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PubMed2006

Mechanism of drug resistance in clonally related clinical isolates of Vibrio fluvialis isolated in Kolkata, India.

The molecular mechanisms of drug resistance in 19 strains of Vibrio fluvialis isolated from 1998 to 2002 in Kolkata, India, were investigated. Class 1 integrons were detected in eight strains, and four strains were found to carry SXT integrases. In the presence of carbonyl cyanide m-chlorophenylhydrazone or reserpine, all nalidixic acid- and ciprofloxacin-resistant strains became sensitive, suggesting that drug efflux plays a major role in quinolone resistance in V. fluvialis. It was further seen that strains which had MICs of > 25 microg/ml for nalidixic acid had a sense mutation (Ser to Ile) at position 83 of the quinolone resistance-determining region of gyrA. All except one of the integron- and SXT integrase-bearing strains belonged to the same ribotype.

Anti-Bacterial Agents↗
PubMed2003

Escherichia coli with a self-transferable, multiresistant plasmid coding for metallo-beta-lactamase VIM-1.

An Escherichia coli strain exhibiting decreased susceptibility to carbapenems was isolated from a hospitalized patient in Greece. The strain carried a self-transferable plasmid coding for metallo-beta-lactamase VIM-1. bla(VIM-1), along with aacA7, dhfrI, and aadA, was included as a gene cassette in a novel class 1 integron. A Citrobacter freundii ampC-derived gene, not associated with the integron, was also located in the same plasmid.

Anti-Bacterial Agents↗
PubMed2004

Complex multiple antibiotic and mercury resistance region derived from the r-det of NR1 (R100).

The sequence of the 45.2-kb multidrug and mercury resistance region of pRMH760, a large plasmid from a clinical isolate of Klebsiella pneumoniae collected in 1997 in Australia, was completed. Most of the modules found in the resistance determinant (r-det), or Tn2670, region of NR1 (also known as R100), isolated from a Shigella flexneri strain in Japan in the late 1950s, were present in pRMH760 but in a different configuration. The location was also different, with the Tn2670-derived region flanked by the transposition module of Tn1696 and a mercury resistance module almost identical to one found in the plasmid pDU1358. This arrangement is consistent with a three-step process. First, the r-det was circularized via homologous recombination between the IS1 elements and reincorporated at a new location, possibly in a different plasmid, via homologous recombination between the 5'-conserved (5'-CS) or 3'-CS of the In34 integron in the r-det and the same region of a second class 1 integron in a Tn1696 relative. Subsequently, resolvase-mediated recombination between the res sites in the r-det and a second mercury resistance transposon removed one end of the Tn1696-like transposon and part of the second transposon. Other events occurring within the r-det-derived portion have also contributed to the formation of the pRMH760 resistance region. Tn2 or a close relative that includes the bla(TEM-1b) gene had moved into the Tn21 mercury resistance module with subsequent deletion of the adjacent sequence, and all four 38-bp inverted repeats corresponding to Tn21 family transposon termini have been interrupted by an IS4321-like element.

Anti-Bacterial Agents↗
PubMed2005

Multidrug-resistant Salmonella enterica serovar Muenchen from pigs and humans and potential interserovar transfer of antimicrobial resistance.

Salmonella serovars are important reservoirs of antimicrobial resistance. Recently, we reported on multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium strains among pigs with resistance to ampicillin, kanamycin, streptomycin, sulfamethoxazole, and tetracycline (resistance [R] type AKSSuT) and resistance to amoxicillin-clavulanic acid, ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline (R type AxACSSuT). In the present study, 67 isolates (39 from humans and 28 from pigs) of clinically important Salmonella serovar Muenchen were characterized. Among the porcine isolates, 75% showed resistance to seven antimicrobials: ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, tetracycline, amoxicillin-clavulanic acid, and kanamycin (R type ACSSuTAxK). One isolate from humans showed resistance to 10 of the 12 antimicrobials: ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, tetracycline, amoxicillin-clavulanic acid, kanamycin, gentamicin, cephalothin, and ceftriaxone (R type ACSSuTAxKGCfCro). Pulsed-field gel electrophoresis revealed no clonality between the porcine and the human strains. The porcine and the human MDR strains carried class 1 integrons of 2.0 and 1.0 kb, respectively. Genes specific to the porcine strain included aadA2, aphA1-Iab, and tetA(B). DNA sequencing revealed that the porcine isolates carried bla(OXA-30) on a class 1 integron. Genes specific to the human strain included bla(TEM), strA, strB, cmlA, tetA(A), and aadA2. No bla(CMY-2) gene was detected. Serovar Muenchen strains of porcine and human origin were able to transfer resistance genes to laboratory strain Escherichia coli MG1655 by conjugation. Plasmid restriction with four restriction enzymes, EcoRI, BamHI, HindIII, and PstI, showed that the conjugative plasmids from porcine Salmonella serovar Muenchen and Typhimurium R-type MDR strains isolated from the same farms at the same time were similar on the basis of the sizes and the numbers of bands and Southern hybridization. The plasmid profiles among the Salmonella serovar Muenchen isolates from the two host species were different. This is the first report to show a high frequency of MDR Salmonella serovar Muenchen strains from pigs and a human strain that is similar to the MDR isolates with the AmpC enzyme previously reported among Salmonella serovars Newport and Typhimurium strains. The MDR strains from the two host species independently represent public health concerns, as Salmonella serovar Muenchen is among the top 10 causes of salmonellosis in humans.

