Downloaded from at Pennsylvania State University on September 18, 2016

Transcription

1 Correspondence 217 Doggett, R. G. Incidence of mucoid Pseudomonas aeruginosa from clinical sources. Applied Microbiology 18: 9367 (1969). Doggett, R. G., Harrison, G. M., Stillwell, R. N. & Wallis, E. S. An atypical Pseudomonas aeruginosa associated with cystic fibrosis of the pancreas. Journal of Pediatrics 68:21521 (1966). Govan, J. R. W. Mucoid strains of Pseudomonas aeruginosa: the influence of culture medium on the stability of mucus production. Journal of Medical Microbiology 8: (1975). Marks, M. I., Prentice, R., Swarson, R., Cotton, E. K. & Eickhoff, T. C. Carbenicillin and gentamicin: Pharmacologic studies in patients with cystic fibrosis and pseudomonas pulmonary infections. Journal ofpediatrics 79: 8228 (1971). Williams, R. J. & Govan, J. R. W. Pyocine typing of mucoid strains of Pseudomonas aeruginosa isolated from children with cystic fibrosis. Journal of Medical Microbiology 6: (1973). Zierdt, C. H. & Schmidt, P. J. Dissociation in Pseudomonas aeruginosa. Journal of Bacteriology 87: (1964). A rapid micromethod for the determination of the sensitivity to antibiotics of Pseudomonas aeruginosa Sir, There are four major antibiotics usually effective against Pseudomonas aeruginosa: carbenicillin, gentamicin, tobramycin and colistin (which is at present the most active in vitro). In order to determine the sensitivity to antibiotics the disc method is in current use but it occasionally gives uninterpretable results with the high molecular weight antibiotic, colistin (Scavizzi & Rykner, 1974) questionable results with aminoglycosides, in particular gentamicin (Traub, 1970), and false resistance to carbenicillin if the discs are not stored in vacuum dessicator at 20 (Griffith, 1973). The development of a simple and fast but at the same time reproducible dilution method for the determination of the minimum inhibitory concentrations (MTCs) is therefore of obvious value for Ps. aeruginosa. The breakdown of Larginine by arginine dihydrolase, that takes place in the absence of free oxygen has been utilized in this system. Two hundred and six recent clinical isolates of Ps. aeruginosa of variable degrees of resistance to the different antibiotics were used in this study. The following antibiotic powders were used: gentamicin (Unilabo Laboratories); tobramycin (Lilly Laboratories); colistin methane sulphonate (Roger Bellon Laboratories): carbenicillin (Beecham Laboratories). Taking into account the potency of these powders, 2048 ug/ml solutions in distilled water were prepared and stored for 1 week at 4 C. The following liquid medium was used with the microdilution method: Mueller Hinton broth (Difco) (21 g); Larginine (10 g); Phenol red (02 g); distilled water (1000 ml); (ph:64). With the solid dilution method MuellerHinton agar (Merieux Institute) was used. Calcium and magnesium ions concentrations of culture mediums were measured by the laboratory of biochemistry of Bicetre Hospital. The MuellerHinton agar contained 12±3 mg/1 of Mg 2 and 48±4 mg/1 of Ca 2 and the complete liquid media contained 14± 3 mg/1 of Mg 2 and 45 ±5 mg/1 of Ca 8. (Only traces of Ca 2 and Mg 2 were present in the Larginine and distilled water). For the micromethod, final bacterial suspension was distributed at the rates of 0075 ml (approximately 10 3 bacteria) per cup. For the agar dilution method, 10 3 bacteria per spot were inoculated on the agar by "Steers" replicating apparatus. The agar dilution method and the microdilution technique were carried out as previously described (Ericsson & Sherris, 1971; Scavizzi & Rykner, 1972; Rykner & Scavizzi, 1972). Reading of the results for the microtechnique took place after 18 h of incubation by examining the row of cups containing the series of twofold dilutions of the antibiotic. The colour change from orange/yellow (ph 64) to violet/red (ph 8) of the ph indicator was read as the end point. For a given strain and antibiotic, the MIC corresponds to the concentration contained in the last cup in which there is no colour change. Even though aqueous solutions of tobramycin are known to be markedly basic, solutions in phosphate buffer, ph 64, did not change the MICs obtained for 10 strains, but prevented a slight colour change in the cups on addition of the highest concentrations of the antibiotic. Gentamicin is known to be more active at alkaline ph but adjustment of gentamicin medium and culture medium to 75 did not change the results obtained with 10 strains. For all strains and antibiotics, we found no discrepancy between culture and indicator colour change. The reading of the end point was distinct. Figure 1 shows the results obtained with gentamicin. For carbenicillin and tobromycin, similar results were obtained. All strains, but one, proved sensitive to colistin. On these figures, the results obtained with the two methods are comparable. The authors, who studied micromethods until now, have used aerobic growth of

