Nucleotide sequence of the aacC2 gene, a gentamicin resistance determinant involved in a hospital epidemic of multiply resistant members of the family Enterobacteriaceae.

A gentamicin resistance determinant of a conjugative plasmid from Enterobacter cloacae was cloned on a 3.2-kilobase fragment in the PstI site of pBR322. Substrate profiles for eight aminoglycosides at three concentrations showed that the resistance was due to aminoglycoside-(3)-N-acetyltransferase isoenzyme II. Insertion mapping by the gamma-delta transposon revealed that the size of the gene was approximately 1 kilobase. Nucleotide sequencing of the aacC2 gene identified an open reading frame of 858 base pairs, preceded by a promoter and a ribosome-binding site. From these data the molecular mass of the protein was calculated to be 30.6 kilodaltons. A comparison of the nucleotide sequence of the aacC2 gene with those published for the aacC3 and aacC4 genes showed complete homology of the aacC2 gene and the presumed aacC3 gene. An internal restriction fragment of the gene used as a probe in colony hybridization demonstrated the presence of the aacC2 gene in 86% of 86 multiply resistant isolates of the family Enterobacteriaceae obtained during an 18-month hospital epidemic. This corroborates our earlier data on the enzyme identification by the susceptibility profiling method.     

Cloning and expression in Escherichia coli of a gene encoding nonenzymatic chloramphenicol resistance from Pseudomonas aeruginosa.

High-level chloramphenicol resistance in Pseudomonas aeruginosa may be due to enzymatic inactivation, ribosomal mutation, or a permeability barrier. We investigated the nonenzymatic resistance mechanism encoded by Tn1696, a transposon found in P. aeruginosa. A 1-megadalton DNA fragment from Tn1696 was cloned which mediated expression of chloramphenicol resistance in Escherichia coli. Comparison of the effects of chloramphenicol on in vitro translation revealed no difference between the susceptible recipient strain and the resistant transformant containing the cloned gene. The rate of chloramphenicol uptake was slower in the resistant strain, suggesting a permeability barrier to the antibiotic. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membranes demonstrated the absence of a 50,000-dalton protein in the resistant strain. DNA homology was evident between Tn1696 and chloramphenicol-resistant isolates of Haemophilus influenzae possessing altered outer membrane permeability. We conclude that chloramphenicol resistance encoded by Tn1696 is due to a permeability barrier and hypothesize that the gene from P. aeruginosa may share a common ancestral origin with these genes from other gram-negative organisms.     

Cloning the gentamicin resistance gene from a Pseudomonas aeruginosa plasmid in Escherichia coli enhances detection of aminoglycoside modification.

Cloning the gene for gentamicin resistance from Pseudomonas aeruginosa plasmid pMG35 on the high-copy-number Escherichia coli cloning vehicle pMK20 allowed detection of 6'-N-acetyltransferase activity that was not readily detected when the parent plasmid was present either in P. aeruginosa or E. coli.     

Plasmid-mediated gentamicin resistance of Pseudomonas aeruginosa and its lack of expression in Escherichia coli.

We isolated 11 nonconjugative plasmids mediating resistance to aminoglycoside antibiotics, including gentamicin, from Pseudomonas aeruginosa strains. Their genetic properties were investigated in both P. aeruginosa and Escherichia coli transformants. The plasmid molecular weights ranged from 11 x 10(6) to 24 x 10(6). A low level or complete absence of gentamicin resistance was observed when these plasmids were introduced into E. coli, but gentamicin resistance was restored when the plasmids were transferred back to P. aeruginosa from E. coli. Aminoglycoside-modifying enzyme activity was detected in P. aeruginosa harboring these plasmids, but was absent or greatly reduced in E. coli strains. This lack of expression may explain the observed decrease in aminoglycoside resistance.     

Analysis of the aac(3)-VIa gene encoding a novel 3-N-acetyltransferase.