Animals↗
PubMed2005

The aadB gene cassette is associated with blaSHV genes in Klebsiella species producing extended-spectrum beta-lactamases.

Integrons were detected in 37 (72.5%) of 51 Klebsiella spp. producing extended-spectrum beta-lactamases by PCR with primers that targeted integrase genes and cassette regions. PCR and amplicon sequencing of the cassette regions revealed aadB and aadA2 gene cassettes that confer resistance to a range of aminoglycosides. aadB was associated with a class 1 integron on a 28-kb plasmid, pES1, that also contained bla(SHV-12) and IS26.

Aminoglycosides↗
PubMed2006

Establishing clonal relationships between VIM-1-like metallo-beta-lactamase-producing Pseudomonas aeruginosa strains from four European countries by multilocus sequence typing.

Ten multidrug-resistant Pseudomonas aeruginosa strains producing VIM-1-like acquired metallo-beta-lactamases (MBLs), isolated from four European countries (Greece, Hungary, Italy, and Sweden), were analyzed for genetic relatedness by several methodologies, including fliC sequence analysis, macrorestriction profiling of genomic DNA by pulsed-field gel electrophoresis (PFGE), random amplification of polymorphic DNA (RAPD), and multilocus sequence typing (MLST). The four approaches yielded consistent results overall but showed different resolution powers in establishing relatedness between isolates (PFGE>RAPD>MLST>fliC typing) and could usefully complement each other to address issues in the molecular epidemiology of P. aeruginosa strains producing acquired MBLs. In particular, the recently developed MLST approach was useful in revealing clonal relatedness between isolates when this was not readily apparent using RAPD and PFGE, and it suggested a common ancestry for some of the VIM-1-like MBL-positive P. aeruginosa strains currently spreading in Europe. The MBL producers belonged in three clonal complexes/burst groups (BGs). Of these, one corresponded to the previously described BG4 and included serotype O12 strains from Hungary and Sweden, while the other two were novel and included serotype O11 or nonserotypable strains from Greece, Sweden, and/or Italy. Comparison of the integrons carrying blaVIM-1-like cassettes of various isolates revealed a remarkable structural heterogeneity, suggesting the possibility that multiple independent events of acquisition of different blaVIM-containing integrons had occurred in members of the same clonal lineage, although a contribution of integrase-mediated cassette shuffling or other recombination mechanisms during the evolution of similar strains could also have played a role in determining this variability.

Bacterial Proteins↗
PubMed2003

Plasmid-mediated antimicrobial resistance in Salmonella enterica.

The selective pressure imposed by the use of antimicrobials in both human and veterinary medicine promotes the spread of multiple antimicrobial resistance. The dissemination of antimicrobial resistance in Salmonella enterica strains, causing severe enteritis in human, has been reported worldwide and is largely attributed to conjugative DNA exchange. In the present review, the relevance of plasmids to the dissemination of antimicrobial resistance in S. enterica is discussed. Recent examples of plasmid-mediated resistance to expanded-spectrum cephalosporins are reported to illustrate the severity of current situation in enteric pathogens. The exchanges between plasmid(s) and the bacterial chromosome and the integration of resistance genes into specialised genetic elements, called integrons, play a major role in acquisition and dissemination of resistance genes. The evolution of a plasmid through the acquisition of integrons is reported, describing novel mechanisms for short-term accumulation of resistance determinants in plasmids circulating in Salmonella.

Animals↗
PubMedDate not supplied

[Genetic variety of Acinetobacter baumannii in an intensive care unit for burn patients].

