VOL. XXX NO. 8 THE JOURNAL OF ANTIBIOTICS 655 RESISTANCE OF BACTERIA TO THE NEWER AMINOGLYCOSIDE ANTIBIOTICS: AN EPIDEMIOLOGICAL AND ENZYMATIC STUDY

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1 VOL. XXX NO. 8 THE JOURNAL OF ANTIBIOTICS RESISTANCE OF BACTERIA TO THE NEWER AMINOGLYCOSIDE ANTIBIOTICS: AN EPIDEMIOLOGICAL A ENZYMATIC STUDY MARLYSE DEVAUD, FRITZ H. KAYSER and URSUL.A HUBER Institute of Medical Microbiology, University of Zurich CH-88 Zurich, Switzerland (Received for publication May 8, 77) Four hundred and thirty-five Staphylococcus aureus and of gramnegative bacteria were tested for susceptibility to gentamicin, sisomicin, tobramycin, netilmicin and amikacin. None of the staphylococcal were resistant to the drugs, except two that were resistant to amikacin. Eighteen percent of the gram-negative bacteria were resistant to gentamicin, % to tobramycin, 4% to sisomicin, 7 % to netilmicin and % to amikacin. Tests from representative showed that these differences were mainly due to the production of aminoglycoside-modifying enzymes. The lower incidence of resistant to sisomicin compared to gentamicin was due to a slightly better antimicrobial activity of sisomicin. Results of experiments on the efficiency of enzymatic phosphorylation, acetylation and adenylylation, and the inactivation of aminoglycoside antibiotics were not always in concurrence with the degree of phenotypic expression of resistance. Thus, the aminoglycoside '-phosphotransferase of staphylococci modified amikacin, but the were susceptible to the drug. Similarly, the aminoglycoside "-nucleotidyltransferase from members of Enterobacteriaceae and Pseudomonas efficiently adenylylated and inactivated netilmicin, but the were susceptible to netilmicin. On the other hand, there can be factors, intrinsically inherent in some, contributing to their phenotypic expression of resistance level. It is inferred, therefore, that the contribution of enzymes to the degree of resistance to aminoglycoside antibiotics in a given strain can be evaluated only by comparative examination of resistance in the wild type strain and its corresponding enzyme-negative variant. In recent years gentamicin, with or without the concomitant administration of beta-lactam antibiotics, has been used with increasing frequency in the treatment of hospital-acquired, bacterial infections. This resulted in increasing emergence of gentamicin-resistant. gram-negative The situation is clearly demonstrated in Table, which contains the total number of examined at our institution from 74 to 7 and the percentage of gentamicin-resistant isolates. Since the Institute of Medical Microbiology in Zurich has to perform diagnostic microbiology for the University Hospital and for about smaller hospitals in the area and has to serve also as a Public Health Laboratory for the Kanton of Zurich, these data can be considered to represent the prevailing situation in the eastern part of Switzerland. The frequency of % of gentamicin-resistant cultures isolated from hospitalized patients in 7 is appallingly high. Because resistance to gentamicin occurred mostly in having resistance to many other antibiotics a comparative examination of the newly developed or recently introduced aminoglycoside antibiotics seemed appropriate and necessary. This paper presents the result of examination of susceptibility of 8 gram-positive and gramnegative bacterial of clinical origin to five selected aminoglycoside antibiotics. In addition, data on susceptibility to modification of these antibiotics by enzymes isolated from representative gentamicin-resistant, are presented. These data help in explaining differences in resistance

2 THE JOURNAL OF ANTIBIOTICS AUG. 77 Table. Number of examined and percentage of gentamicin resistant cultures of clinical isolates of gram-positive and gram-negative bacteria. Bacteria Relation to hospitalizationa 74 (May-December) rb % 7 Rb % 7 jtb % stllph _vlococcus aureus 7, 4,8 4, Staple lococcus epidernlidis 8, 47,8 8,4 Escherichia coli 4,, 4,74, 4,74 Ci trobacte r freundii 4 8 Klebsiella spp. Enterobucter spp. 7 7, , I Serratia spp Protcus nlirabilis 48,,4 Proteus spp. indole-positive Proteus inconstalls 4 7 Salmonella spp Pseudonroncrs spp. 8,,4, 4 Further non-fermentative gram-negative rods Total,4 7, 4,7,7,8,7 a: Status of prior hospitalization of outpatients not determined. b: R=gentamicin resistance. to gentamicin, sisomicin, tobramycin, netilmicin and amikacin existing in our collection of. Bacterial Strains Materials and Methods These were isolated in 7 from different clinical materials. They were comprised of Staphylococcus aurcus (4); Eschericlria coli (4); Klebsiella pneumoniae (7); Klebsiella ozaenae (4); Serratia marecscens (4); Elrterobaeicr cloacae (); Enterobacter agglomerans (); Citrobacter freundii (); Proteus vulgaris (); Proteus mirabilis (); Proteus rettgeri (); Proteus morganii (); Proteus inconstatts (); Salmonella spp. (); Shigella spp. (); Pseudomonas aeruginosa (7); Pseudomonas mcrltophilia (); Pseudomonas spp. (); Acinetobacter spp. (); Alcaligenes spp. (). All were identified according to standard procedures. Staph. aureus was a gentarnicin-resistant strain, isolated by PORT HOUSE et al.) in England.

