β-Lactamase of the Legionnaires' bacterium

Thornsberry, Clyde; Kirven, L. A.

doi:10.1007/bf02601708

Cited by 49 publications

(25 citation statements)

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“…Several antibiotics active against L. pneumophila growing on artificial medium have been found to lack efficacy against L. pneumophila in vivo (8)(9)(10)(11). It is noteworthy that one such antibiotic, penicillin G, was reported by Johnson et al (22) to be taken up very poorly by rabbit alveolar macrophages; in contrast, erythromycin and rifampin, which are active against L. pneumophila both in vivo and under cell-free conditions, were taken up readily by rabbit alveolar macrophages (8)(9)(10)(11)22). In fact, erythromycin and rifampin were among the few antibiotics tested that were concentrated in rabbit alveolar macrophages-erythromycin 20-fold and rifampin twofold (22).…”

Section: Discussionmentioning

confidence: 99%

“…Both clinical experience and in vivo studies indicate that these antibiotics are efficacious in treating Legionnaires' disease (6)(7)(8)(9). L. pneumophila grown under cell-free conditions on artificial media is inhibited from multiplying by these antibiotics and is killed by concentrations of these antibiotics that are the same or somewhat higher than the minimal inhibitory concentration (MIC)' (10,11).…”

Section: Introductionmentioning

confidence: 99%

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Intracellular multiplication of Legionnaires' disease bacteria (Legionella pneumophila) in human monocytes is reversibly inhibited by erythromycin and rifampin.

Horwitz¹,

Silverstein²

1983

J. Clin. Invest.

144

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A B S T R A C T We have previously reported that virulent egg yolk-grown Legionella pneumophila, Philadelphia 1 strain, multiplies intracellularly in human blood monocytes and only intracellularly under tissue culture conditions. In this paper, we have investigated the effect of erythromycin and rifampin on L. pneumophila-monocyte interaction in vitro; erythromycin and rifampin are currently the drugs of choice for the treatment of Legionnaires' disease.The intracellular multiplication of L. pneumophila is inhibited by erythromycin and rifampin, as measured by colony-forming units, whether the antibiotics are added just before or just after infection of monocytes with L. pneumophila, or 2 d after infection when L. pneumophila is in the logarithmic phase of growth in monocytes. Intracellular multiplication of L. pneumophila is inhibited by 1.25 ,tg/ml but not <0.125 ,ig/ml erythromycin and 0.01 ,ug/ml but not <0.001 ,tg/ml rifampin. These concentrations of antibiotics are comparable to those that inhibit extracellular multiplication of L. pneumophila under cell-free conditions in artificial medium; the minimal inhibitory concentration is 0.37 Ag/ml for erythromycin and 0.002 ,gg/ml for rifampin. Multiplication of L. pneumophila in the logarithmic phase of growth in monocytes is inhibited within 1 h of the addition of antibiotics. Intracellular bacteria inhibited from multiplying by antibiotics are not killed. By electron microscopy, the bacteria appear intact within membrane-bound vacuoles, studded with ribosomelike structures.L. pneumophila multiplying extracellularly on artificial medium is killed readily by relatively low con- centrations of erythromycin and rifampin; the minimal bactericidal concentration is 1 ,tg/ml for erythromycin and 0.009 ,ug/ml for rifampin. In contrast, L. pneumophila multiplying intracellularly is resistant to killing by these concentrations of erythromycin and rifampin or by concentrations equal to or greater than peak serum levels in humans. Extracellular L. pneumophila in stationary phase is also resistant to killing by erythromycin and rifampin. These findings, taken together with our previous work, indicate that, in vivo, L. pneumophila is resistant to killing by erythromycin and rifampin.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Intracellular multiplication of Legionnaires' disease bacteria (Legionella pneumophila) in human monocytes is reversibly inhibited by erythromycin and rifampin.