The genetic structure of A. baumannii hospital isolates, formed in the course of 2002 - 2004 in an intensive care unit for burn patients (St. Petersburg) was studied. The prolonged circulation of only some clonal strains was shown, 35% of the isolates belonged to dominating strains. Phenotypically, all cultures were characterized by resistance to the preparations of the cephalosporin row and gentamicin. The presence of class 1 integron with variable segment sized 2.5 kbp was found in the genotype of four isolates with the use the polymerase chain reaction. The restriction analysis revealed its similarity with integron, detected earlier (in 1989 - 2001 ) in A. baumannii in European hospitals abroad.

Acinetobacter Infections↗
PubMedDate not supplied

[Genetic environment of CTX-M-2 in Klebsiella pneumoniae isolates from hospitalized patients in Uruguay].

We studied two CTX-M-2-producing Klebsiella pneumoniae clinical strains, K96005 and K13, isolated from hospitalized patients in Uruguay, during 1996 and 2003, respectively. The genomic surroundings of bla(CTX-M-2) were characterized by PCR-mapping and DNA sequencing. Our results show that blaCTX-M-2 is included in a complex class-1 integron (InK13), associated with an orf513 in both isolates. The genetic array of the integron, aac(6')-lb, bla(OxA,2), orfD (gene cassette region), associated with an orf513-bla(CTX-M-2), seems to be widely disseminated over the Rio de la Plata region.

Base Sequence↗
PubMed2006

[Characterization of Salmonella typhimurium multidrug resistance and the reversal of antimicrobial resistance].

Salmonella typhimurium 17Y, isolated from one diseased pig that was clinically diagnosed as pig salmonellosis, was a multiresistance strain with resistance to 14 antibiotics among tested 19 antibiotics. In this study, the resistance to 11 antimicrobials was reversed by high temperature and high concentration (0.5%) of SDS, resulting in the sensitive strain 17S1. PCR results showed that the resistant genes BlaTEM, blaOXA-1, cat 1, tet (B), aacC2 located on the plasmid. Furthermore, PCR detected the class I integron which carries dhfrX II for trimethoprim resistance, aadA18b for aminoglycoside resistance and sull for sulfamethoxazole resistance. The integron was identified to exist in the plasmid. Because the target genes gyrA and parC for quinolone category were detected by PCR from both resistant and sensitive strains, it was determined that the genes gyrA and parC were located in the bacterial genome. The gene sequencing of gyrA and parC revealed that a point mutation AAC --> GAC resulting in one amino acid replacement of N87D in gyrA occurred for the sensitive strain 17S1. It was demonstrated that the amino acid 87 was a hot point for mutation in quinolone resistance determining region (QRDR). The finding suggests that the amino acid replacement of N87D is responsible for the quinolone susceptibility. In addition, the 100 continuous passages of the sensitive strain showed that the drug sensitive status was stable. However, when the drug pressure maintained for a long time, the resistance was induced again. Meanwhile, 6 salmonella plasmid virulence genes (spvA-D, R and rck) were eliminated with the resistance reversal, indicating that the virulence plasmid was cured. Reasonably, the bacterial virulence decreased shown by 10- fold increase of LD50 for the sensitive strain, and the statistical significant decline of in vivo spread and growth (P < 0.05) in mice. Taken altogether, the multidrug resistance of Salmonella typhimurium was determined by its plasmid. The plasmid elimination with SDS reversed most of the resistance (11/14) and decreased the bacterial virulence. Therefore, strategy to eliminate the plasmids would be an effective way to deal with the multiresistance issue. However, drug control in routine clinical practice would not be neglected at any time.

Animals↗
PubMed1993

Genetic organization of antibiotic resistance genes (aac(6')-Ib, aadA, and oxa9) in the multiresistance transposon Tn1331.

The multiresistance transposon Tn1331 has two 520 bp direct repeats flanking a DNA region similar to the variable portion of the integrons. This region harbors three antibiotic resistance genes, aac(6')-Ib, aadA, and a beta-lactamase gene which encodes an enzyme that was characterized as an oxacillinase-carbenicillinase and was named OXA-9. Within the direct repeat located upstream of these genes there is a copy of the TEM beta-lactamase promoter. Determination of ampicillin resistance levels of Escherichia coli harboring various recombinant clones and an insertion mutant showed that all three genes are transcribed from this promoter in a polycistronic mRNA. These results also indicated that the oxa9 gene possesses another functional promoter, located immediately upstream of this gene. The transcription start site of the mRNA driven by this promoter was identified by primer extension. Within the DNA sequence harboring all three antibiotic resistance genes it is possible to distinguish insert units as defined before in the variable region of the integrons (R. Hall, D. Brookes, and H. W. Stokes (1991) Mol. Microbiol. 5, 1941-1959).

