Millipore Technical Publications |
 | ||
Development of a culture media for air monitoring in antibiotic production area | ||
| Authors: | Serge Ohresser, Stéphanie Sacherer; | |
| ||
| Year: | 2001 | |
| | |
INTRODUCTION
Microbiological air monitoring during aseptic production of beta-lactam antibiotics should be conducted with agar medium containing a beta-lactamase for neutralization of antibiotic, to avoid any false negative. The Millipore MAirT media containing Pnase (Penicillinase) is only efficient against antibiotics belonging to the Penicillines group. A new media was optimized, that can mediate the effects of beta-lactam antibiotics from the three other classes and prevent false negative environmental monitoring data by neutralizing a broader range of antibiotics.MATERIALS AND METHODS
Selection of the most susceptible microorganism
Three strains (Escherichia coli ATCC 8739, Staphylococcus aureus ATCC 6538, Micrococcus luteus ATCC 9341) were tested for their susceptibility to the antibiotics listed in table I (Sensi-DiscTM, Becton-Dickinson), using the classical Disc diffusion test. Bacteria were spreaded on TSA agar plates at a concentration of 106 cfu/ml and paper discs with various antibiotics were deposited on the agar. After incubation for 18 hours, inhibition zones around the discs were measured. Results are expressed in diameter size, including the disc (5 mm); a value of zero indicates that bacterial culture cloth was in contact with the disc.Selection of the most efficient enzyme
Using the same method as below, three enzymes were tested for their ability to protect the bacteria against the antibiotic effect. Each enzyme was incorporated into the agar medium before casting the plate, either alone or in combination. A suspension of Micrococcus luteus (the most susceptible strain following the selection step described below) was spreaded on TSA agar plates at a concentration of 106 cfu/ml and paper discs with various antibiotics were deposited on the agar. After incubation for 18 hours, inhibition zones around the discs were measured. Results are expressed in diameter size, including the disc (5 mm); a value of zero indicates that bacterial culture cloth was in contact with the disc.Results
| Group | Name | Inhibition Zone (mm) Without b-lactamase | Inhibition Zone (mm) With Enzyme I* | Inhibition Zone (mm) With Enzyme II§ | Inhibition Zone (mm) With Enzyme I + II# | Inhibition Zone (mm) With Enzyme III£ |
| Penicillins | Penicillin | 56 | 0 | 0 | 0 | 0 |
| Oxacillin | 46 | ND | 45 | 14 | 0 | |
| Ampicillin | > 60 | 0 | 0 | 0 | 0 | |
| Azlocillin | > 60 | 0 | 30 | 0 | 0 | |
| Piperacillin | > 60 | 0 | 30 | 0 | 0 | |
| Mezlocillin | > 60 | 0 | 25 | 0 | 0 | |
| Ticarcillin | > 60 | 0 | > 60 | 9 | 0 | |
| Mecillinam | 21 | ND | ND | 9 | 0 | |
| Cephalosporines 1st generation | Cephalotin | 47 | 27 | 0 | 0 | 10 |
| Cefazolin | 37 | 25 | 0 | 0 | 13 | |
| Cephalexin | 47 | ND | ND | ND | 35 | |
| Cephalosporines 2nd generation | Cefoxitin | 41 | 41 | 34 | 23 | 38 |
| Cefamandole | 53 | 23 | 18 | 12 | 9 | |
| Cephalosporines 3rd generation | Cefoperazone | 44 | 16 | 16 | 10 | 8 |
| Cefotaxime | 48 | 22 | 38 | 16 | 14 | |
| Cefotetan | 34 | 30 | 32 | 30 | 27 | |
| Cefpirone | 49 | ND | ND | ND | 29 | |
| Cefsulodin | 21 | 14 | 17 | 11 | 0 | |
| Ceftazidime | 38 | 38 | 38 | 33 | 23 | |
| Ceftriaxone | 45 | 25 | 45 | 18 | 9 | |
| Penemes | Imipenem | > 60 | 30 | > 60 | 22 | 9 |
| Meropenem | > 60 | ND | > 60 | 18 | 0 | |
| Monobactames | Aztreoname | 23 | 23 | 23 | 22 | 20 |
Table I. Selection of the most efficient enzyme.
* Enzyme I was a b-lactamase extracted from bacillus cereus, displaying a major bI activity (penicillinase activity) of 6.5 UbI/plate and a minor activity type II (cephalosporinase activity) of 1.5 UbII/plate.§ Enzyme II was a b-lactamase type II extracted from Enterobacter cloacae, displaying a major activity type II of 7.5-12.5 UbII/plate and a minor activity type I of 0.1-0.3 UbI/plate.
# The mix contained the same quantity of Enzyme I as below and twice the quantity of enzyme II.
£ Enzyme III was a b-lactamase type I extracted from bacillus cereus, displaying a major activity type I of 11.1 UbI/plate and a minor activity type II of 1.1 UbII/plate.
| Group | Name | Inhibition Zone (mm) Without b-lactamase | Inhibition Zone (mm) With Enzyme III Before g-sterilization | Inhibition Zone (mm) With Enzyme III After g-sterilization |
| Penicillins | Penicillin | 56 | 0 | 0 |
| Oxacillin | 46 | 0 | 0 | |
| Ampicillin | > 60 | 0 | 0 | |
| Azlocillin | > 60 | 0 | 0 | |
| Mezlocillin | > 60 | 0 | 0 | |
| Piperacillin | > 60 | 0 | 0 | |
| Ticarcillin | > 60 | 0 | 0 | |
| Mecillinam | 21 | 0 | 0 | |
| Cephalosporines 1st generation | Cephalotin | 47 | 10 | 10 |
| Cefazolin | 37 | 13 | 11 | |
| Cephalexin | 47 | 35 | 34 | |
| Cephalosporines 2nd generation | Cefoxitin | 41 | 38 | 37 |
| Cefamandole | 53 | 9 | 9 | |
| Cephalosporines 3rd generation | Cefoperazone | 44 | 8 | 8 |
| Cefotaxime | 48 | 14 | 14 | |
| Cefotetan | 34 | 27 | 26 | |
| Cefpirone | 49 | 29 | 29 | |
| Cefsulodin | 21 | 0 | 0 | |
| Ceftazidime | 38 | 23 | 23 | |
| Ceftriaxone | > 60 | 9 | 0 | |
| Penemes | Imipenem | 45 | 9 | 8 |
| Meropenem | > 60 | 0 | 0 | |
| Monobactames | Aztreoname | 23 | 20 | 19 |
Table II. Influence of gamma-sterilization on enzyme activity.
CONCLUSION