Horwitz¹,

Silverstein²

1983

J. Clin. Invest.

144

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“…There was no consistent difference in susceptibility between Center for Disease Control-supplied stock strains and recent clinical isolates, but there were marked differences with some agents. Susceptibility testing needs to be standardized in view of the influence of inoculum size, strain variation, and the medium used.Susceptibility testing of Legionella pneumophila, the etiological agent of Legionnaires disease, has been previously reported only with small numbers of isolates (8,9,12,14,16,17) and not with charcoal-yeast extract (CYE) agar, which has become the standard medium for L. pneumophila (6). In addition, most strains tested have been passaged multiple times after isolation on media which do not optimally support their growth.…”

mentioning

confidence: 99%

“…Susceptibility testing of Legionella pneumophila, the etiological agent of Legionnaires disease, has been previously reported only with small numbers of isolates (8,9,12,14,16,17) and not with charcoal-yeast extract (CYE) agar, which has become the standard medium for L. pneumophila (6). In addition, most strains tested have been passaged multiple times after isolation on media which do not optimally support their growth.…”

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confidence: 99%

Susceptibility of Legionella pneumophila to twenty antimicrobial agents

Edelstein¹,

Meyer²

1980

Antimicrob Agents Chemother

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Thirty-three isolates of Legionella pneumophila, all except one of which were clinical isolates, were tested against 20 antimicrobial agents by using an agar dilution technique. Erythromycin, rifampin, and rosaramycin were the most active agents tested. Aminoglycosides, chloramphenicol, and cefoxitin also inhibited the organisms at low concentrations. Other agents, including moxalactam, cefoperazone, and cephalosporins, exhibited moderate to little activity. Tetracycline, doxycycline, and minocycline were apparently inactivated by charcoal-yeast extract medium. There was slight inoculum dependence noted with most of the antimicrobials tested, particularly the /3-lactam agents. There was no consistent difference in susceptibility between Center for Disease Control-supplied stock strains and recent clinical isolates, but there were marked differences with some agents. Susceptibility testing needs to be standardized in view of the influence of inoculum size, strain variation, and the medium used.Susceptibility testing of Legionella pneumophila, the etiological agent of Legionnaires disease, has been previously reported only with small numbers of isolates (8,9,12,14,16,17) and not with charcoal-yeast extract (CYE) agar, which has become the standard medium for L. pneumophila (6). In addition, most strains tested have been passaged multiple times after isolation on media which do not optimally support their growth. Because of the availability of a large number of clinical isolates, which were almost entirely recently isolated and had not been passaged multiple times on nonoptimal media, susceptibility testing was performed with 20 antimicrobial agents; these include some new agents, e.g., moxalactam and cefoperazone, which have not been reported in previous studies. MATERLALS AND METHODS Md.) and 1% hemoglobin (MHIH).The remainder of the strains had not been passaged on MHIH and had been passaged from 2 to 10 times on CYE agar only. All strains were kept at 5°C on CYE slants and subcultured monthly. Before the susceptibility studies were done, the strains were subcultured onto CYE agar plates and incubated for 48 h at 35°C in 2.5% CO2 and 65% relative humidity.Inocula were prepared by harvesting growth from a 48-h plate with a sterile wooden stick into approximately 1 ml of Mueller-Hinton broth. The turbidity was adjusted to that of 0.5 MacFarland barium sulfate standard (18). We have found previously that such a suspension of L. pneumophila contains from 7 x 10' to 1 x 108 colony-forming units (CFU)/ml. Dilutions, 1:10 and 1:100, of this suspension were then made with Mueller-Hinton broth and mixed with a Vortex mixer before transfer. Assuming that the volume delivered to each spot by the Steers replicator was 0.001 ml and that a suspension of L. pneumophila adjusted to the turbidity of a 0.5 MacFarland barium sulfate standard contains -10' CFU/ml, the final plate inoculum sizes were -105 CFU/spot for the largest inoculum, i104 CFU/spot for the 1:10 dilution, and -103 CFU/spot for the 1:100 dilution. All

