Two new dfr genes in trimethoprim-resistant integron-negative Escherichia coli isolates.
ABSTRACT: Two Escherichia coli isolates resistant to trimethoprim but negative for integrons carried two new resistance genes, dfrA24 and dfrA26, remotely similar to one another and to the cassette-independent genes dfrA8 and dfrA9. The dfrA24 gene was not associated with known mobile elements, while dfrA26 was associated with the CR1 common region.
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:Sulfonamide-trimethoprim-resistant Aeromonas salmonicida and motile Aeromonas spp. from diseased fish of the GERM-Vet study carried the sul1 gene together with mostly cassette-borne trimethoprim resistance genes, including the novel gene dfrA28. The seven dfrA and dfrB genes identified were located mostly in class 1 integrons which commonly harbored other gene cassettes.
Project description:The most common mechanism of trimethoprim (TMP)-resistance is the acquisition of dihydrofolate reductase enzyme resistant to this drug. Previous molecular characterization of TMP-genes resistance in Chilean isolates of Shigella sonnei searching for dfrA1 and dfrA8, showed solely the presence of dfrA8 (formerly dhfrIIIc). However, these genetic markers were absent in S. sonnei strains further isolated during an outbreak in 2009. To identify the TMP-resistance gene in these strains, a genomic DNA library from a TMP-resistant (TMP(R)) S. sonnei representative strain for the outbreak was used to clone, select and identify a TMP-resistance marker. The TMP(R) clone was sequenced by primer walking, identifying the presence of the dfrA14 gene in the sul2-strA'-dfrA14-'strA-strB gene arrangement, harbored in a native 6779-bp plasmid. The same plasmid was isolated by transforming with a ~4.2 MDa plasmid extracted from several TMP(R) S. sonnei strains into Escherichia coli. This plasmid, named pABC-3, was present only in dfrA14-positive strains and was homologous to a previously described pCERC-1, but different due to the absence of an 11-bp repetitive unit. The distribution of dfrA1, dfrA8, and dfrA14 TMP-resistance genes was determined in 126 TMP(R) S. sonnei isolates. Most of the strains (96%) carried only one of the three TMP-resistance genes assessed. Thus, all strains obtained during the 2009-outbreak harbored only dfrA14, whereas, dfrA8 was the most abundant gene marker before outbreak and, after the outbreak dfrA1 seems have appeared in circulating strains. According to PFGE, dfrA14-positive strains were clustered in a genetically related group including some dfrA1- and dfrA8-positive strains; meanwhile other genetic group included most of the dfrA8-positive strains. This distribution also correlated with the isolation period, showing a dynamics of trimethoprim genetic markers prevalent in Chilean S. sonnei strains. To our knowledge, dfrA14 gene associated to a small non-conjugative plasmid was detected for the first time in Shigella. Apparently, the strain causing the outbreak must have been introduced, changing drastically the genetic distribution of trimethoprim resistance in Chilean S. sonnei strains.
Project description:In this study, a 498-bp dhfrXII gene coding for trimethoprim resistance was found inserted in a cassette-like manner in the recombinationally active locus, the integron, borne on a transposon Tn21-like element. The dhfrXII cassette is distinct from those cassettes earlier observed in integrons and was found here upstream of two similarly inserted cassettes. The second one carried the new unidentified orfF, which is 85% identical to the orfD cassette in R46. The third cassette contained the aadA2 gene mediating spectinomycin resistance. The plasmid carrying this Tn21-like element was originally isolated from a trimethoprim-resistant urinary tract pathogen, Escherichia coli, from Turku City Hospital, Turku, Finland. By colony hybridization and polymerase chain reaction, this group of three cassettes, including dhfrXII, was detected in four additional E. coli strains of similar origin and in four Shigella strains isolated in Finland but originating from Asia. The dihydrofolate reductase produced from dhfrXII showed an unusual drug resistance in that 50% of the enzymatic activity remained at a trimethoprim concentration of 1 mM.
Project description:An unusual In0-like class 1 integron containing a common region that includes the putative recombinase gene named orf513 (CR1) and bla(CTX-M-2) was characterized from Escherichia coli. The integron contained an unusual gene cassette array, estX-aadA1, embedded between the 5'-conserved segment (5'-CS) and 3'-CS1 regions and was flanked by mer-Tn21 sequences downstream of the tni truncated module. This element constitutes one of the few examples of CR1-bearing class 1 integrons that has been fully characterized.
Project description:Twenty-five plasmid-specified antimicrobial resistance determinants common to gram-negative bacilli from nosocomial infection were investigated from 31 Stenotrophomonas maltophilia isolates. Twenty-four clones were identified by pulsed-field gel electrophoresis, and in three clones that exhibited an increased trimethoprim-sulfamethoxazole MIC, the sul1 determinant was found. These results support not only the higher spread of class 1 integrons compared to other mechanisms but also the potential limitation of using trimethoprim-sulfamethoxazole for therapy of severe S. maltophilia infections.