Biochemical analysis (G. A. Papanicolaou, R. S. Hare, R. Mierzwa, and G. H. Miller, abstr. 152, Program Abstr. 29th Intersci. Conf. Antimicrob. Agents Chemother., 1989) demonstrated the presence of a novel 3-N-acetyltransferase in Enterobacter cloacae 88020217. This organism was resistant to gentamicin, and the MIC of 2'-N-ethylnetilmicin for it was fourfold lower than that of 6'-N-ethylnetilmicin, a resistance pattern which suggested 2'-acetylating activity. However, high-pressure liquid chromatography analysis demonstrated that the enzyme acetylated sisomicin in the 3 position. We have cloned the structural gene for this enzyme from a large (> 70-kb) conjugative plasmid present in E. cloacae. Subcloning experiments have localized the aac(3)-VIa gene to a 2.1-kb Sau3A fragment. The deduced AAC(3)-VIa protein showed 48% amino acid identity to the AAC(3)-IIa protein and 39% identity to the AAC(3)-VII protein. Examination of the 5'-flanking sequences demonstrated that the aac(3)-VIa gene was located 167 bp downstream of the aadA1 gene and was present in an integron. In addition, the aac(3)-VIa gene is also downstream of a 59-base element often seen in an integron environment. Primer extension analysis has identified a promoter for the aac(3)-VIa gene downstream of both the aadA1 gene and a 59-base element.     

Diversity of determinants encoding carbenicillin, gentamicin, and tobramycin resistance in nosocomial Pseudomonas aeruginosa.

Plasmid pFMH1010, an 89-megadalton R plasmid, is endemic among members of the family Enterobacteriaceae at Hines Veterans Administration Hospital, Hines, Ill. It encodes resistance to nine antibiotics, including resistance to carbenicillin (Cb), gentamicin (Gm), and tobramycin (Tm). Pseudomonas aeruginosa strains resistant to carbenicillin, gentamicin, and tobramycin were isolated from five patients at Hines Veterans Administration Hospital from whom Serratia marcescens strains harboring pFMH1010 were also obtained. The P. aeruginosa strains were investigated to determine whether their Cb, Gm, and Tm characteristics derived from pFMH1010. One of the isolates, Ps559, was shown by Southern hybridization to contain approximately 76% of pFMH1010. Several lines of evidence suggested that the pFMH1010 sequences in Ps559 are integrated in the chromosome. Southern hybridization also demonstrated that the beta-lactam resistance of pFMH1010 is most probably due to the presence of sequences homologous with Tn3 and that these sequences are retained in Ps559. In two other Pseudomonas isolates, resistance to carbenicillin, gentamicin, tobramycin, and kanamycin was encoded by R plasmids unrelated to pFMH1010. In the last two isolates, resistance to gentamicin and tobramycin and several other antibiotics appeared to be chromosomally encoded, and it was rescuable from one of these strains by RP4-mediated mobilization.     

Nucleotide sequence analysis of a gene from Burkholderia (Pseudomonas) cepacia encoding an outer membrane lipoprotein involved in multiple antibiotic resistance.

Antibiotic-resistant Burkholderia (Pseudomonas) cepacia is an important etiologic agent of nosocomial and cystic fibrosis infections. The primary resistance mechanism which has been reported is decreased outer membrane permeability. We previously reported the cloning and characterization of a chloramphenicol resistance determinant from an isolate of B. cepacia from a patient with cystic fibrosis that resulted in decreased drug accumulation. In the present studies we subcloned and sequenced the resistance determinant and identified gene products related to decreased drug accumulation. Additional drug resistances encoded by the determinant include resistances to trimethoprim and ciprofloxacin. Sequence analysis of a 3.4-kb subcloned fragment identified one complete and one partial open reading frame which are homologous with two of three components of a potential antibiotic efflux operon from Pseudomonas aeruginosa (mexA-mexB-oprM). On the basis of sequence data, outer membrane protein analysis, protein expression systems, and a lipoprotein labelling assay, the complete open reading frame encodes an outer membrane lipoprotein which is homologous with OprM. The partial open reading frame shows homology at the protein level with the C terminus of the protein product of mexB. DNA hybridization studies demonstrated homology of an internal mexA probe with a larger subcloned fragment from B. cepacia. The finding of multiple antibiotic resistance in B. cepacia as a result of an antibiotic efflux pump is surprising because it has long been believed that resistance in this organism is caused by impermeability to antibiotics.     

Nucleotide sequence and characterization of erythromycin resistance determinant that encodes macrolide 2'-phosphotransferase I in Escherichia coli.

The DNA fragment (3.3 kb) containing the erythromycin resistance determinant was cloned from Escherichia coli Tf481A and sequenced. Deletion and complementation analyses indicated that the expression of high-level resistance to erythromycin requires two genes, mphA and mrx, which encode macrolide 2'-phosphotransferase I and an unidentified hydrophobic protein, respectively.     

