Molecular Characterisation of Trimethoprim Resistance in Escherichia coli and Klebsiella pneumoniae during a Two Year Intervention on Trimethoprim Use

BACKGROUND: Trimethoprim resistance is increasing in Enterobacteriaceae. In 2004-2006 an intervention on trimethoprim use was conducted in Kronoberg County, Sweden, resulting in 85% reduction in trimethoprim prescriptions. We investigated the distribution of dihydrofolate reductase (dfr)-genes and integrons in Escherichia coli and Klebsiella pneumoniae and the effect of the intervention on this distribution. METHODOLOGY/PRINCIPAL FINDINGS: Consecutively isolated E. coli (n = 320) and K. pneumoniae (n = 54) isolates phenotypically resistant to trimethoprim were studied. All were investigated for the presence of dfrA1, dfrA5, dfrA7, dfrA8, dfrA12, dfrA14, dfrA17 and integrons class I and II. Isolates negative for the seven dfr-genes (n = 12) were also screened for dfr2d, dfrA3, dfrA9, dfrA10, dfrA24 and dfrA26. These genes accounted for 96% of trimethoprim resistance in E. coli and 69% in K. pneumoniae. The most prevalent was dfrA1 in both species. This was followed by dfrA17 in E. coli which was only found in one K. pneumoniae isolate. Class I and II Integrons were more common in E. coli (85%) than in K. pneumoniae (57%). The distribution of dfr-genes did not change during the course of the 2-year intervention. CONCLUSIONS/SIGNIFICANCE: The differences observed between the studied species in terms of dfr-gene and integron prevalence indicated a low rate of dfr-gene transfer between these two species and highlighted the possible role of narrow host range plasmids in the spread of trimethoprim resistance. The stability of dfr-genes, despite large changes in the selective pressure, indirectly suggests a low fitness cost of dfr-gene carriage

Molecular basis for trimethoprim and sulphonamide resistance in Gram negative pathogens

Resistance to antibiotics is becoming an important global public health problem. Infectious diseases are still among the major causes of disease and death in many parts of the world and the possibilities to treat bacterial infections are now threatened due to the spread of antibiotic resistance. Trimethoprim and sulphonamides are widely accessible and affordable antibiotics which act by inhibiting the folic acid synthesis in bacteria. Trimethoprim and its combination with the sulphonamide sulphamethoxazole, are considered first- and second-line treatment for many infections, such as uncomplicated and severe urinary tract infections. Gram negative bacteria usually become resistant to these drugs by acquiring genes encoding additional enzymes which are not inhibited by the drugs. Such enzymes are encoded by dfr- and sul-genes which can efficiently be spread among bacteria. The majority of the dfr-genes are carried as gene cassettes in a genetic element called integron. Integrons can insert and express gene cassettes conferring resistance to various different antibiotics and are thus important tools for spread of resistance in pathogenic bacteria. In the present studies, a material of 105 Gram negative bacterial strains from urinary samples has been thoroughly investigated. Additional studies have also been performed on other selected materials of E. coli bacteria. The presence of integrons, dfr-genes and sulgenes has been mapped. A subset of detected integrons has been sequenced to explore the carriage of antibiotic resistance gene cassettes in these elements. In strains where no known integron, dfr- or sul-gene could be detected, further studies by means of shotgun cloning experiments were performed to understand the underlying mechanisms for resistance and the spread of such determinants. Two multiplex PCR screening methods for the five most frequently occurring dfr-genes have also been developed and tested in these studies. Gene cassettes in the integrons studied in these strains only contained genes encoding resistance to trimethoprim and two rarely used antibiotics of the aminoglycoside type. The acquisition of other antibiotic resistance determinants must hence be explained by additional genetic dissemination mechanisms in these isolates. The new su13 gene was in the present studies for the first time described in pathogens from humans. The gene was also shown to be present in several isolates from healthy pigs and a spread of this gene from animals to humans was suggested. As many as four new dfr-genes, dfr2d, dfrA22, dfrA24 and dfrA26, were described in the investigated material. The first two of these are carried as gene cassettes in integrons while the third is spread by an unknown mechanism and the fourth was shown to be connected to the recently described elements called the common regions. Common regions have been found to capture and spread a large number of various antibiotic resistance genes unrelated to gene cassettes. As confirmed by the description of dfrA26 in these studies, almost all dfr-genes now fall into the two categories as disseminated either by integrons or by common regions. The importance of the association of resistance to trimethoprim and probably also sulphonamides with common regions can be expected to increase in the future

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No. of <i>E. coli</i> (n = 320) isolates resistant to trimethoprim only and in combination with other antibiotics and in relation to the presence of integrons.

<p>TMP = trimethoprim, AMP = ampicillin, NAL = nalidixic acid, MEC = mecillinam, CFR = cefadroxil and NIT = nitrofurantoin.</p

Primers not previously published used in the PCR screenings.

<p>Primers not previously published used in the PCR screenings.</p

The relationship between <i>dfr-</i>genes and integron class I and II in <i>E. coli</i> (n = 320) and <i>K. pneumoniae</i> (n = 54).

a<p>Including 10 isolates positive for both Igr1 and 2.</p>b<p>Including one isolate positive for both Igr 1 and 2.</p

No. of <i>K. pneumoniae</i> (n = 54) isolates resistant to trimethoprim only and in combination with other antibiotics and in relation to the presence of integrons.

<p>TMP = trimethoprim, AMP = ampicillin, NAL = nalidixic acid, MEC = mecillinam, CFR = cefadroxil and NIT = nitrofurantoin.</p

Changes in patterns of antibiotic use in Chinese public hospitals (2005-2012) and a benchmark comparison with Sweden in 2012

Changes in patterns of antibiotic use in Chinese hospitals before and after intensive nationwide interventions are reported and compared with Chinese national targets and antibiotic use in Swedish hospitals. Chinese data were collected quarterly and yearly from selected patient prescriptions/medical records and medicines inventory control systems from 15 hospitals (2005-2012). Swedish data were extracted from a 2010-point prevalence survey and 2009-2012 sales data from seven university hospitals. An interrupted time series with segmented regression analysis was used to measure changes in patterns of antibiotic use in Chinese hospitals before and after the interventions. Following the 2011 interventions, significant reductions in antibiotic use in Chinese hospitals were seen: the proportion of prescriptions with antibiotics decreased 4.7% (P=0.03) and the proportion of medical records with antibiotic prescription decreased 73% (P=0.04). The proportions of prescriptions and medical records with antibiotics in Chinese hospitals in 2012 were 10% and 50%, respectively, and remained much higher than Swedish hospitals (1.1% in DDD for outpatients and 34% in number of patients for inpatients). Inpatient consumption in Chinese hospitals dropped significantly from 910 DDD/1000 inpatient days in 2008 to 473 in 2012 (588 in Swedish hospitals). Antibiotics are being used less frequently in Chinese hospitals, broad-spectrum antibiotics are still preferred, and overall usage is higher than Sweden. A significant reduction in overall inpatient antibiotic consumption was observed after the interventions. It is not possible to identify whether the-changes have resulted in less inappropriate antibiotic use. Further studies are needed. (C) 2015 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved