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A new rifampin resistance gene, arr-2, has been found in Pseudomonas aeruginosa. The ARR-2 protein shows 54% amino acid identity to the rifampin ADP-ribosylating transferase encoded by the arr gene from Mycobacterium smegmatis. This arr-2 gene is located on a gene cassette within a class I integron.
A conjugative IncL/M plasmid (pSEM) conferring resistance to gentamicin, amikacin, kanamycin, sulfonamides, and expanded-spectrum cephalosporins was found in pathogenic strains of Salmonella enterica serotype Typhimurium. Resistance to aminoglycosides was encoded by a sul1-type class 1 integron (In-t3). An extended-spectrum beta-lactamase gene, bla(SHV-5), was identified 3. 5 kb downstream of the integrase (intI1) gene of In-t3. Nucleotide sequence analysis of the 5.3-kb bla(SHV-5)-In-t3 region of pSEM highlighted striking similarities with IncL/M plasmids isolated from nosocomial gram-negative pathogens, conferring resistance to expanded-spectrum cephalosporins and aminoglycosides.
In a previous survey of 357 trimethoprim-resistant isolates of aerobic gram-negative bacteria from commensal fecal flora, hybridization experiments showed that 25% (90 of 357) of the isolates failed to hybridize to specific oligonucleotide probes for dihydrofolate reductase types 1, 2b, 3, 5, 6, 7, 8, 9, 10, and 12. Subsequent cloning and sequencing of a plasmid-borne trimethoprim resistance gene from one of these isolates revealed a new dihydrofolate reductase gene, dfr13, which occurred as a cassette integrated in a site-specific manner in a class 1 integron. The gene product shared 84% amino acid identity with dfr12 and exhibited a trimethoprim inhibition profile similar to that of dfr12. Gene probing experiments with an oligonucleotide probe specific for this gene showed that 12.3% (44 of 357) of the isolates which did not hybridize to probes for other dihydrofolate reductases hybridized to this probe. Immediately downstream of dfr13, a new cassette, an aminoglycoside resistance gene of the class AADA ¿ANT(3")(9)-I, which encodes streptomycin-spectinomycin resistance, was identified. This gene shares 57% identity with the consensus aadA1 (ant(3")-Ia) and has been called aadA4 (ant(3")-Id). The 3' end of the aadA4 cassette was truncated by IS26, which was contiguous with a truncated form of Tn3. On the same plasmid, pUK2381, a second copy of IS26 was associated with sul2, which suggests that both integrase and transposase activities have played major roles in the arrangement and dissemination of antibiotic resistance genes dfr13, aadA4, bla(TEM-1), and sul2.
A transferable beta-lactamase produced by a multidrug-resistant clinical isolate of Enterobacter cloacae was studied. The bla gene was carried by a large (>80-kb) transmissible plasmid. Nucleotide sequence analysis of cloned fragments revealed that it was part of a gene cassette carried by a class 1 integron along with other resistance genes, including aac(6')-Ib. The encoded beta-lactamase, designated IBC-1, was a novel class A enzyme that hydrolyzed ceftazidime and cefotaxime and was inhibited by tazobactam and, to a lesser extent, by clavulanate. Also, imipenem exhibited potent inhibitory activity against IBC-1. The enzyme consisted of 287 amino acid residues, including Ser-237, cysteines at positions 69 and 237a, and Arg-244, which may be implicated in its interaction with beta-lactams. In amino acid sequence comparisons, IBC-1 displayed the highest similarity with the chromosomal penicillinase of Yersinia enterocolitica, a carbenicillinase from Proteus mirabilis GN79, the species-specific beta-lactamases of Klebsiella oxytoca, and the carbapenemase Sme-1. However, a phylogenetic association with established beta-lactamase clusters could not be conclusively shown.
