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Characterization of clinical enterococcal isolates in Swedish hospitals: studies on genetic relatedness and high-level gentamicin resistance
Linköping University, Department of Molecular and Clinical Medicine, Clinical Microbiology. Linköping University, Department of Molecular and Clinical Medicine, Infectious Diseases. Linköping University, Faculty of Health Sciences.
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

During the last few decades, and in parallel with increasing resistance to multiple antibiotics, enterococci have become one of the leading pathogens that cause nosocomial infections. High-level gentamicin resistant (HLGR) enterococci have become frequent. Thus, there are compelling reasons to control the transmission of enterococci with HLGR. Many different typing methods have been used for epidemiological typing of enterococci. Pulsed-field gel electrophoresis (PFGE) has been shown to be the most discriminating typing method and is currently considered the "gold standard" for typing of enterococci. However, PFGE is an expensive method and remains time-consuming, which may be of critical importance when comparing data obtained from numerous isolates.

The aim of this thesis was to characterize clinical enterococcal isolates from patients admitted to Swedish hospitals, with special focus on HLGR strains and their genetic relatedness. Our purpose was also to develop a faster PFGE protocol for typing of enterococci, as well as to investigate if the Phene Plate (PhP) system can be used as a rapid screening method for detection of genetically related isolates of enterococci. If this could be achieved, it would be possible to minimize the number of isolates subjected to PFGE typing, which would save time and money.

In paper I, we performed a thorough investigation of eight different parameters of importance for the separation of DNA fragments by PFGE. This resulted in a modified PFGE protocol for typing of enterococci with much enhanced resolution. HLGR E. faecalis isolates obtained from patients admitted to eight Swedish ICUs during 1996 and 1998 (paper II), and isolates obtained from patients with bacteremia in the County of Östergötland during the period 1994-2001 (paper III) were characterized by our modified PFGE method. We found that the majority (69%) of isolates from ICUs in the eastern and central parts of southern Sweden, belonged to one dominating cluster, and the same cluster was also found in blood isolates from Östergötland. In nearly all cases, HLGR was due to the presence of the aac(6 ')Ie-aph(2 '')la gene situated on a Tn5281-like transposon (paper II). In the County of Östergötland, HLGR E. faecalis was first isolated from blood cultures in 1996, and the first blood isolates of HLGR E. faecium were found in 1999. During the study period, only 4 HLGR E. faecium isolates were observed, and all of them showed unique PFGE patterns.

To evaluate the efficiency of the gentamicin disk diffusion method for detection of HLGR in clinical isolates of enterococci, all enterococcal blood isolates from paper III were studied with a 30-µg gentamicin disk as recommended by SRGA, and the method was found to have 100% sensitivity and specificity when compared with PCR.

A "biochemical fingerprinting" method (PhP) was compared with PFGE for epidemiological characterization of enterococci. In earlier studies of the PhP method, enterococci were collected mainly from the environment or from normal human flora. Our study indicates that PhP may be a useful screening method for clinical E. faecalis isolates, with a relatively high concordance with PFGE, but that it is less useful for E. faecium since the concordance for E. faecium was low. To fully evaluate PhP as a tool for epidemiological characterization of enterococci from clinical settings, and to address questions regarding the validity of PFGE, further studies using additional genotyping methods, preferably newer typing systems based on DNA sequencing such as MLST, are warranted.

Place, publisher, year, edition, pages
Linköping: Linköping Universitet , 2005. , 80 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 899
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-30067Local ID: 15528ISBN: 91-85299-12-X (print)OAI: oai:DiVA.org:liu-30067DiVA: diva2:250888
Public defence
2005-05-27, Berzeliussalen, Hälsouniversitet, Linköping, 09:00 (Swedish)
Opponent
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2012-10-02Bibliographically approved
List of papers
1. Modified pulsed-field gel electrophoresis protocol for typing of enterococci
Open this publication in new window or tab >>Modified pulsed-field gel electrophoresis protocol for typing of enterococci
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2002 (English)In: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, E-ISSN 1600-0463, Vol. 110, no 12, 869-874 p.Article in journal (Refereed) Published
Abstract [en]

Controlling the spread of vancomycin-resistant enterococci (VRE) is an important task in hospital epidemiology. Pulsed-field gel electrophoresis (PFGE) has become the golden standard for molecular epidemiological characterisation of enterococcal isolates. For separation of DNA fragments by PFGE, different electrophoresis conditions have been recommended, but none of these protocols allows a satisfactory separation of both small and large DNA fragments of enterococci simultaneously. In this study we have speeded up the preparation of chromosomal DNA and defined new electrophoresis conditions that enhance separation of small and large DNA fragments for subtyping of enterococci with a 24 h PFGE.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26482 (URN)10.1034/j.1600-0463.2002.1101205.x (DOI)11034 (Local ID)11034 (Archive number)11034 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-10-02Bibliographically approved
2. Genetic relatedness among Enterococcus faecalis with transposon-mediated high-level gentamicin resistance in Swedish intensive care units
Open this publication in new window or tab >>Genetic relatedness among Enterococcus faecalis with transposon-mediated high-level gentamicin resistance in Swedish intensive care units
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2003 (English)In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 52, no 2, 162-167 p.Article in journal (Refereed) Published
Abstract [en]