2 218 Correspondence >8 8 I 7 i 6 5 e I 4 o 3 * 2 A = V i : I «2 I >8 Log2 for MIC (reference method) Figure 1. MIC values of gentamicin for Ps. aeruginosa. Comparison of results obtained with micromethod and with agar dilution method. Pseudomonas by placing the culture in contact of air. This method leads to a loss of fluid (Tilton et al., 1973). The result is an increased concentration of the antibiotic. So, by using the breakdown of Larginine, we could close the plates with sealing tape. With the microdilution method in liquid medium, we have used MuellerHinton medium: the number of ATP molecules and, therefore, the growth obtained from L arginine are low. This is why the trypticase medium used in the previous technique (Scavizzi & Rykner, 1972) was replaced by MuellerHinton broth and found to be more suitable and permits a good growth of Ps. aeruginosa. In addition, the high peptone content promotes alkalinization and facilitates the colour change of ph indicator. The slight differences obtained in the ionic concentrations of culture media don't result in significant variations of MIC of aminoglycosides and polymyxins within, aeruginosa. The two media were not therefore adjusted to obtain the Ca l and Mg t concentrations recommended by Barth Reller et al. (1974) since the object of this study was to compare two dilution methods and not to compare the MICs to the serum levels of the antibiotics. The results of Tilton et al. (1973) showed that there were less variations of MIC when the inoculum varies from 10* to 10 5 organisms/ml than with higher inocula. We used in the micromethod 10 3 organisms/cup similar to that employed by MacLowry et al. (1970). In t t I I I ::: 1 * addition the agar dilution method uses 10 3 organisms/spot (Ericsson & Sherris, 1971). So the inocula were comparable for the two techniques. The reading of the end point for the microtechnique is as clear as in the previous work (Scavizzi & Rykner, 1972). It was made after 18 h incubation because a reading at 8 h does not give such clear and definite results. Tilton et al. (1973) have already obtained variations in MIC of four to five twofold dilutions by varying incubation time from 8 to 15 h. In studies where a microtechnique was compared to a classical dilution method (Marymont & Wentz, 1966; Harwick et al, 1968; MacLowry & Marsh, 1968), results separated by 2 log a MIC were accepted as being in agreement because of the technical error. Using the same criteria, we find similar agreement: with carbenicillin, 84 out of 91 results were in agreement (923%); with gentamicin 78 out of 85 (918%) and with tobramycin 43 out of 46 (935 %). Comparison of the two methods by statistical analysis will be undertaken in another paper. We wish to express our gratitude to Dr E. Rottman, of the Laboratory of Bacteriology of Bicetre Hospital, for her valuable collaboration, and to F. Rodrique of the Laboratory of Biochemistry of Bicetre Hospital, who has measured the Ca! and Mg 2 concentrations in the medium. G. RYKNER Laboratoire de Bacteriologie Hopital de Bicetre, Paris, France M. R. SCAVIZZI Laboratoire Central de Bacteriologie, Institut Gustave Roussy, Villejuif& Departement de Bacteriologie Medicate, Institut Pasteur, Paris, France References Barth Reller, L., Schoenknecht, F. D., Kenny, M. A. & Sherris, J. C. Antibiotic susceptibility testing of Pseudomonas aeruginosa: selection of a control strain and criteria for magnesium and calcium content in media. Journal of Infectious Diseases 130: (1974). Erricsson, H. M. & Sherris, J. C. Antibiotic sensitivity testing. Report of an international collaborative study. Acta Pathologica Microbiologica Scandinavia, sect. B., suppl. 217: 190 (1971).