Two clonally unrelated Pseudomonas aeruginosa clinical strains, RON-1 and RON-2, were isolated in 1997 and 1998 from patients hospitalized in a suburb of Paris, France. Both isolates expressed the class B carbapenem-hydrolyzing beta-lactamase VIM-2 previously identified in Marseilles in the French Riviera. In both isolates, the bla(VIM-2) cassette was part of a class 1 integron that also encoded aminoglycoside-modifying enzymes. In one case, two novel aminoglycoside resistance gene cassettes, aacA29a and aacA29b, were located at the 5' and 3' end of the bla(VIM-2) gene cassette, respectively. The aacA29a and aacA29b gene cassettes were fused upstream with a 101-bp part of the 5' end of the qacE cassette. The deduced amino acid sequence AAC(6')-29a protein shared 96% identity with AAC(6')-29b but only 34% identity with the aacA7-encoded AAC(6')-I1, the closest relative of the AAC(6')-I family enzymes. These aminoglycoside acetyltransferases had amino acid sequences much shorter (131 amino acids) than the other AAC(6')-I enzymes (144 to 153 amino acids). They conferred resistance to amikacin, isepamicin, kanamycin, and tobramycin but not to gentamicin, netilmicin, and sisomicin.
A multidrug-resistant Salmonella enterica serotype [4,5,12:i:-] clone carried a class 1 integron harboring dfrA12 and aadA2 gene cassettes and bla(TEM-1), aac(3)-IV, cmlA1, and tetA genes located in large plasmids of about 140 kb (carrying spv) or 120 kb (lacking spv). Several segregants, lacking multidrug resistance, contained a plasmid smaller than the parental one and no longer hybridized with probes for the lost resistances. The genes mediating resistance to ampicillin, chloramphenicol, and tetracycline in the [4,5,12:i:-] clone are different from those found in the pentadrug-resistant serotype Typhimurium DT104 clone.
Integrons were detected in 59 of 120 (49%) urinary isolates of Enterobacteriaceae by PCR using degenerate primers targeted to conserved regions of class 1, 2, and 3 integrase genes. PCR sequencing analysis of the cassette arrays revealed a predominance of cassettes that confer resistance to the aminoglycosides and trimethoprim.
Pseudomonas aeruginosa clinical isolate CY-1, which was resistant to ceftazidime, harbored a conjugative ca. 250-kb plasmid that contained a class 1 integron with two gene cassettes encoding OXA-32, an OXA-2- type beta-lactamase, and the aminoglycoside acetyltransferase AAC(6')Ib(9). OXA-32 differed from OXA-2 by an Leu169Ile amino acid substitution (class D numbering). Site-directed mutagenesis established that Ile169 is responsible for resistance to ceftazidime but not to cefotaxime.
Nineteen of 27 ceftazidime-resistant Enterobacter cloacae isolates from a neonatal intensive care unit in Thessaloniki, Greece, had genes coding for the novel extended-spectrum beta-lactamase IBC-1; 18 of those 19 harbored similar conjugative plasmids and belonged to two distinct genetic lineages. A synergy test with ceftazidime and imipenem enabled us to identify five unrelated bla(IBC-1)-carrying E. cloacae isolates from other wards of the hospital. It seems that this integron-associated gene is capable of dispersing both by clonal spread and by gene dissemination.
The genetic organization of the region coding for CTX-M-2 in Salmonella enterica serovar Infantis was determined by PCR mapping. This gene seems to have been mobilized from the Kluyvera ascorbata chromosome to a complex sulI-type integron, similar to In6 and In7.
Examination of the bla(CTX-M-2) gene in plasmid pMAR-12 by sequencing and PCR analysis revealed that the bla gene and the surrounding DNA, which is closely related (99% homology) to the Kluyvera ascorbata chromosomal DNA that contains the bla(KLUA-1) gene, are located in a complex sul1-type integron, termed In35, that includes Orf513. It is possible that bla(CTX-M-2) was acquired by plasmid pMAR-12 through an uncharacterized recombinational event in which Orf513 could be involved.