3 VOL. XXX NO. 8 THE JOURNAL OF ANTIBIOTICS 7 Staph. aureus S was a gentamicin-susceptible mutant of. Both were provided by Dr. BROWN. E. coli W77 (phjr) was a gift of Dr. SMITH.) This strain is the source of the adenylylating enzyme, widely used for enzymatic assay of gentamicin. All the rest of the were of wild type, isolated at our institution. Antibiotics and Chemicals. Laboratory standard preparation of gentarnicin C complex, sisomicin and netilmicin were generously supplied by Schering Corp. (Bloomfield, N.Y.); tobramycin by E. Lilly and Co. (Indianapolis, Ind.), and arnikacin by Bristol Lab. (Syracuse, N. Y.). Radio-labelled chemicals were bought from the Radiochemical Centre (Amersham, England). Dithiothreitol, ATP and acetyl-coa were obtained from Calbiochem (Los Angeles, Calif.), and lysostaphin from Becton, Dickinson and Co. (Orangeburg, N. Y.). Inorganic salts of analytical grade were bought from Merck (Darmstadt, Germany), and agarose from Behringwerke AG (Marburg, Germany). Antibiotic Susceptibility Testing. Disc susceptibility testing was performed by the slightly modified single-disc method of BAUER et a.) and the U.S. Food and Drug Administration,4,) using MUELLER-HINTON agar of Baltimore Biological Laboratories (BBL) (Cockeysville, Md.) supplemented with %, (v/v) old human blood from the blood bank. An inhibition zone of mm or more around the ug gentamicin disc, for a given strain, was interpreted as being susceptible to the drug." The minimal inhibitory concentration (MIC) of drugs was determined on MUELLER-HINTON agar (BBL) according to the standard methods proposed by ERICSSON and SHERRIS." Production of Enzyme. The shock method of NossAL and HEPPEL8) was used for gram-negative organisms. The bacterial under study were serially subcultured on Brain Heart Infusion (BHI) agar (Difco), containing pg gentamicin/ml, and inoculated into a medium composed of Bacto-tryptone (Difco), g; Bacto yeast extract (Difco), g; NaCI, g; glucose, 4 g; and an amount of water sufficient to make I liter. The ph was adjusted to 7.. A hundred ml of the medium was inoculated with one ml of a 4-hoursold culture and incubated in a shaking water bath at 7'C until the end of logarithmic growth phase. Cells were harvested and washed twice with 4 ml of a buffer containing. M Tris-HCl and. M NaCl, ph 7.8 and were suspended in ml of. M Tris-HCl buffer, ph 7. containing. M EDTA and % (w/v) sucrose. After it stood at room temperature for minutes, the suspension was stirred with a magnetic stirrer for minutes and centrifuged for minutes at, x g and 4 C. The supernatant was discarded and the remaining sucrose was removed carefully from the wall of tubes with cotton swabs. The pellet of cells was suspended in 4 ml of an ice-cold solution of. M MgCl, stirred for minutes and centrifuged for minutes at, x g and 4oC. The supernatant (osmotic shockate) was carefully decanted to a flask and stabilized with. M dithiothreitol. Enzymatic extracts of staphylococci were obtained either by the shock method or by lysing the cells with lysostaphin as previously described.) Enzymatic Assays. These were similar to those described by BENVENISTE et al.), DAVIES et al.) and HAAS and DAVIES.) Inactivation by adenylylation of aminoglycosides was tested in a reaction mixture consisting of 4 /Il enzymatic extract; /smol of Tris-maleate buffer, ph 7.4;.4 /cmol MgCl;.,umol dithiothreitol; 4 nmol (-H)ATP (specific activity, uci/,umol) and I nmol of drug base in a total volume of lil. Inactivation by phosphorylation was tested in a reaction mixture containing 4 n- mol of (y-p)atp (specific activity, - 4 mci/mmol). Acetylation was examined in tests comprising. nmol (-C)acetyl-CoA (specific activity,. mci/mmol) and.4 nmol drug base. Appropriate controls were devoid of either the labelled coenzymes, or the drug base or the enzymatic extract. After incubating for. hours at C, d were assayed for residual antibiotic by agar diffusion) in Antibiotic Medium (Difco) containing agarose instead of agar. Bacillus subtilis ATCC was used as test organism, in some cases, and the enzyme-producing strain was used in certain others. Another pl were examined for the extent of adenylylation, phosphorylation or acetylation by mea-

