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Extended-spectrum beta-lactamase producing Enterobacteriaceae: aspects on detection, epidemiology and multi-drug resistance
Linköping University, Department of Clinical and Experimental Medicine, Clinical Microbiology. Linköping University, Faculty of Health Sciences.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Beta-lactam antibiotics are the largest and most commonly used group of antimicrobial agents in Sweden as well as world-wide. They show very good tolerability and many of the drugs can be administrated orally. Bacteria expressing extended-spectrum beta-lactamases (ESBLs), enzymes hydrolysing penicillins and cephalosporins, may not respond to therapy using some of these antibiotics. The isolates are also often co-resistant to other antimicrobial agents, thus further limiting treatment options. Often parenterally administrated carbapenems is one of few safe treatment options left.

In this thesis we have investigated the occurrence of ESBL producing Enterobacteriaceae in clinical isolates from Östergötland, Sweden, from 2002 until end of 2007 and the occurrence of multiresistance among ESBL producing E. coli. During these investigations we developed a simple method well suited for high-throughput analysis, for detection and sub typing of common ESBL genes.

During the six year period, the prevalence of ESBL producing Enterobacteriaceae in Östergötland was very low, <1%, but increasing. The number of patients with ESBL producing E. coli increased significantly from 5 to 47 per year; K. pneumoniae remained between one and four per year. The genes found were dominated by CTX-M group 1 (67%), followed by group 9 (27%). There has been no reason to suspect an outbreak of nosocomial origin. The total consumption of antimicrobial agents was 10.7-12.1 DID per year in primary care; 1.14-1.30 DID per year in hospital care.

Of eight oral agents tested, only three showed a generally high susceptibility; mecillinam (91%), nitrofurantoin (96%) and fosfomycin (99%). The corresponding figures for the fifteen tested parenterally administrated drugs were; amikacin (96%), tigecycline (99%), colistin (99%) and ≥99% susceptibility for the carbapenems.

Sixty eight percent of the isolates were multiresistant. The most common multiresistance pattern was ESBL phenotype with decreased susceptibility to trimethoprim, trimethoprimsulfamethoxazole, ciprofloxacin, gentamicin and tobramycin. A significant difference in susceptibility between CTX-M groups, in favor of group 9 over group 1, was seen for many of the antibiotics tested; amoxicillin-clavulanic acid, aztreonam, cafepime, ceftibuten, ceftazidime, ciprofloxacin, gentamicin, piperacillin-tazobactam, temocillin, and tobramycin.

In conclusion this thesis shows that the prevalence of ESBL producing Enterobacteriaceae in Östergötland was very low but increasing, and the total consumption of antimicrobial agents was stable. A majority of the isolates were multiresistant and a significant difference in susceptibility between CTX-M groups, in favor of group 9 over group 1, was seen for many of the antimicrobial agents tested.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2012. , 53 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1300
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-76134ISBN: 978-91-7519-938-2 (print)OAI: oai:DiVA.org:liu-76134DiVA: diva2:512611
Public defence
2012-04-26, Berzeliussalen, ingång 65, plan 09, Campus US, Linköpings universitet, Linköping, 13:00 (Swedish)
Opponent
Supervisors
Available from: 2012-03-28 Created: 2012-03-28 Last updated: 2012-03-28Bibliographically approved
List of papers
1. Molecular identification of blaCTX-M and blaOXY/K1 beta-lactamase genes in Enterobacteriaceae by sequencing of universal M13-sequence tagged PCR-amplicons.
Open this publication in new window or tab >>Molecular identification of blaCTX-M and blaOXY/K1 beta-lactamase genes in Enterobacteriaceae by sequencing of universal M13-sequence tagged PCR-amplicons.
2009 (English)In: BMC Infectious Diseases, ISSN 1471-2334, E-ISSN 1471-2334, Vol. 9, no 1, 7- p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Plasmid encoded blaCTX-M enzymes represent an important sub-group of class A beta-lactamases causing the ESBL phenotype which is increasingly found in Enterobacteriaceae including Klebsiella spp. Molecular typing of clinical ESBL-isolates has become more and more important for prevention of the dissemination of ESBL-producers among nosocomial environment.

