In Vitro Comparison of Netilmicin, a Semisynthetic Derivative of Sisomicin, and Four Other Aminoglycoside Antibiotics

One hundred isolates of Pseudomonas and Enterobacteriaceae, of which 85 were chosen because of their resistance to gentamicin or amikacin, were tested for susceptibility to netilmicin (SCH 20569), a new semisynthetic derivative of sisomicin, and to four other aminoglycosides. Tests were performed in Mueller-Hinton agar and, with 43 of these isolates, also in Mueller-Hinton broth. Most isolates of Escherichia coli, Klebsiella, Enterobacter, Citrobacter, and Serratia that were gentamicin resistant proved to be susceptible to netilmicin and amikacin. Tests of representative isolates of this group showed that they owed their resistance to the production of aminoglycoside-adenylylating enzymes. Four isolates of Serratia, detected by their resistance to amikacin, were also highly resistant to netilmicin but were susceptible to gentamicin. These isolates produced aminoglycoside-acetylating enzymes. Gentamicin-resistant Proteus and Providencia were, in general, highly resistant to netilmicin but were susceptible to amikacin. These isolates also produced aminoglycoside-acetylating enzymes. Most gentamicin-resistant strains of Pseudomonas were resistant to netilmicin, either by enzymatic aminoglycoside modification or by other undefined mechanisms. Thus, like amikacin, netilmicin extends the aminoglycoside susceptibility pattern of Enterobacteriaceae to include gentamicin-resistant isolates that produce aminoglycoside-adenylylating enzymes. It is ineffective against strains, some of them susceptible to amikacin, gentamicin, or tobramycin, that produce aminoglycoside-acetylating enzymes. Images

Antibiotics. The aminoglycosides used in this study were laboratory standard preparations. Gen-139 tamicin (a mixture of approximately equal parts gentamicin Cl, C0a, and C2), sisomicin, and netilmicin were supplied by J. A. Waitz of the Schering Corp.; tobramycin was supplied by R. S. Griffith, Eli Lilly & Co.; amikacin (BB-K8), a semisynthetic kanamycin derivative, was obtained from G. E. Wright, Bristol Laboratories.
Quantitative determination of aminoglycoside susceptibility. The standard assay for microbial inhibition was performed with each of the five aminoglycosides by an agar dilution technique. For this purpose serial twofold dilutions of the aminoglycosides in duplicate Mueller-Hinton agar (BBL) plates were inoculated with 10-2 and 10-5 dilutions of an overnight growth at 37 C in Mueller-Hinton broth (Difco) (approximately 104 and 10' organisms) by means of a Steers-Foltz apparatus. The minimal inhibitory concentration (MIC) was defined as the lowest concentration of antibiotic that prevented any visible growth of the organisms after overnight incubation at 37 C. The MIC values would have been unchanged in 97% of the tests performed if the MIC end point had been defined as a 90% reduction in number of colonies formed. Tests performed with inocula of 104 and 10' organisms showed the same MIC of netilmicin, or one differing by only one dilution, with 85 of the 100 strains examined.
Twenty-five isolates of P. aeruginosa, one P. putida, and seventeen isolates of Enterobacteriaceae representative of the different susceptibility patterns shown in the agar dilution tests were assayed with gentamicin and netilmicin for MIC and for minimal bactericidal concentration (MBC) by a broth dilution procedure. For this purpose, serial twofold dilutions of aminoglycosides in duplicate tubes containing 1 ml of Mueller-Hinton broth were inoculated with 0.1 ml of a 10-3 dilution of an overnight growth (approximately 105 organisms). The MIC was defined as the lowest concentration ofantibiotic in those tubes showing no turbidity after 18 h of incubation at 37 C. MIC was also determined after 42 h. After 18 h of incubation, the contents of nonturbid tubes were subcultured with a 0.01-ml loop to antibiotic-free Mueller-Hinton agar. The MBC was defined as the lowest concentration of antibiotic that permitted the growth of 10 or fewer colonies.
Single lots ofMueller-Hinton broth and agar were used for these studies. Samples of each were analyzed for total Mg2+ and Ca2+ concentrations with an atomic absorption spectrophotometer by S. Natelson, Department of Biochemistry, Michael Reese Hospital. The agar contained (per liter) 63 mg of Ca2+ and 18 mg of Mg2+, whereas the broth contained 7 and 3 mg/liter, respectively.