Amino Acid Sequence↗
PubMed1994

DNA sequence of direct repeats of the sulI gene of plasmid pSa.

The restriction enzyme and genetic map of the antibiotic-resistance region of plasmid pSa is related to Tn21 integrons by the insertion of 5.4 kb containing a chloramphenicol resistance gene (catII) and a 1.1-kb direct repeat. We report here the nucleotide sequences of both copies of the repeat with adjoining sequences. They were identical for 1065 bp and contained the entire coding sequence of the sulfanilamide resistance gene, sulI. Since only the first copy of the repeat confers sulfonamide resistance, this leads to the conclusion that no promoter was available for the second copy. The sequence of the pSa sulI gene was identical to several published sulI sequences from other plasmids. The first junction point of the catII-containing insert was identical to the sequence for pDG0100; the second junction occurred farther into the 3'-conserved segment of integrons than does that of pDG0100. A recent report of these junction sequences for pSa and pDG0100 differs from our sequences by one nucleotide. Two additional differences were an insert of 41 bases and a single base insertion between sulI and ORF341 in our sequence. Our sequenced regions have been assigned GenBank Accession Nos. UO4277 and UO4278 for the first and second sulI genes of pSa, respectively.

Amino Acid Sequence↗
PubMed1992

A new example of physical linkage between Tn1 and Tn21: the antibiotic multiple-resistance region of plasmid pCFF04 encoding extended-spectrum beta-lactamase TEM-3.

The genetic environment of plasmid-borne blaTEM mutant genes, encoding nine distinct TEM-type extended-spectrum beta-lactamases, was studied in transconjugants from clinical isolates of enterobacteria. Colony hybridization with probes specific for tnpA and tnpR of Tn3, tnpA and tnpI of Tn21, aacA4, and IS15, and restriction endonuclease analysis of plasmid DNA indicated that the structural genes for the enzymes were always associated with intact or deleted variants of the Tn3 family. Four of the nine blaTEM variants, which account for 62% of 222 isolates in a molecular epidemiological study, were associated with replicons indistinguishable from the epidemic Inc7-M plasmid pCFF04 that carries the blaTEM-3 gene. This suggests that mutant genes were selected from the same prototype plasmid carrying penicillinase genes blaTEM-1 or -2. A 6.6 kb DNA fragment of pCFF04 containing blaTEM-3 was characterized by amplification mapping and sequencing. The results obtained indicated that blaTEM-3 was present on a copy of Tn1 interrupted at the start codon of the transposase by a DNA sequence reminiscent of the inverted repeats of class II transposons. This partial Tn1 copy was in turn, inserted into the transposase gene of a Tn21-like transposon containing an integron expressing an aacA4 gene. The presence of an integron can account for the various assortments of aminoglycoside resistance genes found associated with blaTEM-3.

Amino Acid Sequence↗
PubMed2002

Distinct recent lineages of the strA- strB streptomycin-resistance genes in clinical and environmental bacteria.

We report the linkage of the strA-strB streptomycin-resistance genes with Class 1 integron sequences on pSTR1, a 75-kb multiple antibiotic-resistance plasmid from Shigella flexneri. strA-strB had previously been detected only within Tn 5393, a Tn 3-family transposon, and on small nonconjugative broad-host-range plasmids such as RSF1010. The geographic range of Tn 5393 was also extended to Pseudomonas spp. isolated from apple trees in New Zealand and soil in the USA. Comparative sequence analyses indicated that strA-strB from Tn 5393 and nonconjugative plasmids constitute distinct recent lineages with strA-strB from pSTR1 intermediate between the other two. The carriage of strA-strB within an integron, a transposon, and on broad-host-range plasmids has facilitated the world-wide dissemination of this determinant among at least 21 bacterial genera.

Animals↗
PubMed2026

Whole genome sequencing reveals the co-existence of blaPER-7, blaADC-52 and blaOXA-91 in multidrug resistant ST164pas/ST234oxfAcinetobacter baumannii strains in Bangladesh.