METHODS: Multiple displacement amplified DNA derived from 20 K. pneumoniae and 34 K. oxytoca clinical isolates with an ESBL-phenotype was used in a universal CTX-M PCR amplification assay. Identification and differentiation of blaCTX-M and blaOXY/K1 sequences was obtained by DNA sequencing of M13-sequence-tagged CTX-M PCR-amplicons using a M13-specific sequencing primer.

RESULTS: Nine out of 20 K. pneumoniae clinical isolates had a blaCTX-M genotype. Interestingly, we found that the universal degenerated primers also amplified the chromosomally located K1-gene in all 34 K. oxytoca clinical isolates. Molecular identification and differentiation between blaCTX-M and bla OXY/K1-genes could only been achieved by sequencing of the PCR-amplicons. In silico analysis revealed that the universal degenerated CTX-M primer-pair used here might also amplify the chromosomally located blaOXY and K1-genes in Klebsiella spp. and K1-like genes in other Enterobacteriaceae.

CONCLUSION: The PCR-based molecular typing method described here enables a rapid and reliable molecular identification of blaCTX-M, and blaOXY/K1-genes. The principles used in this study could also be applied to any situation in which antimicrobial resistance genes would need to be sequenced.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-17021 (URN)10.1186/1471-2334-9-7 (DOI)19161622 (PubMedID)
Note
Original Publication:Hans-Jurg Monstein, Maria Tärnberg and Lennart Nilsson, Molecular identification of blaCTX-M and blaOXY/K1 beta-lactamase genes in Enterobacteriaceae by sequencing of universal M13-sequence tagged PCR-amplicons., 2009, BMC infectious diseases, (9), 1, 7.http://dx.doi.org/10.1186/1471-2334-9-7Copyright: BioMed Centralhttp://www.biomedcentral.com/Available from: 2009-03-03 Created: 2009-03-03 Last updated: 2017-12-13Bibliographically approved
2. Molecular identification of blaSHV, blaLEN and blaOKP β-lactamase genes in Klebsiella pneumoniae by bi-directional sequencing of universal SP6- and T7-sequence-tagged blaSHV-PCR amplicons
Open this publication in new window or tab >>Molecular identification of blaSHV, blaLEN and blaOKP β-lactamase genes in Klebsiella pneumoniae by bi-directional sequencing of universal SP6- and T7-sequence-tagged blaSHV-PCR amplicons
2009 (English)In: Molecular and Cellular Probes, ISSN 1044-7431, E-ISSN 1095-9327, Vol. 23, no 3-4, 195-200 p.Article in journal (Refereed) Published
Abstract [en]

Plasmid encoded blaSHV enzymes represent an important sub-group of class A β-lactamases causing an ESBL-phenotype which is increasingly found in Enterobacteriaceae including Klebsiella pneumoniae. The chromosomally encoded β-lactamase blaLEN and blaOKP enzymes, which so far only have been found in K. pneumoniae, do not hydrolyse extended-spectrum cephalosporins. In the present study, multiple displacement amplified DNA derived from 20 K. pneumoniae clinical isolates with a blaSHV-like genotype was used in a universal SHV PCR assay using SP6- (forward) and T7- (reverse) sequence-tagged primers. Identification and differentiation of blaSHV, blaLEN and blaOKP genes was obtained by bi-directional amplicon sequencing using SP6- and T7-specific primers. Three well characterised K. pneumoniae strains having a SHV-genotype were included in the study. The bi-directional amplicon sequencing, covering 800 bp (93%) of the blaSHV, blaLEN and blaOKP enzyme encoding sequences, allowed for an unequivocal discrimination of SHV, LEN and OKP genes. Moreover, sequencing revealed the presence of blaSHV allelic variants in six K. pneumoniae isolates in which the amplicons had to be cloned accordingly. Based on deduced amino-acid sequences, a dendrogram was constructed. Seventeen out of 20 K. pneumoniae isolates with an ESBL-phenotype formed a SHV-like cluster, two were LEN-like, and one isolate was OKP-like. The PCR-based molecular typing method described here enables a rapid, reliable and cost-effective identification and differentiation of blaSHV, blaOKP and blaLEN genes.

