Molecular Characterisation of Trimethoprim Resistance in Escherichia coli and Klebsiella pneumoniae during a Two Year Intervention on Trimethoprim Use (original) (raw)
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Clinical Microbiology and Infection, 2007
Two multiplex PCR (mPCR) methods were developed to screen large collections of trimethoprimresistant Escherichia coli isolates for the most prevalent resistance determinants. Five common integroncarried genes (dfrA1, dfrA5, dfrA7, dfrA12 and dfrA17) were selected as PCR targets. Primers and conditions for standard mPCRs and real-time mPCRs were selected and tested. Two protocols using essentially the same primer pairs were established. The standard mPCR protocol also included an internal control targeting the E. coli 16S rRNA gene. Both protocols proved to be sensitive and specific for detection of the five selected genes. Screening of three different collections of clinical urinary and blood isolates (n = 368) with the two multiplex methods revealed that the five dfr genes accounted for 75-86% of trimethoprim resistance. The standard mPCR is useful and accessible for most laboratories, while the real-time mPCR requires additional equipment and expensive reagents, but is very convenient for high-throughput screening of large collections of bacterial isolates.
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.
1999
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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.
Genetic characterization of trimethoprim resistance in Haemophilus influenzae
Antimicrobial agents and chemotherapy, 1996
We previously demonstrated that trimethoprim (Tmp) resistance in Haemophilus influenzae is mediated by chromosomally encoded dihydrofolate reductase (DHFR) with a modified primary structure and distinct kinetic properties. To gain insight into the relationship of the DHFR structure and the level of Tmp resistance that it confers on the host bacterium, we cloned and characterized the folH genes of one Tmp-susceptible and two Tmp-resistant H. influenzae strains. Differences were observed between Tmp-susceptible and Tmp-resistant isolates both in the promoter region and in the coding sequences. The effect of differences between H. influenzae folH genes on Tmp susceptibility was investigated in Escherichia coli. Various folH gene hybrids were constructed, and their influence on Tmp susceptibility was determined. Resistance in E. coli mediated by folH from H. influenzae strain R1047 was associated with alterations in the promoter and the central part of folH. In contrast, the E. coli Tmp...
1996
Resistance to antimicrobial agents is an increasing problem, especially in developing countries where resistant strains have undermined the effectiveness of antimicrobial agents such as trimethoprim. Since the commensal faecal flora are strongly implicated as a reservoir for resistant organisms. A survey was conducted in South Africa to determine the incidence of resistance in aerobic Gram-negative commensal faecal flora. Faecal specimens from 272 out of 361 (75%) healthy volunteers carried trimethoprimresistant bacteria; 357 trimethoprim-resistant strains were isolated. Trimethoprim resistance was transferable by conjugation in 55% of the isolates. The majority of the isolates were resistant to other antimicrobial agents including ampicillin 71.4% and tetracycline 88%. Most of these resistance phenotypes co-transferred with trimethoprim resistance. Analysis of 189 plasmids revealed 107 different restriction profiles which indicated that there is a large gene pool of trimethoprim-re...