Quinolone accumulation in Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus.

The accumulation of quinolones by Escherichia coli JF568, Pseudomonas aeruginosa PAO1, and Staphylococcus aureus ATCC 29213 was measured by a modified fluorometric assay (J. S. Chapman and N. H. Georgopapadakou, Antimicrob. Agents Chemother. 33:27-29, 1989). The quinolones examined were fleroxacin, pefloxacin, norfloxacin, difloxacin, A56620, ciprofloxacin, ofloxacin, and Ro 09-1168. In all three organisms, uptake was complete in less than 5 min and was proportional to extracellular quinolone concentrations between 2 and 50 micrograms/ml, which is consistent with simple diffusion. Washing cells with quinolone-free buffer decreased accumulation by up to 70% in E. coli and P. aeruginosa but not in S. aureus. Similarly, incubation with the uncouplers 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone increased accumulation up to fourfold in E. coli and P. aeruginosa, though not in S. aureus, suggesting endogenous, energy-dependent efflux. High quinolone hydrophobicity was generally associated with decreased accumulation in E. coli and P. aeruginosa (except in the case of pefloxacin) but was associated with increased accumulation in S. aureus (except in the case of difloxacin). Ciprofloxacin had the highest accumulation in E. coli and P. aeruginosa, while pefloxacin had the highest accumulation in S. aureus.     

Cloning and sequence of the gene encoding a novel cefotaxime-hydrolyzing beta-lactamase (CTX-M-9) from Escherichia coli in Spain

A new CTX-M-type beta-lactamase (CTX-M-9) has been cloned from a clinical cefotaxime-resistant Escherichia coli strain. Despite the close identity that exists between the CTX-M-9 and Toho-2 beta-lactamases (88%), the 35 amino acids located between residues Ala-185 and Ala-219 are totally different in both enzymes. Outside of this region there are only six amino acids substitutions between both proteins.     

Novel 3-N-aminoglycoside acetyltransferase gene,aac(3)-Ic,from a Pseudomonas aeruginosa integron

A novel gene, aac(3)-Ic, encoding an AAC(3)-I aminoglycoside 3-N-acetyltransferase, was identified on a gene cassette inserted into a Pseudomonas aeruginosa integron that also carries a bla(VIM-2) and a cmlA7 gene cassette. The aac(3)-Ic gene product is 59 and 57% identical to AAC(3)-Ia and AAC(3)-Ib, respectively, and confers resistance to gentamicin and sisomicin.     

Cloning and characterization of a 3-N-aminoglycoside acetyltransferase gene, aac(3)-Ib, from Pseudomonas aeruginosa.

A novel gene encoding an aminoglycoside 3-N-acetyltransferase, which confers resistance to gentamicin, astromicin, and sisomicin, was cloned from Pseudomonas aeruginosa Stone 130. Its sequence was determined and found to show considerable similarity to an aac(3)-I gene previously cloned from R plasmids from Enterobacter, Pseudomonas, and Serratia spp. We have designated the genes from the R plasmids and this work aac(3)-Ia and aac(3)-Ib, respectively. The two aac(3)-I genes share 74% nucleotide identity, and their deduced protein products are 88% similar. These data suggest that the genes derive from a common ancestor. Homology between the flanking sequences of both aac(3)-I genes and other resistance determinants known to reside in integron environments was also observed. Intragenic probes specific for either aac(3)-Ia or aac(3)-Ib were used in hybridization studies with a series of gentamicin-, astromicin-, and sisomicin-resistant clinical isolates. Of 59 clinical isolates tested, no isolates hybridized with both probes, 30 (51%) hybridized with the aac(3)-Ia probe, 12 (20%) hybridized with the aac(3)-Ib probe, and 17 (29%) did not hybridize with either probe. These data suggest the existence of at least one other aac(3)-I gene.     

Indole and (E)-2-hexenal, phytochemical potentiators of polymyxins against Pseudomonas aeruginosa and Escherichia coli.

Combinations of polymyxins and phytochemicals were tested for antimicrobial activity against two gram-negative bacteria. Various degrees of potentiation were found against Pseudomonas aeruginosa and Escherichia coli with (E)-2-hexenal and indole. Three-compound combinations were found to further increase the activity of polymyxin B sulfate and colistin methanesulfonate against both bacteria. Combinations with colistin against P. aeruginosa resulted in the highest degree of potentiation, with a 512-fold increase in colistin antimicrobial activity. These results indicate the potential efficacy of phytochemical combinations with antibiotics to enhance total biological activity.     