We studied 45 isolates of Enterococcus faecalis with high-level gentamicin resistance (HLGR), all but one concomitantly resistant to ciprofloxacin, and 25 ciprofloxacin-resistant isolates without HLGR for genetic relatedness using pulsed-field gel electrophoresis (PFGE). E. faecalis were isolated from patients admitted to intensive care units at eight hospitals in southern Sweden from December 1996 through December 1998. Genomic analysis by PFGE resulted in three clusters of genetically related isolates (designated clusters I, II and III) and 23 unique clones. Cluster I was found predominantly in the eastern and central parts of southern Sweden and clusters II and III in south-western Sweden. Among the 45 isolates with HLGR, 69% belonged to cluster I, 20% to cluster II, and 11% had unique PFGE patterns, which suggests that the majority of isolates with HLGR are closely related. Among the 25 ciprofloxacin-resistant isolates without HLGR, 68% had unique PFGE patterns, 12% belonged to cluster I and 20% to cluster III, which suggests the ciprofloxacin-resistant isolates are not related. All isolates with HLGR contained the aac(6)Ie-aph(2)Ia gene, which was carried on a Tn5281-like transposon in all isolates except one. We conclude that HLGR in E. faecalis was mainly due to dissemination of genetically related clones during the time studied, and that HLGR in these isolates was due to the presence of the aac(6)Ie-aph(2)Ia gene.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26483 (URN)10.1093/jac/dkg315 (DOI)11035 (Local ID)11035 (Archive number)11035 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-10-02Bibliographically approved
3. Genetic relatedness of Enterococcus faecalis isolates with high-level gentamicin resistance from patients with bacteraemia in the south east of Sweden 1994-2001
Open this publication in new window or tab >>Genetic relatedness of Enterococcus faecalis isolates with high-level gentamicin resistance from patients with bacteraemia in the south east of Sweden 1994-2001
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2004 (English)In: Scandinavian Journal of Infectious Diseases, ISSN 0036-5548, Vol. 36, no 6-7, 405-409 p.Article in journal (Refereed) Published
Abstract [en]

High-level gentamicin resistant (HLGR) enterococci (Enterococcus faecalis and Enterococcus faecium) have become a substantial nosocomial problem in many countries. In this study, we investigated the prevalence of HLGR enterococci and their genetic relatedness in blood culture isolates from patients with bacteraemia admitted to the 3 hospitals in Östergötland, a county in the south east of Sweden, during 1994–2001. 36 of 250 E. faecalis (14%) and 4 of 106 E. faecium isolates (4%) were shown by PCR to carry the aac(6′)-Ie-aph(2″)-Ia aminoglycoside modifying gene and these isolates were also classified as HLGR enterococci by the gentamicin antibiotic disk diffusion method. A majority of HLGR E. faecalis isolates (83%) belonged to the same cluster of genetically related isolates, according to the pulsed-field gel electrophoresis (PFGE) patterns, whereas all 4 HLGR E. faecium isolates had unique PFGE patterns. In conclusion, our study showed that in contrast to studies from many other countries, the presence of HLGR enterococci was more common in E. faecalis than in E. faecium and appeared the first time in 1996 and 1999, respectively. Bacteraemia with HLGR enterococci in Östergötland was mainly due to the spread of a cluster related of E. faecalis strains.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-24621 (URN)10.1080/00365540410020622 (DOI)6802 (Local ID)6802 (Archive number)6802 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2012-10-02Bibliographically approved
4. Phene Plate (PhP) biochemical fingerprinting: a screening method for epidemiological typing of enterococcal isolates?
Open this publication in new window or tab >>Phene Plate (PhP) biochemical fingerprinting: a screening method for epidemiological typing of enterococcal isolates?
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2005 (English)In: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, E-ISSN 1600-0463, Vol. 113, no 9, 603-612 p.Article in journal (Refereed) Published
Abstract [en]

Pulsed-field gel electrophoresis (PFGE) is currently considered the gold standard for genotyping of enterococci. However, PFGE is both expensive and time-consuming. The purpose of this study was to investigate whether the PhP system can be used as a reliable clinical screening method for detection of genetically related isolates of enterococci. If so, it should be possible to minimize the number of isolates subjected to PFGE typing, which would save time and money. Ninety-nine clinical enterococcal isolates were analysed by PhP (similarity levels 0.90–0.975) and PFGE (similarity levels ≤3 and ≤6 bands) and all possible pairs of isolates were cross-classified as matched or mismatched. We found that the probability that a pair of isolates (A and B) belonging to the same type according to PhP also belong to the same cluster according to PFGE, i.e. p(APFGE=BPFGE • APhP=BPhP), and the probability that a pair of isolates of different types according to PhP also belong to different clusters according to PFGE, i.e. p(APFGE≠BPFGE • APhP≠BPhP), was relatively high for E. faecalis (0.86 and 0.96, respectively), but was lower for E. faecium (0.51 and 0.77, respectively). The concordance which shows the probability that PhP and PFGE agree on match or mismatch was 86%–93% for E. faecalis and 54%–66% for E. faecium, which indicates that the PhP method may be useful for epidemiological typing of E. faecalis in the current settings but not for E. faecium.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-33468 (URN)10.1111/j.1600-0463.2005.apm_217.x (DOI)19490 (Local ID)19490 (Archive number)19490 (OAI)
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2012-10-02Bibliographically approved

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