To compare the effects of the different antibiotics in tests of each type (i.e., agar MIC, broth MIC or MBC), the results of each test were recorded separately as susceptible if the organisms were inhibited in an MIC test or killed in an MBC test by 5 Ag or less of gentamicin, tobramycin, sisomicin, or netilmicin per ml and by 10 ug or less of amikacin per ml. Strains that required 20 pg or more of the former antibiotics per ml or 40 ,ug of amikacin per ml for inhibition or killing were considered resistant. Organisms requiring 10 pLg of one of the former agents ANTIMICROB. AGzNTs CHEMOTHER.
per ml or 20 ,ug of amikacin per ml for inhibition or killing were considered intermediate. These values were selected on the basis of the therapeutic drug concentrations attained in serum by treatment with gentamicin, tobramycin, sisomicin, and amikacin. The resistant, susceptible, and intermediate values selected for netilmicin were the same as those for gentamicin and sisomicin, in view of the close chemical relation between these agents.
Enzymatic assays. From prior unpublished studies on 32 of the gentamicinor amikacin-resistant strains used in this study, information was available on results of adenylylation and acetylation assays by sonic extracts of the bacteria against aminoglycosides selected from the following: gentamicin C,, C1a, and C2, gentamicin A, sisomicin, tobramycin, amikacin, kanamycin A, kanamycin B, neomycin B, and paromomycin. The assays were performed according to published methods (1,2,6,9). Bacteria whose extracts gave at least five times the counts per minute of a similarly processed extract of a comparable antibiotic-susceptible strain were considered to acetylate or adenylylate the substrate significantly.

RESULTS
Enterobacteriaceae: MICs and MBCs. Aminoglycoside susceptibility pattems were similar among the 36 isolates ofE. coli, K. pneumoniae, and Enterobacter studied by the agar dilution method (Fig. 1). In general, the majority of the gentamicin-resistant isolates were susceptible to netilmicin and amikacin. They were resistant to tobramycin and were resistant or intermediate to sisomicin. Only 8% of the isolates were resistant to netilmicin, and 14% were resistant to amikacin. Fig. 2 shows that 24 of the 36 isolates of Enterobacteriaceae were resistant to gentamicin but susceptible to netilmicin, whereas only one isolate was resistant to netilmicin but susceptible to gentamicin. Five isolates that were either intermediate or resistant to gentamicin had similar MICs with netilmicin. The susceptibility of two gentamicin-resistant C. freundii isolates, not shown in Fig. 1, resembled the pattern predominant in this group of bacteria: these isolates were susceptible to netilmicin and amikacin.
Two patterns of susceptibility to aminoglyco- Proteus-Providencia organisms. Note that the scale is logarithmic. The boundary between the two shaded lines indicates results that differ by twofold. The boundary between the shaded and the unshaded zones indicates results that differ by fourfold. sides were found by agar dilution tests among isolates of Serratia marcescens (Fig. 3). Three isolates that were resistant to gentamicin were susceptible to netilmicin. Four isolates that were susceptible to gentamicin were resistant to netilmicin. One isolate was nearly equally susceptible to both agents. Seven  mycin and sisomicin (Fig. 1). The gentamicinresistant isolates were susceptible to amikacin, whereas the four gentamicin-susceptible isolates that were resistant to netilmicin were also resistant to amikacin. Most (Fig. 1). One P. mirabilis isolate was equally susceptible to all the agents tested.
Thirteen isolates of the E. coli, Klebisella, Enterobacter group, two of the Proteus isolates, and two of the Providencia isolates were tested by the tube dilution method. In 88% of the tests, MICs after 18 h of incubation at 37 C were identical to or only one dilution different from the agar dilution MIC (Fig. 4). There was 88 and 86% agreement (within one dilution) between 18-h and 42-h MICs and MBCs, respectively.
