Standard and real-time multiplex PCR methods for detection of trimethoprim resistance dfr genes in large collections of bacteria (original) (raw)
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Journal of Medical Microbiology, 2010
A total of 456 non-repetitive Escherichia coli isolates from human clinical specimens (urinary, n5134; cervix, vagina and prostate, n552; blood, pus and wounds, n545), healthy animals (cattle, n545; poultry, n520) and diseased animals (cattle, n553; swine, n564; poultry, n543) obtained in Lithuania during the period 2005-2008 were studied for trimethoprim (TMP) resistance and the prevalence of dfr genes. A TMP resistance rate in the range of 18-26 % respective to the origin was found in clinical isolates, 23-40 % in isolates from diseased animals and 9-20 % in isolates from healthy animals. Of 112 TMP-resistant isolates, 103 carried at least one of the six dfrA genes (dfrA1, dfrA5, dfrA8, dfrA12, dfrA14 and dfrA17) as determined by multiplex PCR and RFLP. The dfrA1 and dfrA17 genes were found most frequently in clinical isolates (17 and 19 isolates, respectively), whilst dfrA1 and dfrA14 genes dominated in isolates of animal origin (25 and 13 isolates, respectively). The dfrA5, dfrA12 and dfrA8 genes were detected at lower frequencies. The association with class 1/class 2 integrons was confirmed for 73-100 % of dfr genes found in most groups of isolates, except for the isolates from diseased swine. In this group, the majority of dfr-positive isolates (67 %, 8/12) carried dfrA8 (6/12) or dfrA14 genes (2/12) that were not associated with integrons. Non-integron location was also confirmed for the remaining dfrA8 genes (six clinical isolates and one isolate from diseased cattle) and for dfrA14 genes (two isolates from diseased cattle and swine each). All cassetteindependent dfrA14 genes were found to be located within the strA gene. This study on the prevalence and distribution of TMP resistance genes among E. coli isolates of human and animal origin in Lithuania demonstrates that dfr genes are carried most frequently as gene cassettes within class 1 and/or class 2 integrons. However, TMP resistance in some of the isolates was found to be mediated by non-integron-associated dfrA8 and dfrA14 genes, indicating the existence of alternative sources for the spread of resistance.
A 2-year prospective intervention on the prescription of trimethoprim reduced the use by 85% in a health care region with 178,000 inhabitants. Here, we performed before-and-after analyses of the within-population distribution of trimethoprim resistance in Escherichia coli. Phylogenetic and population genetic methods were applied to multilocus sequence typing data of 548 consecutively collected E. coli isolates from clinical urinary specimens. Results were analyzed in relation to antibiotic susceptibility and the presence and genomic location of different trimethoprim resistance gene classes. A total of 163 E. coli sequence types (STs) were identified, of which 68 were previously undescribed. The isolates fell into one of three distinct genetic clusters designated BAPS 1 (E. coli phylogroup B2), BAPS 2 (phylogroup A and B1), and BAPS 3 (phylogroup D), each with a similar frequency before and after the intervention. BAPS 2 and BAPS 3 were positively and BAPS 1 was negatively associated with trimethoprim resistance . In before-and-after analyses, trimethoprim resistance frequency increased in BAPS 1 and decreased in BAPS 2. Resistance to antibiotics other than trimethoprim increased in BAPS 2. Analysis of the genomic location of different trimethoprim resistance genes in isolates of ST69, ST58, and ST73 identified multiple independent acquisition events in isolates of the same ST. The results show that despite a stable overall resistance frequency in E. coli before and after the intervention, marked within-population changes occurred. A decrease of resistance in one major genetic cluster was masked by a reciprocal increase in another major cluster.
Antimicrobial Agents and Chemotherapy, 1991
Among a colection of cdlna Escherchia cofi isolates, the type I dihydrofolate reductase (DHFR) mediating trimethoprim resistance was generally observed to be chromosomafly determined. Only a minority of isolates carried the type I DHFR gene simultusy on a plasmid. The majority of E. coil isolates studied also hybridized with a probe spec for the transposition gene tnsC of transposon Tn7; and Wn most of these isolates, Tn7 was found to be inserted into a preferd site in the E. co,i chromosome. A minority of isolates that harbored the type I DHFR gene in the chromosome lacked a complete Tn7. Some of these harbored the type I gene inerted in a simar to that containing the gene for streptomycin resistance in Tn2l. In the other Isolates that were negative for a complete Tn7, the sequences upstream of the type I DHFR gene were demonstrated to be homologous to those type I DHFR gene in Tn7. This could Indicate that the antibiotic resisne region of Tn7 may occur independently of this transposon. In two Isolates, no sequences resembling Tn7 or Tn2l were found adjacent to the type I DHFR gene.