Keyword
Klebsiella pneumoniae; ESBL genes; Concurrent bi-directional SP6- and T7-sequence-tagged; PCR amplicon sequencing
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-17067 (URN)10.1016/j.mcp.2009.01.005 (DOI)
Note
Original Publication: Maria Tärnberg, Lennart E. Nilsson and Hans-Jürg Monstein, Molecular identification of blaSHV, blaLEN and blaOKP β-lactamase genes in Klebsiella pneumoniae by bi-directional sequencing of universal SP6- and T7-sequence-tagged blaSHV-PCR amplicons, 2009, Molecular and Cellular Probes, (23), 3-4, 195-200. http://dx.doi.org/10.1016/j.mcp.2009.01.005 Copyright: Elsevier Science B.V., Amsterdam http://www.elsevier.com/ Available from: 2009-06-09 Created: 2009-03-05 Last updated: 2017-12-13Bibliographically approved
3. Prevalence of extended-spectrum beta-lactamase-producing Enterobacteriaceae and trends in antibiotic consumption in a county of Sweden
Open this publication in new window or tab >>Prevalence of extended-spectrum beta-lactamase-producing Enterobacteriaceae and trends in antibiotic consumption in a county of Sweden
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2010 (English)In: Scandinavian Journal of Infectious Diseases, ISSN 0036-5548, E-ISSN 1651-1980, Vol. 42, no 11-12, 831-838 p.Article in journal (Refereed) Published
Abstract [en]

In the last decade extended-spectrum beta-lactamase (ESBL)-producing bacteria have become an increasing problem. Our aims were to investigate the prevalence of ESBL-producing Enterobacteriaceae and trends in antibiotic use in the county of Ostergotland, Sweden. From 2002 through 2007 there were 224 ESBL-producing Escherichia coli and 23 Klebsiella pneumoniae isolates with an ESBL-phenotype identified among all Enterobacteriaceae isolated at the clinical laboratory. Trends in antibiotic consumption expressed as defined daily doses (DDD) per 1000 inhabitants and day (DID) were studied. The prevalence of ESBL-producing isolates among Enterobacteriaceae in our region is still low (andlt; 1%). Patients with ESBL-producing E. coli increased significantly (p andlt; 0.001) from 5 in y 2002 to 47 in y 2007. CTX-M group 1 was the dominant enzyme group in both E. coli and K. pneumoniae. Antibiotic susceptibility testing of ciprofloxacin, gentamicin and trimethoprim-sulfamethoxazole revealed that 58% of E. coli and 50% of K. pneumoniae isolates were multi-resistant. Antibiotic use remained unchanged from 2001 through 2009, but there was a trend towards increased use of drugs with low ESBL selection potential, which was probably due to the increased prevalence of ESBL producers.

Place, publisher, year, edition, pages
Informa Healthcare, 2010
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-63145 (URN)10.3109/00365548.2010.498017 (DOI)000284168300006 ()
Available from: 2010-12-13 Created: 2010-12-13 Last updated: 2017-12-11Bibliographically approved
4. In vitro activity of beta-lactam antibiotics against CTX-M-producing Escherichia coli
Open this publication in new window or tab >>In vitro activity of beta-lactam antibiotics against CTX-M-producing Escherichia coli
Show others...
2011 (English)In: European Journal of Clinical Microbiology and Infectious Diseases, ISSN 0934-9723, E-ISSN 1435-4373, Vol. 30, no 8, 981-987 p.Article in journal (Refereed) Published
Abstract [en]