P. aeruginosa: MICs and MBCs. Nearly all of the gentamicin-resistant isolates were resistant or intermediate to netilmicin in agar dilution MIC tests (Fig. 3). Fifty percent of the isolates gave either identical or twofold different MICs with gentamicin and netilmicin whereas 22% gave fourfold different results. Nine (25%) isolates had greater than fourfold higher MICs with gentamicin, whereas only one (3%) had a greater than fourfold higher MIC with netilmicin. Amikacin was the most effective agent tested: only 55% were resistant or intermediate (Fig. 5). The superiority ofamikacin was enhanced if we compared the frequency of resistant isolates alone. Eight percent were resistant (MIC -40 ,zg/ml) to amikacin, compared with 81% to gentamicin, 92% to netilmicin, 72% to sisomicin, and 50% to tobramycin (MIC >20 ,ug/ml).
The MICs for the 25 isolates ofP. aeruginosa tested by the broth dilution method with gentamicin and netilmicin were generally lower than those obtained by the agar dilution method and enhanced the differences in MICs between those two agents (Fig. 4). Forty-eight percent of the isolates tested were scored as resistant to gentamicin, whereas only 8% were scored as resistant to netilmicin (Fig. 5). For isolates in which we obtained definite end points (i.e., -80 and ¢0.6 ,ug/ml), the mean ratio of the agar dilution MIC to the broth dilution MIC was 6.
Judged by the MBC test, most of these 25 isolates ofP. aeruginosa would be classified as resistant both to gentamicin and netilmicin ( Table 1). The MBCs were four-to eightfold higher than the tube dilution MICs. They were, however, comparable to the MIC values obtained by agar dilution. one isolate ofP. putida, MICs were 10, 20, and 40 ug of amikacin, tobramycin, and netilmicin per ml, respectively. By the broth dilution method, the MICs of gentamicin and netilmicin were 40 and <0.6 Ag/ml, respectively, for this isolate. The MBC for this isolate was 10 ug of netilmicin per ml.
Tests with organisms capable of modifying aminoglycosides. From prior unpublished work, supplemented by some determinations on netilmicin, we had data on the aminoglycoside-adenylylating and -acetylating activity of 32 strains from the group submitted for susceptibility determinations. The strains had been selected for assay of these enzymatic activities as part of an investigation of mechanisms of gentamicin resistance. The available data gave evidence of a correlation between the enzymatic activities of the isolates examined and the results of the susceptibility tests.
The 10 Enterobacteriaceae isolates that were resistant or intermediate to gentamicin and produced the enzyme AAD(Z') were susceptible to netilmicin and amikacin ( Table 2). The four gentamicin-resistant isolates of P. aeruginosa that also produce this enzyme had netilmicin MICs that were only 10 to 20 ,ug/ml (resistant or intermediate) ( Table 2) and, in addition, by the broth dilution tests had MICs of <0.6 to 1.2 ,ug/ml (susceptible). All but one of the 14  nylylating isolates included 7 of the 24 Enterobacteriaceae that were resistant to gentamicinand susceptible to netilmicin in Fig. 2 and 4 of the 9 Pseudomonas isolates in Fig. 3 that had greater than fourfold higher MICs with gentamicin than with netilmicin. The remaining 17 Enterobacteriaceae and 5 Pseudomonas isolates in these two groups were not assayed, but we think it likely that they also produced AAD(Z'). Other adenylylating isolates include C. freundii strain WIL and two of the three Serratia shown in Fig. 3 that were resistant to gentamicin and susceptible to netilmicin.
In contrast to the nearly uniform resistance of the acetylating strains to the aminoglycosides cited above, three Providencia strains and one Proteus strain were susceptible to amikacin, and one Proteus and two Pseudomonas isolates were intermediate. The r.maining acetylating isolates were resistant to amikacin, and most of them produced enzymes either known to be AAC(6')-4 or closely resembling it. AAC(6')-4 is the only enzyme known to acetylate amikacin. Five isolates that produced this enzyme were susceptible to gentamicin. They included four isolates of Serratia that were resistant to netilmicin (Fig. 3) and one strain of E. coli K-12 carrying R5 (Fig. 2). In addition, P. aeruginosa GN315, which had a higher MIC to  netilmicin than to gentamicin, carried this enzyme (Fig. 3). AAC(6')4 does not acetylate gentamicin C,, a component ofthe gentamicin complex. The unimpaired activity of gentamicin C,, together with possible residual antibiotic activity of the acetylated components, may account for the effectiveness of gentamicin against these organisms (4). Three isolates resistant to netilmicin (one P.