4 8 THE JOURNAL OF ANTIBIOTICS AUG. 77 suring the radioactivity incorporated by the phosphocellulose paper binding assay described by DAVIES and coworkers.) Radioactivity was counted in a Packard Tri-Carb scintillation spectrometer (Packard Instr., Downers Grove, Ill.) in ml of a toluene based scintillant. For determining the relative efficiency of the aminoglycosides as substrates, enzymatic extracts were appropriately diluted and the reaction mixtures incubated for. and minutes at 'C, after which ul were assayed by the phosphocellulose paper binding method mentioned above. These conditions ensured that linear assays were always occurring. Elimination. Attempts to obtain enzyme-negative variants of Staphylococcus were made as described previously4). To eliminate R-plasmids from gram-negative bacteria, the sodium dodecyl sulfate method was used.) Results Antimicrobial Activity of noglycosides The in vitro activity of aminoglycosidic antibiotics against gram-negative organisms, selected from our collection of fresh clinical isolates, are summarized in Table. The minimal inhibitory concentration (MIC) of the drugs against susceptible are similar to those reported by other investigators `). Resistant were found among bacterial species as outlined in Table. No strain of Salmonella, Shigella, Enterobacter and Citrobacter in the collection was observed to be resistant to the examined drugs. Resistance to gentamicin in these species, however, do occur in our area (see Table ). The susceptibility pattern of 4 Staphylococcus aureus is shown in Table 4. At the time of collection of these (January to March 7), no isolate resistant to gentamicin was observed. Since that time, however, gentamicin-resistant Staphylococcus aureus appeared in Zurich (see Table ). noglycoside Modifying Enzymes In Tables `7, data on tests with capable of modifying aminoglycoside antibiotics are shown. The enzymatic nomenclature and the abbreviations were taken from the Plasmid Nomenclature Group) and from the commission on Chemoresistance, International Society of Chemotherapy (S. MITSUHASHI, personal communication, Gunma University, Maebashi, Japan). The APH(') type 4 was recently found in Staph. aureus and Staph. epidermidis.,) This enzyme phosphorylates the hydroxyl group in position of ring I of aminoglycosides (see Fig. ). In contrast to the '-phosphotransferases of gram-negative organisms, amikacin, also, is phosphorylated and inactivated by the staphylococcal enzyme (see Table ). The drug, however, is a weak substrate, and producing APH(')-4, therefore, were phenotypically susceptible to amikacin. Some of staphylococci that were resistant or moderately resistant to amikacin (i.e. FK7) possessed an inherent mechanism of resistance still unknown. The APH(") was recently observed in gentamicin-resistant staphylococci in England.,4) The enzyme phosphorylates the hydroxyl group in position of ring III of aminoglycosides.) (see Fig. ) Strains producing APH(") exhibited resistance to gentamicin, sisomicin and tobramycin, and intermediate resistance to netilmicin and amikacin (see Table ). In addition to APH("), strain produced an acetylating enzyme with low affinity to tobramycin and amikacin.) The enzyme was still produced by the phosphotransferasenegative mutant. This is the reason for the rather high MIC of tobramycin and amikacin against the mutant strain (see Table ).