The carbenicillin, gentamicin, kanamycin, streptomycin, spectinomycin, sulfonamide, and tobramycin resistance determinants found on Pseudomonas aeruginosa plasmid R151 have previously been shown to translocate to another plasmid, R388, and it was inferred that a transposon, Tn1404, carried the resistance determinants. Sequencing of the cassette array from the plasmid known as R388::Tn1404 revealed two known gene cassettes, oxa10 and aadB, and a previously unidentified cassette determining resistance to streptomycin and spectinomycin, here designated aadA10, in the order oxa10-aadB-aadA10. These cassettes replaced the dfrB2-orfA cassette array of R388, indicating that movement of the resistance determinants from R151 to R388 resulted from recombinational exchange between two class 1 integrons rather than transposition. The AadA10 protein is most closely related to AadA6 (85% identical) and AadA7 (80% identical). The aadA10 cassette found here has only a simple site containing a 7-bp spacer derived from attI1 in place of a 59-base element and is likely to represent a derivative of the complete cassette. IntI1-mediated deletion of the aadA10 cassette was not detected, indicating that this single simple site is either inactive or only weakly active.
Integron In2 integrase (IntI1)-mediated site-specific recombination between two primary sites occurs at a high frequency, while that between a primary and a secondary site occurs at frequencies around 10,000 times lower. Secondary sites consist of a pentanucleotide with only two fully conserved residues (GWTMW). The analysis of IntI1-mediated recombinants in the plasmid pOX38 revealed the existence in this plasmid of a site used at a frequency intermediate between those of primary and secondary sites. Analysis of this site showed two potentially relevant structural features: first, a set of two consensus pentanucleotides, separated by 5 bp and in opposite orientations, forming what will be called a double site; and second, a longer sequence with some extent of sequence symmetry with the double site at its 3' end. A recombinant plasmid, pSU18P, containing a double site was constructed. Examination of R388-pSU18P recombinants showed that double sites were used preferentially over single pentanucleotides by IntI1. Comparisons of the nucleotide sequences of known 59-bp elements showed that in most cases there was a double site at each element end. Mutagenesis of the F hot spot was carried out to make it look more like the consensus 59-bp element. The improved sites showed recombination frequencies and specificities almost comparable to those observed at IntI1 primary sites.
IntI1 integrase is a member of the prokaryotic DNA integrase superfamily. It is responsible for mobility of antibiotic resistance cassettes found in integrons. IntI1 protein, as well as IntI1-COOH, a truncated form containing its carboxy-terminal domain, has been purified. Electrophoretic mobility shift assays were carried out to study the ability of IntI1 to bind the integrase primary target sites attI and aadA1 attC. When using double-stranded DNA as a substrate, we observed IntI1 binding to attI but not to attC. IntI1-COOH did not bind either attI or attC, indicating that the N-terminal domain of IntI1 was required for binding to double-stranded attI. On the other hand, when we used single-stranded (ss) DNA substrates, IntI1 bound strongly and specifically to ss attC DNA. Binding was strand specific, since only the bottom DNA strand was bound. Protein IntI1-COOH bound ss attC as well as did the complete integrase, indicating that the ability of the protein to bind ss aadA1 attC was contained in the region between amino acids 109 and 337 of IntI1. Binding to ss attI DNA by the integrase, but not by IntI1-COOH, was also observed and was specific for the attI bottom strand, indicating similar capabilities of IntI1 for binding attI DNA in either double-stranded or ss conformation. Footprinting analysis showed that IntI1 protected at least 40 bases of aadA1 attC against DNase I attack. The protected sequence contained two of the four previously proposed IntI1 DNA binding sites, including the crossover site. Preferential ssDNA binding can be a significant activity of IntI1 integrase, which suggests the utilization of extruded cruciforms in the reaction mechanisms leading to cassette excision and integration.
