Project description:A fifth gene cassette containing an aacC gene, aacCA5, was found in an aacCA5-aadA7 cassette array in a class 1 integron isolated from a multiply drug resistant Salmonella enterica serovar Kentucky strain. The AacC-A5 or AAC(3)-Ie acetyltransferase encoded by aacCA5 is related to other AAC(3)-I enzymes and confers resistance to gentamicin.
Project description:Class 1 integrons accumulate antibiotic resistance genes by site-specific recombination at aatI-1 sites. Captured genes are transcribed from a promoter located within the integron; for class 1 integrons, the first gene to be transcribed and translated normally encodes an aminoglycoside antibiotic resistance protein (either an acetyltransferase [AAC] or adenyltransferase [AAD]). The leader RNA from the Pseudomonas fluorescens class 1 integron contains an aminoglycoside-sensing riboswitch RNA that controls the expression of the downstream aminoglycoside resistance gene. Here, we explore the relationship between integron-dependent DNA recombination and potential aminoglycoside-sensing riboswitch products of recombination derived from a series of aminoglycoside-resistant clinical strains. Sequence analysis of the clinical strains identified a series of sequence variants that were associated with class I integron-derived aminoglycoside-resistant (both aac and aad) recombinants. For the aac recombinants, representative sequences showed up to 6-fold aminoglycoside-dependent regulation of reporter gene expression. Microscale thermophoresis (MST) confirmed RNA binding. Covariance analysis generated a secondary-structure model for the RNA that is an independent verification of previous models that were derived from mutagenesis and chemical probing data and that was similar to that of the P. fluorescens riboswitch RNA. The aminoglycosides were among the first antibiotics to be used clinically, and the data suggest that in an aminoglycoside-rich environment, functional riboswitch recombinants were selected during integron-mediated recombination to regulate aminoglycoside resistance. The incorporation of a functional aminoglycoside-sensing riboswitch by integron recombination confers a selective advantage for the expression of resistance genes of diverse origins.
Project description: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.
Project description:Integrons confer a rapid adaptation capability to bacteria. Integron integrases are able to capture and shuffle novel functions embedded in cassettes. Here, we investigated cassette recruitment in the Vibrio cholerae chromosomal integron during horizontal transfer. We demonstrated that the endogenous integrase expression is sufficiently triggered, after SOS response induction mediated by the entry of cassettes during conjugation and natural transformation, to mediate significant cassette insertions. These insertions preferentially occur at the attIA site, despite the presence of about 180 attC sites in the integron array. Thanks to the presence of a promoter in the attIA site vicinity, all these newly inserted cassettes are expressed and prone to selection. We also showed that the RecA protein is critical for cassette recruitment in the V. cholerae chromosomal integron but not in mobile integrons. Moreover, unlike the mobile integron integrases, that of V. cholerae is not active in other bacteria. Mobile integrons might have evolved from the chromosomal ones by overcoming host factors, explaining their large dissemination in bacteria and their role in antibioresistance expansion.
Project description:In this work, we demonstrate that the fosI gene encodes a predicted small protein with 134 amino acids and determines reduced susceptibility to fosfomycin. It raised the MIC from 0.125 to 6 μg/ml when the pBRA100 plasmid was introduced into Escherichia coli TOP10 and to 16 μg/ml when the gene was cloned into the pBC_SK(-) vector and expressed in E. coli TOP10.
Project description:A sixth gene cassette containing a dfrB-type gene, dfrB6, was found in a dfrB6-aadA1 cassette array in class 1 integrons. This array was isolated from several multiply antibiotic-resistant Salmonella enterica serovar Infantis strains that appear to be clonally related. The DfrB6 dihydrofolate reductase conferred resistance to trimethoprim.