5 VOL. XXX NO. 8 THE JOURNAL OF ANTIBIOTICS Table. Summary of antimicrobial activity of five aminoglycoside antibiotics against grain-negative bacteria (Zurich, 7). Susceptible a Resistant Antibiotic tamicin omicin ramycin ilmicin kacin 4 87 % of Geometric mean of M ICS (,ug/ml) Range (hg/ml). `.. `.. `.. `..4 `. of % of Geometric mean of (,ug/ml) Range of (; eg/ml). ` >. ` >. ` >. ` > `> and netil- a Strains were regarded as susceptible, when the of gentamicin, sisomicin, tobramycin micin were. ug/ml or less, and of amikacin. ug/ml or less. Table. Summary of aminoglycoside-antibiotic resistant gram-negative organisms. Organism Escherichia coli Klebsiella spp. Serratia spp. Proteus mirabilis Proteus spp. (indole positive) Pseudomonas spp. Acinetobacter spp. and Alcaligenes spp. Total resistant to Strains were regarded as resistant, when the of gentamicin (), sisomicin (), tobramycin () and netilmicin () were.,ug/ml or greater, and of amikacin () leg/ml or more Table 4. Summary of antimicrobial activity of five aminoglycoside antibiotics against 4 Staphylococcus aureus (Zurich, 7). Susceptible a Resistant Antibiotics tamicin omicin ramycin ilmicin kacin % of.4 Geometric mean of (hg/ml) Range (ug/ml). `..7 `.. `.. `.. `. of % of. Geometric mean of (fug/ml) Range of (frg/ml) a Strains were regarded as susceptible, when the of gentamicin, sisomicin, tobramycin and netilmicin were. ug/ml or less, and of amikacin. #g/ml or less. The AAC(') type ) transfers the acetyl group from acetyl-coa to the amino group in position of ring I of aminoglycosides (see Fig. ). This enzyme was produced by the Proteus vulgaris strain HK. It efficiently acetylated and inactivated gentamicin, sisomicin, tobramycin and netilmicin, but not amikacin (see Table ). In contrast, the aminoglycoside -N-acetyltransferase7) was only capable of acetylating the deoxystreptamine moiety (ring II in Fig. ) of gentamicin and, slowly, of

6 THE JOURNAL OF ANTIBIOTICS AUG. 77 s (A) The structures of the kanamycins. Positions in ring I are numbered '-' (clockwise), in ring - (counterclockwise) and in ring III "-" (counterclockwise). Position Y is the ' position. The hydroxyl group in ring II is in position. rarnycin is '- deoxykanamycin B. kacin is I-hydroxybutyric acidkanamycin A. (B) The structures of the gentamicins. Numbering is as indicated for the kanamycins. The hydroxyl group in ring III is in position ". omicin is 4','-dehydrogentamicin Ca. ilmicin is -N-acetyl-4','- dehydrogentamicin Ca. (C) The structures of the neomycins. Positions are numbered as in the kanamycins. netilmicin. Strains producing this enzyme, therefore, were susceptible to tobramycin, netilmicin and amikacin (see Table ). Resistance to tobramycin and weak resistance to amikacin, but susceptibility to gentamicin and sisomicin was recently observed in Staph. epidermidis) and Staph. aureus.8) This unusual phenotype was due to the new aminoglycoside 4'-nucleotidyltransferase, adenylylating the OH-group in position 4 of ring I and also perhaps of ring III (see Fig. l) of tobramycin, amikacin and other drugs (U. SCHWOTZER, F. H. KAYSER and W. SCHWOTZER, in preparation). Since the gentamicins, sisomicin and netilmicin do not contain reactive groups in the corresponding position, were susceptible to these drugs (see Table 7). The aminoglycoside "-nucleotidyltransferase) is widely observed in gentamicin-resistant mem bers of Enterobacteriaceae and also in Pseudomonas aeruginosa. The enzyme adenylylates the hydroxyl group in position of ring III (see Fig. ). Accordingly, gentamicin, sisomicin, tobramycin and netilmicin were substrates for this enzyme, but amikacin was not. Strains producing this enzyme, however, were phenotypically susceptible to netilmicin. The low level of resistance of the Pseudomonas strain

7 VOL. XXX NO. 8 THE JOURNAL OF ANTIBIOTICS Table. Relation of aminoglycoside-phosphorylating enzymes and inactivation of antibiotics to MIC. Enzyme noglycoside '-phosphot ransferase type 4 [APH(')-4] [APH (")] Strain Staph. aureus FK 7 Staplt. aureus Antibiotica Neo Wild type strain > MIC (/cg/ml) noglycoside "-phosphotransferase Enzymenegative variant Inactivationb (zone diameter in tun) 8/8 / / / /4 / / /8 /7 / 8/ Extent of phosphorylation (cpm),,8,,8,,, Efficiency of phosphorylationd (%) a Abbreviations see legend of Table ; Neo=neomycin B. b Zone diameter of assayed sample/zone diameter of control sample. A value of mm means no inhibition zone around well. c Counts per minute incorporated and assayed after. hours incubation at C. d The percent is relative to the phosphorylation of neomycin or gentamicin. 