Project description:Background:Ready to eat (RTE) vegetables are easily accessible healthy foods that are commonly consumed globally, including in Indonesia. However, these RTE vegetables contain potential contamination from pathogens and multi-drug resistant bacteria. Therefore, in the present study, we examined the presence of tetracycline-resistant E. coli (TRE) isolates from RTE vegetables. Methods:Susceptibility to antimicrobial agents was determined using the Kirby-Bauer disc diffusion method. Characterisation of antibiotic resistant genes was performed using PCR and sequencing of tetracycline resistant gene, integron and gene cassette from the TRE isolates. Results:The isolates collected in this study were resistant not only to tetracycline, but also to streptomycin. Some isolates also displayed resistance to kanamycin (77.8%), chloramphenicol (11.1%), and ciprofloxacin (5.6%). All of the isolates contained integrons (intI1) and the tetA gene; tetB was not detected in our study. Further analysis showed that some isolates (38.8%) contained the dfrA7 gene cassette, which encodes dihydrofolate reductase, which is responsible for resistance to trimethoprim. Of all the isolates that presented integrons, 11 isolates (61.1%) did not carry gene cassettes. These empty integrons have the potential to convert themselves rapidly into multigraviton strains. Conclusions:TRE isolates contain the tetA gene and integron 1. Only 38.8% of the isolates that have been identified contain the dfrA7 gene cassette, which is responsible for trimethoprim antibiotic resistance. Further identification of genes conferring resistance to other antibiotics is necessary to better characterise antibiotic resistance.
Project description:Salmon farming industry in Chile currently uses a significant quantity of antimicrobials to control bacterial pathologies. The main aims of this study were to investigate the presence of transferable sulfonamide- and trimethoprim-resistance genes, sul and dfr, and their association with integrons among bacteria associated to Chilean salmon farming. For this purpose, 91 Gram-negative strains resistant to sulfisoxazole and/or trimethoprim recovered from various sources of seven Chilean salmonid farms and mainly identified as belonging to the Pseudomonas genus (81.0%) were studied. Patterns of antimicrobial resistance of strains showed a high incidence of resistance to florfenicol (98.9%), erythromycin (95.6%), furazolidone (90.1%) and amoxicillin (98.0%), whereas strains exhibited minimum inhibitory concentrations (MIC90) values of sulfisoxazole and trimethoprim of >4,096 and >2,048 ?g mL-1, respectively. Strains were studied for their carriage of these genes by polymerase chain reaction, using specific primers, and 28 strains (30.8%) were found to carry at least one type of sul gene, mainly associated to a class 1 integron (17 strains), and identified by 16S rRNA gene sequencing as mainly belonging to the Pseudomonas genus (21 strains). Of these, 22 strains carried the sul1 gene, 3 strains carried the sul2 gene, and 3 strains carried both the sul1 and sul2 genes. Among these, 19 strains also carried the class 1 integron-integrase gene intI1, whereas the dfrA1, dfrA12 and dfrA14 genes were detected, mostly not inserted in the class 1 integron. Otherwise, the sul3 and intI2 genes were not found. In addition, the capability to transfer by conjugation these resistance determinants was evaluated in 22 selected strains, and sul and dfr genes were successfully transferred by 10 assayed strains, mainly mediated by a 10 kb plasmid, with a frequency of transfer of 1.4 × 10-5 to 8.4 × 10-3 transconjugant per recipient cell, and exhibiting a co-transference of resistance to florfenicol and oxytetracycline, currently the most used in Chilean salmon industry, suggesting an antibacterial co-selection phenomenon. This is the first report of the characterization and transferability of integrons as well as sul and dfr genes among bacteria associated to Chilean salmon farms, evidencing a relevant role of this environment as a reservoir of these genes.
Project description:BACKGROUND: The aims of this study were to understand the molecular epidemiology of integron-associated gene cassettes in Acinetobacter baumannii across four hospitals in northern Taiwan and to clarify the relationship between the presence of integrons and antibiotic-resistant phenotypes. METHODS: Sixty-five A. baumannii isolates, collected from the patients of four regional hospitals in northern Taiwan in 2009, were tested for the presence of integrons and their associated gene cassettes. The susceptibility difference between integron-positive and integron-negative A. baumannii strains was analyzed. Antibiotic-resistant phenotypes among A. baumannii with different types of gene cassette array combinations were also compared. RESULTS: Around 72% of the A. baumannii isolates carried class 1 integrase genes. Despite this, only three gene cassette arrays were found in the integrons. Integron-positive strains were significantly more resistant to all the tested antibiotics than the integrase-negative strains. All the four types of A. baumannii with different gene cassette array combinations were multidrug-resistant in nature. Gene cassette array aacA4-catB8-aadA1 existed in all the integron-positive A. baumannii isolates. Repetitive-sequence-based PCR (rep-PCR) results revealed the prevalence of one major cluster of imipenem-resistant A. baumannii strains (84%) in the four regional hospitals. CONCLUSIONS: The presence of integrons with associated antimicrobial resistance gene cassettes can be used as a representative marker of multidrug resistance in A. baumannii. Some prevalent gene cassette arrays may exist among epidemiologically unrelated A. baumannii strains.
Project description: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.