Beta-lactam antibiotics have been discussed as options for the treatment of infections caused by multiresistant extended-spectrum beta-lactamase (ESBL)-producing bacteria if the minimum inhibitory concentration (MIC) is low. The objective of this study was to investigate the in vitro activity of different beta-lactam antibiotics against CTX-M-producing Escherichia coli. A total of 198 isolates of E. coli with the ESBL phenotype were studied. Polymerase chain reaction (PCR) amplification of CTX-M genes and amplicon sequencing were performed. The MICs for amoxicillin-clavulanic acid, aztreonam, cefepime, cefotaxime, ceftazidime, ceftibuten, ertapenem, imipenem, mecillinam, meropenem, piperacillin-tazobactam, and temocillin were determined with the Etest. Susceptibility was defined according to the breakpoints of the European Committee on Antimicrobial Susceptibility Testing (EUCAST). MIC(50) and MIC(90) values were calculated. Isolates from CTX-M group 9 showed higher susceptibility to the beta-lactam antibiotics tested than isolates belonging to CTX-M group 1. More than 90% of the isolates belonging to CTX-M group 9 were susceptible to amoxicillin-clavulanic acid, ceftazidime, ceftibuten, piperacillin-tazobactam, and temocillin. The susceptibility was high to mecillinam, being 91%, regardless of the CTX-M group. All isolates were susceptible to imipenem and meropenem, and 99% to ertapenem. This study shows significant differences in susceptibility to different beta-lactam antibiotics among the CTX-M-producing E. coli isolates and a significant difference for many antibiotics tested between the CTX-M-producing groups 1 and 9. The good in vitro activity of other beta-lactam antibiotics compared to carbapenems indicate that clinical studies are warranted in order to examine the potential role of these beta-lactam antibiotics in the treatment of infections caused by multiresistant ESBL-producing E. coli.

Place, publisher, year, edition, pages
Springer Science Business Media, 2011
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-69787 (URN)10.1007/s10096-011-1183-4 (DOI)000292553500008 ()
Available from: 2011-08-10 Created: 2011-08-08 Last updated: 2017-12-08Bibliographically approved
5. In vitro susceptibility of CTX-M-producing Escherichia coli to non-beta-lactam agents
Open this publication in new window or tab >>In vitro susceptibility of CTX-M-producing Escherichia coli to non-beta-lactam agents
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Background: The objective of this study was to investigate the in vitro activity of different antibiotics against CTX-M-producing Escherichia coli, to determine the occurrence of multiresistance and plasmid mediated quinolone resistance among these isolates.

Methods: A total of 198 isolates of E. coli with ESBL phenotype and mainly CTX-M genotype, were studied. The MICs for amikacin, chloramphenicol, ciprofloxacin, colistin, fosfomycin, gentamicin, nalidixic acid, nitrofurantoin, tigecycline, tobramycin, trimethoprim and trimethoprim-sulphamethoxazole were determined with the Etest. Susceptibility was defined according to the breakpoints of the European Committee on Antimicrobial Susceptibility Testing (EUCAST). MIC(50) and MIC(90) values were calculated.

Results: ≥95% of isolates were susceptible to amikacin, nitrofurantoin, colistin, tigecyclin, and fosfomycin. CTX-M group 9 was more susceptible than CTX-M group 1 to ciprofloxacin, gentamicin, and tobramycin. 68% of the isolates were multiresistant, and the most common multi-resistance pattern was ESBL-phenotype with decreased susceptibility to trimethoprim, trimethoprim-sulphamethoxazol, ciprofloxacin, gentamicin and  tobramycin. Only one isolate carried a qnrS1-gene, nine isolates carried aac(6’)-Ib-cr.

Conclusions: The high frequency of multi-resistance found in this study is alarming and it is urgent to find strategies to limit the emergence and spread of these multi-resistant strains.

Keyword
Etest, MIC, ESBL
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-76133 (URN)
Available from: 2012-03-28 Created: 2012-03-28 Last updated: 2012-03-28Bibliographically approved

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