In the 1996-1997 cholera epidemic in Guinea-Bissau, surveillance for antimicrobial resistance showed the emergence of a multidrug-resistant strain of Vibrio cholerae O1 during the course of the epidemic. The strain was resistant to ampicillin, erythromycin, tetracycline, furazolidone, aminoglycosides, trimethoprim, and sulfamethoxazole. Concomitant with the emergence of this strain, we observed a resurgence in the number of registered cholera cases as well as an increase in the case fatality rate from 1.0% before the emergence of the multiple-drug-resistant strain to 5.3% after the emergence of the strain. Our study shows that the strain contained a 150-kb conjugative multiple-antibiotic resistance plasmid with class 1 integron-borne gene cassettes encoding resistance to trimethoprim (dhfrXII) and aminoglycosides [ant(3")-1a]). The finding of transferable resistance to almost all of the antibiotics commonly used to treat cholera is of great public health concern. Studies should be carried out to determine to what extent the strain or its resistance genes have been spread to other areas where cholera is endemic.
The prevalence of integrons in five enterobacterial species was analyzed in 900 blood culture isolates from 1993, 1996, and 1999. Remarkably, the prevalence increased from 4.7% in 1993 to 9.7% in 1996 and finally to 17.4% in 1999 (P < 0.01). Within 7 years the combined percentage of P1 strong promoters and P1 weak plus P2 active promoters with high transcription efficacies has increased from 23.1 to 33.3 and finally 60% (P < 0.05).
From January 2001 to December 2002, 587 strains of gram-negative bacterial isolates demonstrating resistance to ceftazidime and a combination of sulbactam and cefoperazone were subjected to a disk diffusion screening test using sodium mercaptoacetic acid; 431 strains (73.4%) appeared to produce metallo-beta-lactamase (MBL). Of these 431 strains, 357 were found by PCR to carry genes for IMP-1 type MBL (bla(IMP-1)), while only 7 and 67 strains carried the IMP-2 gene (bla(IMP-2)) and the VIM-2 gene (bla(VIM-2)), respectively. Neither VIM-1 nor SPM-1 type MBL genes were found among the strains tested. Of 431 strains, 427 carried the intI1 gene, and 4 strains carrying both the intI1 and intI3 genes were reidentified as Pseudomonas putida harboring bla(IMP-1). Of these four P. putida strains, three strains and one strain, respectively, were separately isolated from two hospitals located in the same prefecture, and the three strains showed very similar pulsed-field gel electrophoresis patterns. Of 357 bla(IMP-1) carriers, 116, 53, 51, 47, and 30 strains were identified as Pseudomonas aeruginosa, Alcaligenes xylosoxidans, P. putida/fluorescens, Serratia marcescens, and Acinetobacter baumannii, respectively. Four strains carrying bla(IMP-2) were reidentified as P. putida. Sixty-three P. aeruginosa strains and four P. putida strains carried bla(VIM-2). Of 427 intI1-positive strains, 180, 53, 51, 47, and 35 were identified as P. aeruginosa, A. xylosoxidans, P. putida/fluorescens, S. marcescens, and A. baumannii, respectively. In the present study, it was confirmed that strains carrying bla(IMP-1) with a class 1 integron are the most prevalent type in Japan, although several intI3 carriers have also been identified sporadically in this country.
Conservative site-specific recombination functions to create biological diversity in prokaryotes. Simple site-specific recombination systems consist of two recombination sites and a recombinase gene. The plasmid R64 shufflon contains seven recombination sites, which flank and separate four DNA segments. Site-specific recombinations mediated by the product of the rci gene between any two inverted recombination sites result in the inversion of four DNA segments independently or in groups. The shufflon functions as a biological switch to select one of seven C-terminal segments of the PilV proteins, which is a minor component of R64 thin pilus. The shufflon determines the recipient specificity in liquid matings of plasmid R64. Other multiple inversion systems as well as integrons, which are multiple insertion systems, are also described in this review.