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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
Linköping University, Department of Clinical and Experimental Medicine, Clinical Microbiology. Linköping University, Faculty of Health Sciences.
Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Clinical Microbiology.
Linköping University, Department of Clinical and Experimental Medicine, Clinical Microbiology. Linköping University, Faculty of Health Sciences. (Landstinget i Östergötland; Centre for Laboratory Medicine; Department of Molecular Biological Techniques; Laboratoriemedicinskt centrum; Molekylärbiologiska tekniklaboratoriet)
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.

Place, publisher, year, edition, pages
2009. Vol. 23, no 3-4, 195-200 p.
Keyword [en]
Klebsiella pneumoniae; ESBL genes; Concurrent bi-directional SP6- and T7-sequence-tagged; PCR amplicon sequencing
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-17067DOI: 10.1016/j.mcp.2009.01.005OAI: oai:DiVA.org:liu-17067DiVA: diva2:201569
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
In thesis
1. Extended-spectrum beta-lactamase producing Enterobacteriaceae: aspects on detection, epidemiology and multi-drug resistance
Open this publication in new window or tab >>Extended-spectrum beta-lactamase producing Enterobacteriaceae: aspects on detection, epidemiology and multi-drug resistance
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:nbn:se:liu:diva-76134 (URN)978-91-7519-938-2 (ISBN)
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

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Tärnberg, MariaNilsson, Lennart E.Monstein, Hans-Jürg

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