Biomedical subjects

M H Perlin

Publications and source records attributed to M H Perlin.

21 records · Page 2Linked to original sources

PubMed1982

Decreased susceptibility to 4'-deoxy-6'-N-methylamikacin (BB-K311) conferred by a mutant plasmid in Escherichia coli.

Escherichia coli MP6 contains a plasmid that encodes aminoglycoside 3'-phosphotransferase II, which phosphorylates kanamycin and confers high-level kanamycin resistance, Amikacin is a minor substrate of this enzyme, but MP6 is susceptible to amikacin. Strain MP10 has a spontaneous mutation in the plasmid of MP6 that increases the aminoglycoside 3'-phosphotransferase II activity not only against kanamycin but also against amikacin. This mutation is also responsible for the appearance of resistance to amikacin in MP10. Resistance to 4'-deoxy-6'-N-methylamikacin (BB-K311) by enzymatic modification has not been reported previously. As with amikacin, MP6 was susceptible to BB-K311 and its aminoglycoside 3'-phosphotransferase II did not phosphorylate this amikacin derivative appreciably. We found that the plasmid-borne mutation in MP10, however, localized by being cloned with a 3.7-megadalton HindIII fragment containing the aminoglycoside 3'-phosphotransferase II gene, resulted in increased phosphorylation of BB-K311 and resistance to it. Thus, the mutation distinguishing MP6 and MP10 has increased the activity of an existing aminoglycoside-modifying enzyme and produced new bacterial resistance to two previously minor substrates of the enzyme.

Amikacin↗

PubMed1981

Localization of an amikacin 3'-phosphotransferase in Escherichia coli.

A plasmid-encoded enzyme reported by us to phosphorylate amikacin in a laboratory strain of Escherichia coli has been localized in the bacterial cell. More than 88% of this amikacin phosphotransferase (APH) activity was retained in spheroplasts formed by ethylenediaminetetraacetate-lysozyme treatment of an APH-containing E. coli transconguant known to form spheroplasts readily. By comparison, the spheroplasts retained 94% of deoxyribonucleic acid polymerase I and 98% of glutamyl-transfer ribonucleic acid synthetase, two internal markers, whereas less than 10% of the activity of a periplasmic marker, acid phosphatase, was present in spheroplasts. Treatment of whole cells of the transconjugant with chemical probes incapable of crossing the plasma membrane obliterated acid phosphatase activity, whereas the internal markers deoxyribonucleic acid polymerase I, glutamyl-transfer ribonucleic acid synthetase, and beta-galactosidase were virtually unaffected after treatment for 5 min; more than 60% of the APH activity remained. As a control, similar chemical treatment of sonic extracts, in which enzymes were not protected by bacterial compartmentalization, produced more extensive reduction in the activities of all test enzymes, including APH. Spheroplasts preincubated with adenosine triphosphatase were shown by thin-layer chromatography to phosphorylate amikacin. Spheroplasts of cells grown in the presence of H(3) (32)PO(4) were shown to utilize internally generated adenosine 5'-triphosphate in the phosphorylation of amikacin. The absence of (32)P-phosphorylated amikacin after incubation of [gamma-(32)P]adenosine 5'-triphosphate with spheroplasts confirmed that exogenous adenosine 5'-triphosphate was not used in the reaction. These results suggest an internal location for APH. This conclusion has implications for the role of such enzymes in aminoglycoside resistance of gram-negative bacteria.

Adenosine Triphosphatases↗

PubMed1979

Amikacin resistance associated with a plasmid-borne aminoglycoside phosphotransferase in Escherichia coli.

Enzymatic phosphorylation of amikacin has not been reported previously in gram-negative bacteria. We found that extracts of MP1, a mutant of Escherichia coli JR66/W677 that is resistant to amikacin, were able to phosphorylate this aminoglycoside more rapidly than were extracts of the parental strain. Conjugal transfer of resistance from MP1 to a recipient strain was accompanied by acquisition in the transconjugants of amikacin phosphotransferase activity and of a 57-megadalton plasmid present in the donor. Partial purification of the phosphotransferase activity on amikacin-Sepharose 4B yielded an enzyme with a substrate spectrum similar to that of the 3'-neomycin-kanamycin phosphotransferase II found E. coli, except that it was also active against amikacin. A mutant of MP1, MP5, had increased susceptibility to amikacin and reduced phosphotransferase activity. MP9, a mutant MP5, was more resistant to amikacin and had increased phosphotransferase activity. The mutations leading to these alterations of amikacin susceptibility and amikacin phosphotransferase activity were transferable with the same plasmid that was associated with amikacin resistance and phosphotransferase activity in MP1. These studies demonstrate that resistance to amikacin in a laboratory strain of E. coli is due to an aminoglycoside phosphotransferase coded by a transferable plasmid-borne gene.

Amikacin↗