Expression in Escherichia coli of a New Multidrug Efflux Pump, MexXY, from Pseudomonas aeruginosa

Two new genes (mexXY) similar to mexAB, mexCD, and mexEF and mediating multidrug resistance were cloned from the chromosome of Pseudomonas aeruginosa. Elevated ethidium extrusion was observed with Escherichia coli cells harboring the plasmid carrying mexXY. This MexXY system confers higher resistance to fluoroquinolones than the MexAB and MexCD systems, and E. coli TolC or P. aeruginosa OprM is necessary for the function of the MexXY system.     

Cloning and expression of a tetracycline resistance determinant from Campylobacter jejuni in Escherichia coli.

The tetracycline resistance gene (tet) from the Campylobacter jejuni plasmid pFKT1025 was cloned into both pUC18 and pBR322 and was expressed when the chimeric plasmids were introduced into Escherichia coli. The location of the tet determinant on the chimeric plasmids was determined by BAL 31 deletion mapping within a 2.25-kilobase (kb) RsaI-HincII fragment. A protein of approximately 70 kilodaltons was consistently produced by E. coli maxicells harboring the cloned tet determinant. A 500-base-pair restriction fragment from within the 2.25-kb tet region was shown to hybridize only to DNA from tetracycline-resistant strains of C. jejuni and C. coli, but not to the DNA of organisms known to carry the streptococcal tetM determinant. No homology was noted between the DNA of 10 tetracycline-resistant isolates of campylobacter and the streptococcal tetL, tetM, or tetN determinants when tested under conditions of high stringency. However, homology was noted between a 5.0-kb HincII restriction fragment containing the tetM determinant and two C. jejuni tet R factors under conditions of reduced stringency.     

Isolation, characterization, and DNA sequence analysis of an AAC(6'')-II gene from Pseudomonas aeruginosa.

The gene encoding a 6'-N-acetyltransferase, AAC(6')-II, was cloned from Pseudomonas aeruginosa plasmid pSCH884. This gene mediates resistance to gentamicin, tobramycin, and netilmicin but not amikacin or isepamicin. The DNA sequence of the gene and flanking regions was determined. The 5'- and 3'-flanking sequences showed near identity to sequences found abutting a variety of different genes encoding resistance determinants. It is likely that the current structure arose by the integration of the 572-base-pair sequence containing the AAC(6')-II gene into a Tn21-related sequence at the recombinational hot spot, AAAGTT. We have compared the sequence of the AAC(6')-II gene to genes of other 6'-N-acetyltransferases. An AAC(6')-Ib protein (encoded by the aacA4 gene; G. Tran Van Nhieu and E. Collatz, J. Bacteriol. 169:5708-5714, 1987) that results in resistance to amikacin but not gentamicin was found to share 82% sequence similarity with the AAC(6')-II protein. We speculate that these two genes arose from a common ancestor and that the processes of selection and dissemination have led to the observed differences in the spectrum of aminoglycoside resistance.     

Detection of a second mechanism of resistance to gentamicin in animal strains of Escherichia coli.

One mechanism of plasmid-mediated resistance to gentamicin in Escherichia coli strains isolated from animals is due to the synthesis of the aminoglycoside 3-N-acetyltransferase type IV. A second mechanism of plasmid-mediated resistance to gentamicin was detected in animal strains of E. coli in France and is due to the production of the aminoglycoside 3-N-acetyltransferase type II. The molecular relationships among plasmids encoding this enzyme were studied.     

Sequence and characterization of a novel chromosomal aminoglycoside phosphotransferase gene, aph (3')-IIb, in Pseudomonas aeruginosa.

A novel, probably chromosomally encoded, aminoglycoside phosphotransferase gene was cloned on a 2,996-bp PstI fragment from Pseudomonas aeruginosa and designated aph (3')-IIb. It coded for a protein of 268 amino acids that showed 51.7% amino acid identity with APH (3')-II [APH(3') is aminoglycoside-3' phosphotransferase] from Tn5. Two other open reading frames on the cloned fragment showed homology to a signal-transducing system in P. aeruginosa.