Biomedical subjects

Lenie Dijkshoorn

Publications and source records attributed to Lenie Dijkshoorn.

At least 19 recordsLinked to original sources

PubMed2006

Horizontal gene transfer in a polyclonal outbreak of carbapenem-resistant Acinetobacter baumannii.

In the last few years, phenotypically carbapenem resistant Acinetobacter strains have been identified throughout the world, including in many of the hospitals and intensive care units (ICUs) of Australia. Genotyping of Australian ICU outbreak-associated isolates by pulsed-field gel electrophoresis of whole genomic DNA indicated that different strains were cocirculating within one hospital. The carbapenem-resistant phenotype of these and other Australian isolates was found to be due to carbapenem-hydrolyzing activity associated with the presence of the blaOXA-23 gene. In all resistant strains examined, the blaOXA-23 gene was adjacent to the insertion sequence ISAba1 in a structure that has been found in Acinetobacter baumannii strains of a similar phenotype from around the world; blaOXA-51-like genes were also found in all A. baumannii strains but were not consistently associated with ISAba1, which is believed to provide the promoter required for expression of linked antibiotic resistance genes. Most isolates were also found to contain additional antibiotic resistance genes within the cassette arrays of class 1 integrons. The same cassette arrays, in addition to the ISAba1-blaOXA-23 structure, were found within unrelated strains, but no common plasmid carrying these accessory genetic elements could be identified. It therefore appears that antibiotic resistance genes are readily exchanged between cocirculating strains in epidemics of phenotypically indistinguishable organisms. Epidemiological investigation of major outbreaks should include whole-genome typing as well as analysis of potentially transmissible resistance genes and their vehicles.

Acinetobacter Infections↗

PubMed2006

AFLP fingerprinting for the analysis of genetic diversity within Necator americanus.

In the present study, we utilised the method of AFLP to screen for genetic variation within and among individuals of the blood-feeding human hookworm Necator americanus (Nematoda) from Africa, Asia and South America. A total of 45 adult worms (i.e. 20 from Ghana, 16 from Colombia and 9 from Nepal) were subjected to analysis using the restriction enzyme/primer combination HindIII+AG/BglII+AC. Cluster analysis divided N. americanus into multiple, genetically distinct groups, consistent with previous findings using ribosomal and mitochondrial DNA data sets. The results demonstrated the usefulness of AFLP fingerprinting for establishing genetic variation within N. americanus and reinforce its applicability to other parasitic helminths of human and/or veterinary health importance.

Animals↗

PubMed2006

Naturally transformable Acinetobacter sp. strain ADP1 belongs to the newly described species Acinetobacter baylyi.

Genotypic and phenotypic analyses were carried out to clarify the taxonomic position of the naturally transformable Acinetobacter sp. strain ADP1. Transfer tDNA-PCR fingerprinting, 16S rRNA gene sequence analysis, and selective restriction fragment amplification (amplified fragment length polymorphism analysis) indicate that strain ADP1 and a second transformable strain, designated 93A2, are members of the newly described species Acinetobacter baylyi. Transformation assays demonstrate that the A. baylyi type strain B2(T) and two other originally identified members of the species (C5 and A7) also have the ability to undergo natural transformation at high frequencies, confirming that these five strains belong to a separate species of the genus Acinetobacter, characterized by the high transformability of its strains that have been cultured thus far.

Acinetobacter↗

PubMed2006

Nosocomial bacteremia due to an as yet unclassified acinetobacter genomic species 17-like strain.

We describe a case of bacteremia due to an as yet unclassified Acinetobacter genomic species 17-like strain. The recognition of this microorganism as non-Acinetobacter baumannii may have important epidemiological implications, as it relieves the hospital of the implementation of barrier precautions for patients infected or colonized as may be necessary with a multiresistant A. baumannii epidemic.

Acinetobacter↗

PubMed2005

Adherence of Acinetobacter baumannii strains to human bronchial epithelial cells.

Acinetobacter baumannii is an important nosocomial pathogen, but the mechanisms contributing to its epidemicity and virulence are largely unknown. The organism is able to colonize skin and mucosal surfaces of the human host. Adherence of microorganisms to host cells is an important virulence factor as it is the initial step of the colonization process. In the present study, adherence of A. baumannii to human bronchial epithelial NCI-H(292) cells was examined by light and scanning electron microscopy. Thirty-seven strains were investigated including 18 from outbreaks, 16 not associated with outbreaks, and three for which an epidemic implication was unknown. Eight and 11 isolates belonged to European clone I and II, respectively. Two types of adherence were observed, dispersed adherence of bacteria to the cell, and adherence of clusters of bacteria at localized areas of the cells. Bacteria with dispersed adherence interacted with the epithelial cells through fimbriae, but were also entrapped by protrusions extending from the epithelial cells. Quantitative adherence varied considerably among strains but there was no significant correlation of the outbreak-associated strains with the percentage of infected cells. There was, however, a correlation between the clonal lineage and the percent of infected cells, with clone II being more adherent than clone I (P<0.05). Ten consecutive isolates from one outbreak were investigated to test whether adherence increased during passage among patients, but this appeared not to be the case. This study showed that A. baumannii adheres to human bronchial epithelial cells in vitro and that A. baumannii strains of clone II had a relatively high capacity for adhering to these cells.