Journal of Rapid Methods & Automation in Microbiology, 2009
Two multiplex polymerase chain reaction (mPCR) assays were developed for simultaneous detection of antimicrobial resistance genes in Escherichia coli and enterococci. Oligonucleotide primer sets were designed and formulated. Primer set I was used to detect resistance genes including tetracycline (tetM and tetA), chloramphenicol (cat1 and cmlA) and sulfonamide (sul1), in 20 E. coli isolates. Primer set II was designed to identify three target genes including tetM, tetL and ermB of tetracycline and erythromycin resistance genes, respectively. In the final volume of 25 mL, optimal concentrations of mPCR primer for investigation of E. coli and enterococci resistance genes were 0.1, 0.4, 0.2, 0.4 and 0.2 mM of tetA, tetM, cat1, cmlA and sul1, and 0.2, 0.2 and 0.1 mM of tetM, tetL and ermB, respectively. The optimal annealing temperatures were 51 and 57C to detect all expected resistance genes of E. coli and enterococci, respectively. The amplicon sizes ranged from 171 to 847 bp for E. coli and 171 to 505 bp for enterococci, differing by at least 83 bp to simplify gel electrophoretic separation. The correlation between dot blot hybridization and mPCR results were investigated. Two of the 20 E. coli isolates showed positive results by mPCR and negative results by hybridization assay. The same results were observed with two different methods among 3 Corresponding 117 20 enterococci isolates indicating that the targets were amplified efficiently. The developed mPCR assay is a simple, rapid and useful method for genotyping detection of multiple resistance genes in single reaction.
1999
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Clinical Infectious Diseases, 2005
Background. Antibiotic resistance is increasingly complicating the management of urinary tract infection. We investigated the extent to which a group of Escherichia coli called clonal group A (CGA), which is associated with resistance to trimethoprim-sulfamethoxazole (TMP-SMZ), accounted for TMP-SMZ resistance among a prospectively collected set of uropathogenic and rectal E. coli isolates from a university population in Michigan. Methods. Resistant and susceptible uropathogenic E. coli isolates (45 each) and 79 randomly selected rectal E. coli isolates were evaluated for CGA status by use of 2 definitions of this group-the enterobacterial repetitive intergenic consensus sequence 2 (ERIC2)-polymerase chain reaction (PCR) pattern A fingerprint and the C288T single nucleotide polymorphism (SNP) in the fumC gene. We compared virulence gene profiles and molecular mechanisms of resistance to TMP-SMZ between isolates classified as CGA by both approaches to better characterize the relationship between isolates. Results. Of the 45 isolates that exhibited ERIC2-PCR pattern A, one-half (23 of 45) were resistant to TMP-SMZ, and 16 contained the C288T SNP. The pattern A isolates were diverse, exhibiting multiple mechanisms of resistance to TMP-SMZ and various combinations of virulence factors. C288T SNP isolates showed less variation, with 15 of 16 resistant to TMP-SMZ and a 1.8-kb class I integron bearing the dfrA17 gene present in 14 of 15 resistant isolates. Twelve of 16 exhibited the same combination of virulence genes. Pulsed-field gel electrophoresis patterns for these 12 isolates were unique. Conclusion. CGA, as defined by the fumC C288T SNP, appears to be distantly clonal but is not an outbreakrelated group. The widespread group has likely evolved through lateral transfer of genes conferring virulence and antibiotic resistance.
The ISME Journal
Trimethoprim (TMP) is a low-cost, widely prescribed antibiotic. Its effectiveness is increasingly challenged by the spread of genes coding for TMP-resistant dihydrofolate reductases: dfrA, and the lesser-known, evolutionarily unrelated dfrB. Despite recent reports of novel variants conferring high level TMP resistance (dfrB10 to dfrB21), the prevalence of dfrB is still unknown due to underreporting, heterogeneity of the analyzed genetic material in terms of isolation sources, and limited bioinformatic processing. In this study, we explored a coherent set of shotgun metagenomic sequences to quantitatively estimate the abundance of dfrB gene variants in aquatic environments. Specifically, we scanned sequences originating from influents and effluents of municipal sewage treatment plants as well as river-borne microbiomes. Our analyses reveal an increased prevalence of dfrB1, dfrB2, dfrB3, dfrB4, dfrB5, and dfrB7 in wastewater microbiomes as compared to freshwater. These gene variants w...
Trimethoprim-resistant Enterobacteriaceae in urinary tract infection
Canadian Medical Association journal, 1975
The incidence of trimethoprim-resistant Enterobacteriaceae has not increased since the introduction of the combination trimethoprim-sulfamethoxazole (TMP-SMX) into the clinical use at our centre in 1973. Using the minimum inhibitory concentration (MIC) as the index of trimethoprim resistance, this ranged from 1.6 to 800 mug/ml; for the majority of isolates it lay between 1.6 and 12.5 mug/ml. About half of these trimethoprim-resistant organisms were sensitive to sulfonamide. In vitro data suggest that organisms resistant to sulfonamide as well as to trimethoprim, where the MIC for the former drug is 3.1 mug/ml or less, will be susceptible to the combination. More resistant organisms, i.e., those for which the MIC of trimethoprim is 6.2 mug/ml or more, often appear quite resistent to the combination. There is no evidence that previous therapy with TMP-SMX is a significant predisposing factor to infection with these organisms, although there is a significant correlation between previou...