3

4 Plate 1. Wise and Pippard

5 Correspondence 219 Griffith, L. J. Pseudomonas resistance due to inactivated susceptibility discs. Antimicrobial Agents and Chemotherapy 4: 6467 (1973). Harwick, J. G., Weiss, P. & Fekety, F. R. Application of microtitration techniques to bacteriostatic and bactericidal antibiotic susceptibility testing. Journal of Laboratory and Clinical Medicine 72: 5116 (1968). MacLowry, J. D., Jaqua, M. J. & Selecpak, S. T. Detailed methodology and implementation of a semiautomated serial dilution microtechnique for antimicrobial susceptibility testing. Applied Microbiology 20: 4653 (1970). MacLowry, J. D. & Marsh, H. H. Semiautomatic microtechnique for serial dilution antibiotic sensitivity testing in the clinical laboratory. Journal of Laboratory and Clinical Medicine 72: 6857 (1968). Marymont, J. H. & Wentz, R. M. Serial dilution antibiotic sensitivity testing with the microtiter system. American Journal of Clinical Pathology 45: (1966). Rykner, G. & Scavizzi, M. R. Determination de la concentration minima inhibitrice des antibiotiques par micromethode. Pathologie et Biologie 20 (2122): 9037 (1972). Scavizzi, M. R. & Rykner, G. Determination de la concentration minima inhibitrice des antibiotiques Mise au point d'une micromethode. Nouvelle Presse Medicate 1: (1972). Scavizzi, M. R. & Rykner, G. Sensibilite des enterobacteries a la colistine. Utilisation d'une micromethode. Pathologie et Biologie 22, no. 4: (1974). Tilton, R. C., Liebermann, L. & Gerlach, E. H. Microdilution antibiotic susceptibility test: examination of certain variables. Applied Microbiology 26 (5): (1973). Traub, W. H. Susceptibility of Pseudomonas aeruginosa to gentamicin sulfate in vitro: lack of correlation between disc diffusion and broth dilution sensitivity data. Applied Microbiology 20: (1970). The alteration of colonial morphology after exposure to mecillinam Sir, We should like to report an interesting phenomenon that we observed when investigating the activity of mecillinam in urine. Seventeen recent clinical isolates of E. coli were cultured overnight in pooled human urine (ph 65, osmolality 502 mmol/1) and then subcultured to two aliquots of similar urine, one containing 100 mg/1 of mecillinam and the other antibiotic free. After 48 h incubation at 37 C the aliquots were subcultured by a series of tenfold dilutions onto MacConkey agar and the morphology of the colonies was examined after a further 18h incubation. The colonies were described as "large" (typical "coliform") with a diameter of 3 to 4 mm, medium 1 to 3 mm and small 1 or less mm. Any organisms which showed colonial variation compared with the antibiotic free control were subcultured and the minimum inhibitory concentration (MIC) of mecillinam was determined by a standard broth dilution technique. Seven of the 17 strains showed marked variation in the colonial morphology, with organisms 5 and 6 showing four distinct colonial variants (see Table I and Plate 1). Organism Large Table I Medium * * Small t t = variant of this morphology noted. =no variant with this morphology. *=two distinct "medium" sized colonies noted. t=cysteine dependent colonies. It was very interesting to note that the small colonies from strains 5 and 6 were proved to be cysteine dependent when tested by the agar well method (Gillespie, 1952). These strains came from different sources and were serologically distinct. With repeated subculture, on antibioticfree media, of the morphological variants there was a tendency to revert to the "large" form, but certain strains still showed considerable morphological variation even after eight subcultures. The cysteine dependent variants also Table II MIC (mg/1) of mecillinam in a strain of E. coli showing morphological variants Original Large Medium (1) Medium (2) Small Organism >