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Molecular identification of blaCTX-M and blaOXY/K1 beta-lactamase genes in Enterobacteriaceae by sequencing of universal M13-sequence tagged PCR-amplicons.
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)
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.
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.

Place, publisher, year, edition, pages
2009. Vol. 9, no 1, 7- p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-17021DOI: 10.1186/1471-2334-9-7PubMedID: 19161622OAI: oai:DiVA.org:liu-17021DiVA: diva2:201174
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
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)
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Supervisors
Available from: 2012-03-28 Created: 2012-03-28 Last updated: 2012-03-28Bibliographically approved

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