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“…Clavulanic acid and CP-45,899, f)-lactamase inhibitors, prevented the hydrolysis of cephalosporins and penicillins. The fl-lactamase activity appears to be different from that found in Enterobacteriaceae and Pseudomonas.The reported in vitro resistance of Legionella pneumophila to penicillins and cephalosporins (7,8) as well as the clinical failure of these agents in the treatment of patients with Legionnaire's pneumonia (1) prompted us to evaluate the ability of this organism to destroy fi-lactam antibiotics and to clarify whether the inactivating activity was due to the presence of a f8-lactamase. …”

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confidence: 99%

Inactivation of beta-lactam antibiotics by Legionella pneumophila

Fu¹,

Neu²

1979

Antimicrob Agents Chemother

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Beta-lactam-inactivating activity has been found in all sero-groups of Legionella pneumophila. The ,B-lactamase activity could be detected in intact cells and released by ethylenediaminetetraacetic acid treatment, indicating that it is located in the periplasmic space. The enzyme acted primarily as a cephalosporinase hydrolyzing cefamandole, cephalothin, cephaloridine, and also penicillin G and ampicillin. Cefoxitin and cefuroxime were not hydrolyzed. Clavulanic acid and CP-45,899, f)-lactamase inhibitors, prevented the hydrolysis of cephalosporins and penicillins. The fl-lactamase activity appears to be different from that found in Enterobacteriaceae and Pseudomonas.The reported in vitro resistance of Legionella pneumophila to penicillins and cephalosporins (7,8) as well as the clinical failure of these agents in the treatment of patients with Legionnaire's pneumonia (1) prompted us to evaluate the ability of this organism to destroy fi-lactam antibiotics and to clarify whether the inactivating activity was due to the presence of a f8-lactamase. MATERIALS AND METHODSOrganisms and antibiotics. Isolates of L. pneumophila were provided by the Center for Disease Control, Atlanta, Ga. The following isolates were examined: Seattle, Vermont, Flint, Knoxville, Philadelphia, Pontiac, Bloomington, Togus, and Los Angeles.Organisms were maintained on Mueller-Hinton agar plates supplemented with 0.04% cysteine and 0.025% ferric phosphate. The antibiotics studied were gifts from their respective manufacturers. Clavulanic acid was provided by Beecham Laboratories and CP-45,899, a penicillanic acid sulfone, was provided by Pfizer Inc. Susceptibility tests. Susceptibility tests were performed in Mueller-Hinton broth containing 0.04% cysteine and 0.1% ferric phosphate. The inoculum used was 105 colony-forming units. This was prepared by making a suspension in broth of a 48-h growth removed from agar plates. Tubes were incubated at 35°C for 72 h in the presence of 5% CO2.Osmotic shockates. Osmotic shockates were prepared by the technique of Neu and Chou, using 0.1 g of bacteria to 10 ml of 1 mM ethylenediaminetetraacetic acid in 10 mM tris(hydroxymethyl)aminomethanehydrochloride buffer, pH 7.0.Enzyme preparation. Three-day-old cultures were removed from agar plates by means of a glass spatula, using potassium phosphate buffer (0.05 M, pH 7). The cells were washed twice in the same buffer and then suspended in buffer at a concentration of 1010 cells per ml. The cells were sonically disrupted by a Branson Sonifier, using 30-s pulses for 3 min with the vessel cooled in an ice bath. All procedures were performed in a hood which incinerated the exhaust. The sonic extracts were centrifuged for 2 h at 4°C and 40,000 x g. The cellular debris was discarded, and the supernatant was dialyzed for 24 h at 4°C against the phosphate buffer. This material was used for the assays.f8-Lactamase assays. Intact organisms, material from osmotically shocked organisms, and the crude extract were tested for f,-lactamase activity with the nitrocefin (5), ...

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β-Lactamase of the Legionnaires' bacterium

Cited by 49 publications

References 7 publications

Intracellular multiplication of Legionnaires' disease bacteria (Legionella pneumophila) in human monocytes is reversibly inhibited by erythromycin and rifampin.

Intracellular multiplication of Legionnaires' disease bacteria (Legionella pneumophila) in human monocytes is reversibly inhibited by erythromycin and rifampin.

Susceptibility of Legionella pneumophila to twenty antimicrobial agents

Inactivation of beta-lactam antibiotics by Legionella pneumophila

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