Project description:An Achromobacter xylosoxydans strain showing broad-spectrum resistance to beta-lactams (including carbapenems) and aminoglycosides was isolated at the University Hospital of Verona (Verona, Italy). This strain was found to produce metallo-beta-lactamase activity and to harbor a 30-kb nonconjugative plasmid, named pAX22, carrying a bla(VIM-1) determinant inserted into a class 1 integron. Characterization of this integron, named In70, revealed an original array of four gene cassettes containing, respectively, the bla(VIM-1) gene and three different aminoglycoside resistance determinants, including an aacA4 allele, a new aph-like gene named aphA15, and an aadA1 allele. The aphA15 gene is the first example of an aph-like gene carried on a mobile gene cassette, and its product exhibits close similarity to the APH(3')-IIa aminoglycoside phosphotransferase encoded by Tn5 (36% amino acid identity) and to an APH(3')-IIb enzyme from Pseudomonas aeruginosa (38% amino acid identity). Expression of the cloned aphA15 gene in Escherichia coli reduced the susceptibility to kanamycin and neomycin as well as (slightly) to amikacin, netilmicin, and streptomycin. Characterization of the 5' and 3' conserved segments of In70 and of their flanking regions showed that In70 belongs to the group of class 1 integrons associated with defective transposon derivatives originating from Tn402-like elements. The structure of the 3' conserved segment indicates the closest ancestry with members of the In0-In2 lineage. In70, with its array of cassette-borne resistance genes, can mediate broad-spectrum resistance to most beta-lactams and aminoglycosides.
Project description:We characterized multidrug-resistant Pseudomonas aeruginosa strains isolated from patients involved in an outbreak of catheter-associated urinary tract infections that occurred in a neurosurgery ward of a hospital in Sendai, Japan. Pulsed-field gel electrophoresis of SpeI-, XbaI-, or HpaI-digested genomic DNAs from the isolates revealed that clonal expansion of a P. aeruginosa strain designated IMCJ2.S1 had occurred in the ward. This strain possessed broad-spectrum resistance to aminoglycosides, beta-lactams, fluoroquinolones, tetracyclines, sulfonamides, and chlorhexidine. Strain IMCJ2.S1 showed a level of resistance to some kinds of disinfectants similar to that of a control strain of P. aeruginosa, ATCC 27853. IMCJ2.S1 contained a novel class 1 integron, In113, in the chromosome but not on a plasmid. In113 contains an array of three gene cassettes of bla(IMP-1), a novel aminoglycoside resistance gene, and the aadA1 gene. The aminoglycoside resistance gene, designated aac(6')-Iae, encoded a 183-amino-acid protein that shared 57.1% identity with AAC(6')-Iq. Recombinant AAC(6')-Iae protein showed aminoglycoside 6'-N-acetyltransferase activity by thin-layer chromatography. Escherichia coli expressing exogenous aac(6')-Iae showed resistance to amikacin, dibekacin, isepamicin, kanamycin, netilmicin, sisomicin, and tobramycin but not to arbekacin, gentamicins, or streptomycin. Alterations of gyrA and parC at the amino acid sequence level were detected in IMCJ2.S1, suggesting that such mutations confer the resistance to fluoroquinolones observed for this strain. These results indicate that P. aeruginosa IMCJ2.S1 has developed multidrug resistance by acquiring resistance determinants, including a novel member of the aac(6')-I family and mutations in drug resistance genes.
Project description:A novel gene encoding an aminoglycoside 2'-N-acetyltransferase (AAC) was cloned from Mycobacterium fortuitum. DNA sequencing results identified an open reading frame that we have called aac(2')-Ib encoding a putative protein with a predicted molecular mass of 24,800 Da. The deduced AAC(2')-Ib protein showed homology to the AAC(2')-Ia from Providencia stuartii. This is the second member of a subfamily of AAC(2')-I enzymes to be identified. No homology was found with other acetyltransferases, including all of the AAC(3) and AAC(6') proteins. The aac(2')-Ib gene cloned in a mycobacterial plasmid and introduced in Mycobacterium smegmatis conferred resistance to gentamicin, tobramycin, dibekacin, netilmicin, and 6'-N-ethylnetilmicin. DNA hybridization with an intragenic probe of aac(2')-Ib showed that this gene was present in all 34 strains of M. fortuitum tested. The universal presence of the aac(2')-Ib gene in M. fortuitum was not correlated with any aminoglycoside resistance phenotype, suggesting that this gene may play a role in the secondary metabolism of the bacterium.