aeruginosa strain Kru, one E. coli, and one K.
pneumoniae) had previously been found not to acetylate or adenylylate gentamicin. In contrast to the organisms that were resistant by virtue of their enzymatic modification ofaminoglycosides, these isolates were uniformly resistant to all five of the aminoglycosides tested, with MICs of nearly identical magnitude. Resistance in these isolates may be caused by restricted access of these agents to their site of action, as has been demonstrated for streptomycin resistance in some strains ofP. aeruginosa (13).
DISCUSSION Our data suggest that many gentamicin-resistant E. coli, Klebsiella, Enterobacter, Citrobacter, and Serratia were susceptible to netilmicin and to amikacin. Gentamicin-resistant Proteus and Providencia were, in general, resistant to netilmicin but were susceptible to amikacin. Amikacin-resistant Serratia were also resistant to netilmicin but were susceptible to gentamicin.
P. aeruginosa and the other Pseudomonas isolates occupy a special position, because their MICs varied with experimental conditions. A systematic difference between broth and agar dilution MIC has been observed with P. aeruginosa and other aminoglycosides, attributed to the higher concentration of the divalent cations Mg2+ and Ca2+ in Mueller-Hinton agar than in Mueller-Hinton broth (7,12). Since the concentration of these cations in agar more closely approximates that of serum, it has been suggested that the data obtained in agar may be of more clinical significance (7,12 Abstr. 93, 1975).
In general, strains harboring a gentamicinadenylylating enzyme were resistant to gentamicin but susceptible to netilmicin. In our experience, these enzymes have been found most frequently in E. coli, Klebsiella, Enterobacter, and Citrobacter. Thus, it seems likely that netilmicin may prove to be a therapeutically useful agent against many isolates of these genera. The P. aeruginosa isolates that harbored adenylylating enzymes conceivably might respond to therapy with netilmicin, especially if found in the urine, since in 80% of the isolates, MICs did not exceed 20 ,ug/ml, even by agar dilution.
Preliminary experiments in our laboratory indicate that netilmicin is not refractory to attack by AAD-(2'), when tested at saturating concentrations of substrate. A possible remaining mechanism for the susceptibility of adenylylating bacteria to this antibiotic is a low affinity of netilmicin for AAD-2(2") at concentrations comparable to the MIC for susceptible bacteria. Confirming preliminary observations of J. A. Waitz (personal communication), we have found that at least one adenylylating enzyme, AAD-(2") from E. coli K-12 carrying JR66, adenylylated netilmicin at concentrations of 1 to 5 Ag/ml to an appreciably lesser extent than gentamicin C1 or the gentamicin complex. An additional mechanism, as yet untested, is that the adenylylated netilmicin retains antibiotic activity. A number of varieties of aminoglycoside acetyltransferases with somewhat different substrate specificities are known to exist in bacteria (4). From our observations and those of others, it seems likely that our collection included at least three different varieties of acetyltransferase. The bacteria that harbored any of these enzymes were, in general, highly resistant to netilmicin. In contrast to the results with the adenylyltransferase enzyme, our preliminary experiments indicate that netilmicin at concentrations of 1 to 5 ,ug/ml is acetylated, as well as gentamicin C, or Cla, by each of the three varieties of acetyltransferases encountered in our collection. Judging by the isolates we have examined, resistance to netilmicin owing to acetyltransferase activity is relatively infrequent in Escherichia, Klebsiella, and Enterobacter, but is common in Proteus, Providencia, and Pseudomonas.
Thus, it appears that netilmicin, like amikacin, may prove effective against Enterobacteriaceae and possibly some Pseudomonas strains that are resistant to gentamicin owing to aminoglycoside-adenylylating enzymes. The therapeutic ineffectiveness of netilmicin against bacteria possessing aminoglycosideacetylating enzymes may be mitigated by the susceptibility of some of these bacteria to amikacin and, in a few instances, to gentamicin and tobramycin. ACKNOWLEDGMENTS This study was supported by a grant from the Schering