8 7 Table. Relation of aminoglycoside-acetylating enzymes and inactivation of antibiotics to MIC. Enzyme noglycoside '-N-acctvItransferase type [AAC(')-] noglycoside -N-acetyltransferase type [AAC()] Strain Proteus vulgaris HK Proteus mirabilis HK8 Antibiotics Wild type strain >...8. MIC (ug/ml) Enzymenegative variant Inactivationb (zone diameter in mm) / / / 4 /8 4/4 / / /4 7/7 4/4 Extent of acetylationc (cpm),7,4,7 4,7, 4, 4, 48 Efficiency of acetylatio nd (%) a Abbreviations see legend of Table. b Zone diameter of assayed sample/zone diameter of control sample. A value of mm means no inhibition zone around well. c Counts per minute incorporated and assayed after. hours of incubation at ` C. d The percent is relative to the phosphorylation of gentamicin. N D =not done. 7 4 to netilmicin and amikacin (see Table 7) was due to a mechanism other than enzymatic modification, inherent in this strain. Variants, which had lost the ability to produce AAD("), did not differ from the wild type culture in the degree of resistance to netilmicin and amikacin. Discussion The antimicrobial activity of the five aminoglycoside antibiotics examined against susceptible are in agreement with other reports `). On the basis of micrograms per milliliter, amikacin

8 THE JOURNAL OF ANTIBIOTICS AUG. 77 Table 7. Relation of aminoglycoside-adenylylating activity and inactivation of antibiotics to MIC. Enzyme noglycoside 4'-nucleotidyltransferase [AAD(4')] [AAD(")] Strain Staph. epidermidis FK E. coli W77 (phjr) Pseudomonas aeruginosa HK MIC (,ag/ml) Wild type strain >.. noglycoside "-nucleotidyltransferase Antibiotics Enzymenegative variant Inactivationb (zone diameter in mm) / 7/8 / / /4 / /8 / / / 7/ /8 /8 / / a Abbreviations see legend of Table. b Zone diameter of assayed sample/zone diameter of control sample. C Counts per minute incorporated and assayed after. hours of incubation at C. d The percent is relative to the adenylylation of tobramycin or gentamicin. =not done. Extent of adenylylationc (cpm) 4,,,87,, 4,,4,,, Efficiency of adenylylationd (%) is the least active agent, especially against Staphylococcus aureus. However, this in vitro disadvantage may be offset clinically by the fact that amikacin reaches higher levels in human serum than the other drugs. kacin is the most effective drug against gentamicin-resistant, followed by netilmicin, sisomicin and tobramycin (see Tables and 4). This discrepancy can be partly explained by the differences in efficiency of these drugs as substrates of the aminoglycoside-modifying enzymes, and by the frequencies of the occurrence of the enzymes among aminoglycoside-resistant. In Tables ` 7, the activity of such enzymes in vitro and the phenotypic character of the corresponding strain are given. As can be seen, the data of test tube experiments correlated well with the susceptibility or resistance of some of the. In certain other cases, however, the results of experiments in vitro and in vivo were not in concurrence. The APH(')-4 of staphylococci, for instance, phosphorylated amikacin. Strains producing this enzyme, however, were phenotypically susceptible to the drug.) (Strain FK7 exhibits an unknown intrinsic mechanism of resistance to amikacin). A simple explanation would be that-because of a slow rate of phosphorylation)-sufficient molecules of the drug pass through the cell wall and membrane and reach the cytoplasm to exert an antibacterial effect. A similar cause could explain the susceptibility to netilmicin of producing the AAC (). In addition, the -N-acetylated netilmicin still had antimicrobial activity, since no inactivation in the microbiological assay was observed. It is more difficult to understand, why producing the AAD(") were susceptible to netilmicin (see Table 7). The enzyme isolated from the E. coli strain efficiently adenylylated the drug. In addition, inactivation in vitro also occurred, but the strain was highly susceptible to netilmicin. On the other hand, the strain of Pseudomonas aeruginosa was inhibited only by.!cg/ml of netilmicin. Enzyme-negative variants, however, did not differ in the level of resistance to this drug. The examples demonstrate that the resistant or susceptible phenotypic expression of bacterial do not depend exclusively on the presence or absence of aminoglycoside-modifying enzymes.

