 7551 | |
| S. cyanea | 7552 | |
| S. glauca | 7444 | |
| Saccharopolyspora spp. | ||
| S. erythraea | 4026 | |
| S. gregorii | 9687 | |
| S. hordei | 8090 | |
| S. taberi | 9383 | |
| Saccharothrix spp. | ||
| S. aerocolonigenes subsp. aerocolonigenes | 4150 | |
| S. aerocolonigenes subsp. staurosporum | 9734 | |
| S. australiensis | 3370 | |
| S. coeruleofusca | 3313 | |
| S. coeruleoviolacea | 9110 | |
| S. cryophylis | 9111 | |
| S. espanaensis | 9112 | |
| S. flava | 3296 | |
| S. longispora | 3314 | |
| S. mutabilis subsp. capreolus | 4248 | |
| S. mutabilis subsp. mutabilis | 3380 | |
| S. syringae | 6844 | |
| S. texasensis | 9113 | |
| S. waywayandensis | 9114 | |
| Streptosporangium spp. | ||
| S. album | 3025 | |
| S. amethystogenes subsp. amethystogenes | 3026 | |
| S. longisporum | 3106 | |
| S. nondiastaticum | 3114 | |
| S. pseudovulgare | 3115 | |
| S. roseum | 3005 | |
| S. violaceochromogenes | 3281 | |
| S. vulgare | 3028 |
Screening for β-lactamase.
Actinomycete strains were grown on a rotary shaking machine (180 rpm) in 500-ml flasks containing 100 ml of a solution containing 1% medium E (Polypepton; Nihon Seiyaku Co.), 0.2% Bacto Yeast Extract (Difco Co.), 0.5% glycerol, and 0.6% CaCO3, (pH 6.8) at 30°C. Nocardioides pyridinolyticus JCM 10369 was grown at 35°C, Actinomadura yumaensis JCM 3369 was grown at 37°C, and Saccharomonospora glauca JCM 7444 and Saccharopolyspora hordei JCM 8090 were cultured at 40°C. With S. cacaoi subsp. cacaoi, medium R (Bacto Peptone, 1%; Bacto Yeast Extract, 0.5%; Bacto Malt Extract, 0.5%; Bacto Casamino Acids, 0.5%; Bacto Beef Extract, 0.2%; glycerol, 0.2%; Tween 80, 0.005%; MgSO4 · 7H2O, 0.1% [pH 7.2]) was also used. After appropriate times of cultivation, 1-ml aliquots were taken out, the mycelia were removed by centrifugation at 10,000 × g for 5 min, and β-lactamase activity in the supernatant was determined spectrophotometrically (8). One unit of the enzyme was defined as the amount that catalyzed the hydrolysis of 1 μmol of benzylpenicillin per h at 30°C and pH 7.0. Growth was monitored by weighing the mycelia after they were dried at 90 to 100°C and then at 170 to 180°C in previously tared test tubes until complete dehydration.
Generally, as actinobacteria other than Streptomyces grow very slowly, the β-lactamase activity was determined every 7 days for over 30 days. The results are summarized in Table 2. β-Lactamase activity was detected in only 6 strains belonging to 4 genera of 127 strains of 36 genera tested. This result is in contrast to results with Streptomyces, most of the strains of which produce β-lactamase (5, 8). This may be related to the fact that β-lactam antibiotics are hardly detected in actinobacteria other than Streptomyces (9).
TABLE 2.
Production of β-lactamases in and MICs for actinobacteria
| Species (medium) | β-Lactamase activity (U/ml) | Culture time (days) | MIC (μg/ml) |
|---|---|---|---|
| Aeromicrobium erythreum JCM 8359 | 0.43 | 3 | 50 |
| Actinomadura cremea subsp. rifamicini JCM 3309 | 0.16 | 10–14 | 200 |
| Saccharomonospora azurea JCM 7551 | 0.11 | 37 | 50 |
| Saccharothrix flava JCM 3296 | 1.04 | 7 | 500 |
| Saccharothrix aerocolonigenes subsp. aerocolonigenes JCM 4150 | 0.92 | 7 | 200 |
| Saccharothrix waywayandensis JCM 9114 | 0.16 | 7 | 50 |
| Streptomyces cacaoi subsp. cacaoi JCM 4352 (medium E) | 1.59 | 8 | 100 |
| Streptomyces cacaoi subsp. cacaoi JCM 4352 (medium R) | 1.50 | 3 | 100 |
Relationship of β-lactamase-producing strains to their locations in a phylogenetic tree.
A phylogenetic tree constructed on the basis of nucleotide sequences of 16S rRNAs with CLUSTAL W software (12) is shown in Fig. 1. A similar tree was obtained with the NucML program (1). Among these species, the genera of the ones that showed β-lactamase activity are underlined. No relationship was observed between the β-lactamase-producing capacity and the location of a strain in the phylogenetic tree. Thus, it is interesting to know why only a few strains express β-lactamase and how the property of producing β-lactamase is transferred.
FIG. 1.
Phylogenetic tree constructed on the basis of nucleotide sequences of 16S rRNAs with CLUSTAL W (12). The GenBank accession numbers are indicated in parentheses. The bootstrap probabilities are indicated at the left. The bar represents 0.1 substitution per site. Escherichia coli was used as an outgroup. The genera of the ones that showed β-lactamase activity are underlined.
Time course of β-lactamase production.
The time course of β-lactamase production and growth of actinomycete strains were determined. In Aeromicrobium erythreum and Saccharothrix flava, β-lactamase was produced at late logarithmic phase and β-lactamase activities were maintained at high levels even at 8 days. A similar pattern of β-lactamase production and growth was observed with the other strains. With Streptomyces, β-lactamase was also produced in late logarithmic phase. In this sense, the production patterns of β-lactamase in Streptomyces and other actinomycetes were similar.
MICs for actinomycete strains.
Each strain was grown in yeast-glucose medium (yeast extract, 1%; d-glucose, 1% [pH 7.2]) for appropriate times and then streaked on yeast-glucose-agar plates containing various amounts of benzylpenicillin. The concentrations of benzylpenicillin potassium (Sigma Co.) used were 0, 0.1, 0.5, 1, 2, 5, 10, 50, 100, 200, 500, and 1,000 μg/ml. Each plate was incubated at the same temperature used for the determination of β-lactamase activity. MICs were obtained from the results of plates without colonies, when full growth was observed on plates without benzylpenicillin.
MICs for actinobacteria producing β-lactamase are shown in Table 2. No relationship was observed between MICs of benzylpenicillin and β-lactamase production. For example, even though Actinomadura verrucosospora JCM 3147 does not produce β-lactamase, the MIC for it is over 1,000 μg/ml, while even though Saccharomonospora azurea and Saccharothrix waywayandensis secrete β-lactamase, the MICs for them are 50 μg/ml.
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