The application of molecular techniques for the study of aminoglycoside resistance.

Aminoglycosides have been cinically used since 1944. Although this class of antibacterial agents has some nephrotoxicity and ototoxtcity issues, they continue to be part of the hospital armamentarium because of then rapid bactericidal activity, especially in combination with ß-lactams. Bacterial resistance to aminoglycosides can be caused by modifying enzymes, changes in cell permeability, and changes in the cellular target. The clinical observation of high levels of aminoglycoside resistance most often results from the acquisition of genes that encode modifying enzymes and are often plasmid-borne. Aminoglycosides are inactivated by three classes of enzymes:

Microbiological and pharmacokinetic studies of acyl demycinosyltylosin and related tylosin derivatives.

A series of tylosins and acyl derivatives of 23-O-demycinosyltylosin (DMT) were initially tested for in vitro antibacterial activity and serum levels in squirrel monkeys (po) and mice (iv). Overall, the DMT compounds were more active in vitro than the tylosins. Two tetraacylated DMTs, Sch 37644 and Sch 38646, were selected from the initial studies for further evaluation and compared to erythromycin and A-56268 (6-O-methyl erythromycin). Sch 37644 and Sch 38646 were 2 to 8-fold less potent in vitro against Gram-positive bacteria than erythromycin and A-56268. In squirrel monkeys, Sch 37644 (AUC, 19.7 micrograms.hour ml) and A-56268 (21.6 micrograms.hour/ml) had similar serum levels following po administration of 20 mg/kg, while Sch 38646 (11.8 micrograms.hour/ml) and erythromycin (1.5 micrograms.hour/ml) had lower levels. In mice administered 200 mg/kg orally, Sch 37644 (AUC, 19.4 micrograms.hour/ml) and Sch 38646 (15.4 micrograms.hour/ml) had higher serum levels than erythromycin (5.7 micrograms.hour/ml). A-56268 was the most active po macrolide in mouse protection studies (PD50S) against Staphylococci and Streptococci, while Sch 37644 and Sch 38646 were similar to erythromycin.

The changing nature of aminoglycoside resistance mechanisms and the role of isepamicin--a new broad-spectrum aminoglycoside. The Aminoglycoside Resistance Study Groups.

Aminoglycoside resistance mechanisms from recent studies were compared with those found in earlier studies in the USA and Europe for three pathogen groups. Among Citrobacter-Enterobacter-Klebsiella, four single mechanisms (AAc(3)-II, AAC(3)-I, ANT(2")-I and AAC(6')-I were found in all studies, but the most recent studies showed a significant increase in combinations of AAC(6')-I with the other common mechanisms. Since AAC(6')-I confers resistance to tobramycin, netilmicin and amikacin, combinations of it with the other gentamicin modifying enzymes conferred broad-spectrum resistance to all clinically available aminoglycosides except isepamicin. Similar changes occurred in Escherichia-Morganella-Proteus-Salmonella-Shigella except that the frequency of combinations was much lower and two additional single mechanisms - AAC(3)-IV and permeability - were also found frequently. Among aminoglycoside-resistant Pseudomonas, three mechanisms, AAC(6')-II, ANT(2")-I and permeability, were always common and remained common. However, combinations of the three mechanisms with each other and with other mechanisms were more common in the recent surveys. Different genes which produce different proteins with the same aminoglycoside-modifying activity are now known. The results of hybridisation studies with two aac(3)-I, 2 aac(6')-II and 4 aac(6')-I gene probes are presented. The most commonly occurring genes were: aac(3)-Ia, aac(3)-IIa, aac(6')-IIa, aac(6')-Ib and, in Serratia, aac(6')-Ic. The activity of isepamicin against amikacin resistant strain which produce AAC(6')-I can be related to differences in the structure of these two similar aminoglycosides at Position 3". Amikacin may form a stable complex with AAC(6')-I enzymes via binding interaction at Position 3 and 3". Isepamicin, which has a secondary amino group at Position 3", may only be able to interact at Position 3 and enzyme-isepamicin complexes are likely to be less stable.

In-vitro activity of Sch 34343 against Gram-negative bacteria producing characterized beta-lactamases.

The activity of Sch 34343 against 13 strains producing large amounts of characterized beta-lactamases was compared with that of imipenem, latamoxef (moxalactam), aztreonam and other third-generation cephalosporins. Sch 34343, like imipenem, was active against all strains, including many resistant to all other beta-lactams. MICs of Sch 34343 determined for 16 different inocula were rarely increased even at very high inocula. Sch 34343 was rapidly bactericidal against Escherichia coli TEM-2, Enterobacter agglomerans (with an induced beta-lactamase) and two strains of Bacteroides fragilis with highly active cephalosporinases. Like cefoxitin, Sch 34343 was only slowly inactivated by concentrated crude penicillinases which inactivated cefotaxime within 1 h. Sch 34343 was even more stable to cephalosporinases than was cefoxitin. Stability of the antibiotics to the different beta-lactamases was also determined by pre-incubating them with dilutions of the beta-lactamases before determination of MICs against E. coli 25922. Very large amounts of all enzymes were required to increase the MICs significantly for Sch 34343 and imipenem. These results indicate the good stability of Sch 34343 to beta-lactamases.

Evaluation of the in-vitro antibacterial activity of Sch 34343.

The in-vitro activity (as measured by geometric mean MICs, mg/l) of Sch 34343 against aerobic and anaerobic bacteria was compared with that of 14 other selected beta-lactam antibiotics including aztreonam, latamoxef (moxalactam), ceftazidime and imipenem. Sch 34343 had good activity (less than 2 mg/l) against most Gram-negative aerobic bacteria whether or not they contained high levels of plasmid-mediated or chromosomally-mediated beta-lactamases. It was slightly less potent against strains of Morganella and Serratia (less than 4 mg/l) and inactive against Pseudomonas (greater than 64 mg/l). A very small inoculum effect was observed against strains containing beta-lactamases indicating stability. Unlike the third-generation cephalosporins, Sch 34343 had excellent activity (less than or equal to 0.18 mg/l) against staphylococci, comparable to that of imipenem and ampicillin. While Sch 34343 had equally good potency (0.17 mg/l) against penicillinase-positive staphylococci, it was inactive against methicillin-resistant staphylococci (greater than or equal to 35 mg/l). Sch 34343 also had good activity against streptococci. The most unusual aspect of the in-vitro activity was its activity against Bacteroides (including Bact. fragilis) and other anaerobes. Sch 34343 had mean MICs less than or equal to mg/l for all Bacteroides and Clostridium spp. tested except CI. difficile (3.4 mg/l).

Correlation between aminoglycoside resistance profiles and DNA hybridization of clinical isolates.

DNA hybridization data and aminoglycoside resistance profiles (AGRPs) were determined for 4,088 clinical isolates from three studies (United States, Belgium, and Argentina). The correlation between susceptibility profiles and hybridization results was determined with nine DNA probes. For each of the seven aminoglycoside resistance profiles which we were able to test, the data suggested at least two distinct genes could encode enzymes which lead to identical resistance profiles. Furthermore, the DNA hybridization data showed that individual strains carried up to six unique aminoglycoside resistance genes. DNA hybridization revealed interesting differences in the frequencies of these genes by organism and by country.

Synthesis and antibacterial activity of 2-alkoxy penems.

The phosphite mediated Oxalimide cyclization reaction was extended to 4-dithiocarbonates of N-oxalyl-2-azetidinones to synthesize 2-alkoxy penems 3. In general, the in vitro antibacterial potency of compounds 3 was weak compared to the highly potent 2-alkylthiopenems 2.