9 VOL. XXX NO. 8 THE JOURNAL OF ANTIBIOTICS The amount of such enzymes within the bacterial cell, the kinetic constants at the site of their location and the antibacterial activity of modified aminoglycosides may all play a significant role. In addition,, certain intrinsic mechanisms of resistance in these can contribute to resistance. Thus, an assessment of the degree of influence by the aminoglycoside-modifying enzymes on the phenotypic expression of a given strain can be made only by the comparative examination of the wild type strain and its corresponding enzyme-negative variant. As mentioned above, the differences in resistance of gram-negative to the five antibiotics (see Table ) are mainly due to the occurrence of various aminoglycoside-modifying enzymes. Out of randomly selected gentamicin-resistant cultures from our collection, nine produced the AAD(") and, all understandably were susceptible to netilmicin and amikacin. Five produced the AAC- (')-, which modifies all the drugs except amikacin. Only one strain produced the AAC(), rendering the strain resistant to gentamicin and sisomicin. To our knowledge, there is no enzyme known to inactivate selectively gentamicin only, and not sisomicin. The differences in the frequency of resistance against these two drugs are due to a slightly better antimicrobial activity of sisomicin. Thus, some with a low degree of resistance to gentamicin may show susceptibility to sisomicin. Acknowlegements These studies were supported by a grant from Schering Corporation. References ) PoRTHousr, A.; D. F. J. BROWN, R. G. SMITH & T. ROGERS: tamicin resistance in Staphylococcus aureus. Lancet 7-: `, 7 ) SMITH, A. L. &. D. H. SMITH: tamicin: adenine mononucleotide transferase: Partial purification, characterization and use in the clinical quantitation of gentamicin. J. Infect. Dis. : ` 4, 74 ) BAui.R, A. W.; W. M. M. KIRBY, J. C. SHERRIS & M. TURCK: Antibiotic susceptibility testing by a standardized single disk method. Amer. J. Clin. Pathol. 4: 4 `4, 4) Federal Register: Rules and Regulations. Antibiotic susceptibility discs. Fed. Resist. 7: `,7 ) Federal Register: Rules and Regulations. Antibiotic susceptibility discs-corrections. Fed. Regist. 8: 7, 7 ) Revised Tentative Standard. Performance standards for antimicrobial disc susceptibility tests, as used in clinical laboratories. National Committee for Clinical Laboratory Standards, Los Angeles, 7 7) ERICSSON, H. M. & J. C. SHERRIS: Antibiotic sensitivity testing. Report of an international Collaborative Study. Acta Path. Microbiol. Scand. Section B, Suppl. 7, 7 8) NOSSAE, N. G. & L. A. HLPPEL: The release of enzymes by osmotic shock from Escherichia coli in exponential phase. J. Biol. Chem. 4: `, ) SANTANAM, P. & F. H. KAYSER: ramycin adenylyltransferase: a new aminoglycoside-inactivating enzyme from Staphylococcus epidermidis. J. Infect. Dis. 4: S `S, 7 ) BENVENISTE, R.; T. YAMADA & J. DAVIES: Enzymatic adenylylation of streptomycin and spectinomycin by R-factor resistant Escherichia coli. Infect. Immun. : `, 7 ) DAMES, J.; M. BRZEZINSKA & R. BENVENISTE: R-Factors: biochemical mechanisms of resistance to aminoglycoside antibiotics. Ann. N.Y. Acad. Sci. 8: `, 7 ) HAAS. M. J. & J. DAVITS: Enzymatic acetylation as a means of determining serum aminoglycoside concentrations. Antimicr. Agents & Chemoth. 4: 47 `4, 7 ) BENNETT, J. V.; J. L. BRODIE, E. J. BENNER & W. M. M. KIRBY: Simplified, accurate method for antibiotic assay of clinical specimens. Appl. Microbiol. 4: 7 ` 77, 4) KAYSER, F. H.; J. WUEST & P. CORRODI: Transduction and elimination of resistance determinants in methicillin-resistant Staplrrlococcus aureus. Antimicr. Agents & Chemoth. : 7 `, 7 ) ADACHI, H.; M. NAKANO, M. INUZUKA & M. TOMEDA: Specific role of sex pili in the effective eliminatory action of sodium dodecyl sulfate on sex and drug resistance factors in Escherichia coli. J. Bacteriol. : 4-4, 7 ) BRIEDIS, D. J. & H. G. ROBSON: Comparative activity of netilmicin, gentamicin, amikacin and tobramycin against Pseudomonas aeruginosa and Enterobacterioceae. Antimicr. Agents & Chemoth. : ` 7,7

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