Development of two DNA probes for differentiating the structural genes of subclasses I and II of the aminoglycoside-modifying enzyme 3'-aminoglycoside phosphotransferase

Abstract

Two DNA probes were developed to screen for the genes encoding 3'-aminoglycoside phosphotransferase activity in gram-negative bacilli. The 3'-I phosphotransferase [APH(3')I] probe was subcloned from Tn903; the APH(3')II probe was subcloned from Tn5. Each probe proved to be specific for genes corresponding to its own APH(3') subclass and did not hybridize with DNA from other classes when tested at high stringency by either Southern hybridization or dot-blot hybridization methods. The APH(3')I probe hybridized to DNA obtained from organisms demonstrating APH(3')I activity as measured by the phosphocellulose paper binding assay (PPBA) as well as to DNA from organisms reported to have both APH(3')I and APH(3')II activity by PPBA. This probe did not hybridize to DNA from organisms showing only APH(3')II activity by PPBA. The APH(3')II probe demonstrated homology with DNA from organisms showing APH(3')II activity by PPBA but not with DNA from organisms showing APH(3')I activity or both APH(3')I and APH(3')II activity by PPBA. We conclude that organisms previously believed to contain both APH(3')I and APH(3')II genes based on PPBA contain in fact only the APH(3')I gene.

Correlation between aminoglycoside resistance profiles and DNA hybridization of clinical isolates

“…At this level of resistance, acetylation and adenylylation are partially eliminated (Tables 3 and 4). The APH(3')-Ia gene (8,30) (Fig. 2) observed in Serratia strains from Belgium, and the AAC(3)-Ia gene was found in 87% (107 of 123) of these strains (Fig.…”

“…The APH(3') AGRPs are characterized by resistance to kanamycin and neomycin (8,10,18,30). At least seven unique APH(3') genes, only some of which are distinguishable by extended resistance profiles to butirosin, lividomycin, amikacin, and gentamicin B (7, 11), have been genetically defined.…”

“…However, it is more difficult to characterize strains containing several aminoglycoside resistance genes solely on the basis of aminoglycoside resistance profiles (AGRPs), since often one resistance profile can partially duplicate and thereby mask the presence of an additional profile. DNA probes to the structural genes encoding several aminoglycoside-modifying enzymes, including AAC(3)-Ia (25), ANT(2")-Ia (24), APH(3')-Ia and APH(3')-Ib (30), AAC(3)-Va (3), and AAC(6')-Ic (14), have been developed. These DNA probes were useful in characterizing the dissemination of specific aminoglycoside resistance genes in hospital epidemics (3,5,13,14,19,24).…”

Molecular cloning of amikacin resistance determinants from a Klebsiella pneumoniae plasmid

“…The BamHI fragment specifying Ak in pJHC-T15.2 will be used as a probe for the localization of acetyltransferasemediated Ak in different bacterial species, as well as other K. pneumoniae strains. The development of probes for the identification of an 2"-O-adenylyltransferase and a phosphotransferase was recently reported (35,37 …”

Dispersal in Campylobacter spp. of aphA-3, a kanamycin resistance determinant from gram-positive cocci

“…In human pathogens, three types (I, II, and III) of APH(3') can be discriminated according to their in vitro substrate profiles (3,15,22), and the corresponding genes aphA-1, aphA-2, and aphA-3 do not cross hybridize (11,24).…”