OBJECTIVE: Acinetobacter baumannii (A. baumannii) has emerged as a critical multidrug-resistant (MDR) pathogen with the capacity to persist in diverse ecological niches. Environmental reservoirs in densely populated settings such as Dhaka, Bangladesh, may play a significant role in sustaining and disseminating antimicrobial resistance (AMR). This study aimed to characterize the genomic and phenotypic features of MDR A. baumannii isolates recovered from urban water bodies. METHODS: Three environmental isolates of A. baumannii were subjected to antimicrobial susceptibility testing, biofilm and serum resistance assays, whole-genome sequencing and analysis. Comprehensive genome analysis was carried out emphasizing on antimicrobial resistance genes, virulence factor genes, multi-locus sequence type, integron, prophage and mobile genetic elements. RESULTS: Phenotypically, all the three isolates showed serum resistance and biofilm forming capacity. All the three isolates were identified as ST164pas/ST234oxf. The antimicrobial resistance genes investigation revealed that all the three isolates had co-existence of beta lactam resistance genes blaPER-7, blaADC-52 and blaOXA-91. The isolates had gyrA (S81L) and parC (V104I/D105E) mutations associated with fluoroquinolone resistance. Several prophage regions were found in the strains and A. baumannii ML1 harbored AMR genes inside prophage regions. All the isolates harbored integron 1 in their genome. Comparative genome analysis of the Bangladeshi ST164pas/ST234oxf strains revealed a high degree of genomic conservation. CONCLUSION: The findings from this study highlighted environmental water bodies as reservoirs for MDR A. baumannii and emphasize the need for targeted One Health surveillance and improved wastewater management to limit resistance dissemination.

Journal Article↗
PubMed2006

Resistance to antibiotics in clinical isolates of Pseudomonas aeruginosa.

OBJECTIVES: To analyse the global resistance to some antibiotics used to treat nosocomial infections by Pseudomonas aeruginosa, specially to carbapenems, and its relationship with the presence of carbapenemases, OXA, VIM and IMP. METHODS: The study included 229 P. aeruginosa isolates from a Hospital in Northern Spain (year 2002). Susceptibility to antimicrobial agents was determined by the analysis of the MIC. Genetic typing was carried out by RAPD-PCR fingerprinting with primer ERIC-2. Genetic experiments to detect class-1 integrons were performed by PCR with primers 5'CS and 3'CS. Detection of carbapenemases was done by phenotypic (Hodge test and DDST) and genotypic methods (PCR with primers for imp, vim1, vim2 and oxa40 genes). RESULTS: 23.9% of isolates were resistant to ceftazidime, 35.9% to cefotaxime, 5.3% to amikacin, 54.9% to gentamicin, 14.6% to imipenem and 6.6% to meropenem. Isolates resistant to imipenem (33) were furtherly tested. Genetic typing didn't show clonal relatedness among the most of the isolates. Class-1 integrons were present in most isolates (sizes 600-1700 bp). Phenotypic methods for carbapenemases showed 5 positive isolates. Genotypic methods showed the presence of two isolates with the oxa40 gene. CONCLUSIONS: Meropenem, amikacin and imipenem were the most active agents to treat infections caused by Pseudomonas aeruginosa. In our study, the presence of carbapenemase enzymes wasn't high. Phenotypic tests cannot be considered as accurate screening tool to detect carbapenemases. This is the fist report of the oxa40 gene in Pseudomonas aeruginosa isolates.

Anti-Bacterial Agents↗
PubMed2003

Antimicrobial resistance and resistance gene determinants in clinical Escherichia coli from different animal species in Switzerland.

Antimicrobial susceptibility testing was performed on a total of 581 clinical Escherichia coli isolates from diarrhea and edema disease in pigs, from acute mastitis in dairy cattle, from urinary tract infections in dogs and cats, and from septicemia in laying hens collected in Switzerland between 1999 and 2001. Among the 16 antimicrobial agents tested, resistance was most frequent for sulfonamides, tetracycline, and streptomycin. Isolates from swine presented significantly more resistance than those from the other animal species. The distribution of the resistance determinants for sulfonamides, tetracycline, and streptomycin was assessed by hybridization and PCR in resistant isolates. Significant differences in the distribution of resistance determinants for tetracycline (tetA, tetB) and sulfonamides (sulII) were observed between the isolates from swine and those from the other species. Resistance to sulfonamides could not be explained by known resistance mechanisms in more than a quarter of the sulfonamide-resistant and sulfonamide-intermediate isolates from swine, dogs and cats. This finding suggests that one or several new resistance mechanisms for sulfonamides may be widespread among E. coli isolates from these animal species. The integrase gene (intI) from class I integrons was detected in a large proportion of resistant isolates in association with the sulI and aadA genes, thus demonstrating the importance of integrons in the epidemiology of resistance in clinical E. coli isolates from animals.

Animal Diseases↗