Acinetobacter Infections↗

PubMed2005

Differential roles of CD14 and toll-like receptors 4 and 2 in murine Acinetobacter pneumonia.

RATIONALE: Acinetobacter baumannii is an opportunistic bacterial pathogen that is increasingly associated with gram-negative nosocomial pneumonia, but the molecular mechanisms that play a role in innate defenses during A. baumannii infection have not been elucidated. OBJECTIVE: To gain first insight into the role of CD14 and Toll-like receptors 4 and 2 in host response to A. baumannii pneumonia. METHODS: Respective gene-deficient mice were intranasally infected with A. baumannii, and bacterial outgrowth, lung inflammation, and pulmonary cytokine/chemokine responses were determined. To study the importance of LPS in the inflammatory response, mice were also challenged with A. baumannii LPS. MEASUREMENTS AND MAIN RESULTS: Bacterial counts were increased in CD14 and Toll-like receptor 4 gene-deficient mice, and only these animals developed bacteremia. The pulmonary cytokine/chemokine response was impaired in Toll-like receptor 4 knockout mice and the onset of lung inflammation was delayed. In contrast, Toll-like receptor 2-deficient animals displayed an earlier cell influx into lungs combined with increased macrophage inflammatory protein-2 and monocyte chemoattractant protein-1 concentrations, which was associated with accelerated elimination of bacteria from the pulmonary compartment. Neither CD14 nor Toll-like receptor 4 gene-deficient mice responded to intranasal administration of LPS, whereas Toll-like receptor 2 knockout mice were indistinguishable from wild-type animals. CONCLUSIONS: Our results suggest that CD14 and Toll-like receptor 4 play a key role in innate sensing of A. baumannii via the LPS moiety, resulting in effective elimination of the bacteria from the lung, whereas Toll-like receptor 2 signaling seems to counteract the robustness of innate responses during acute A. baumannii pneumonia.

Acinetobacter Infections↗

PubMed2005

Rapid epidemiological analysis of Acinetobacter strains by Raman spectroscopy.

From earlier publications, we noticed that Raman spectra could potentially be used for subspecies identification of microorganisms. Here we evaluated the technique for its use as a typing tool of Acinetobacter species, using a collection of well-characterised strains from five hospital outbreaks. The strains were previously analysed using molecular techniques as cell envelope protein profiling and ribotyping. In this study, we have typed the strains by AFLP analysis and Raman spectroscopy. We compared the results using hierarchical cluster analysis, which showed highly similar groupings by both techniques. There seemed to be some misclassification between two sets of outbreak strains in the Raman analysis. We ascribe this to the clonal relationship between the strains of both outbreaks, described earlier. This results from a highly similar biochemical composition of the strains involved, and hence a highly similar Raman spectrum. We conclude that Raman spectroscopy could be an easy-to-use alternative in epidemiological studies of Acinetobacter strains and a promising starting point for the development of epidemiological studies in general.

Acinetobacter↗

PubMed2005

Characterization of a 2.6 kbp variable region within a class 1 integron found in an Acinetobacter baumannii strain isolated from a horse.

OBJECTIVES: A complete gene cassette contained in a class 1 integron from a multidrug-resistant (MDR) isolate of Acinetobacter baumannii cultured from a horse was characterized by molecular methods. METHODS: Template genomic DNA purified from the A. baumannii isolate was investigated by PCR. A gene cassette-associated amplicon was detected and completely characterized. RESULTS: A 2.6 kbp DNA fragment containing four gene cassettes was amplified from the MDR A. baumannii isolate. Sequence analysis showed it was similar to sequences recently reported in Klebsiella pneumoniae, Serratia marcescens and an Escherichia coli plasmid p1658/97 which conferred aminoglycoside resistance. Aminoglycoside resistance-encoding genes aacC1 and aadA1 were located within the 2.6 kbp amplicon, separated by two open reading frames (ORFs) coding for unknown products. This cassette structure (and some variants) was identified in unrelated Acinetobacter spp. from human sources, based on sequence comparisons of the current databases. CONCLUSIONS: Identification of a complete class 1 integron in an equine isolate of A. baumannii suggests that the screening of isolates from animals for these elements should be considered, as this information could influence the selection of chemotherapeutic agents.

Acinetobacter baumannii↗

PubMed2005

Repetitive-DNA-element PCR fingerprinting and antibiotic resistance of pan-European multi-resistant Acinetobacter baumannii clone III strains.

In the present study, it was shown that repetitive-DNA-element PCR fingerprinting using the (GTG)5 primer [(GTG)5-PCR] is a rapid and reliable tool to genotypically differentiate members of the recently described pan-European multi-resistant Acinetobacter baumannii (MAB) clone III from the known MAB clones I and II. The identification of four new representatives of the MAB clone III dating from 1991 to 1993 by (GTG)5-PCR indicates that this clone has persisted in European hospitals since the beginning of the 1990s. Tetracycline (TET) resistance was found to be common among clone III strains, including one strain that also displayed resistance to minocycline. The TET-resistance phenotype in this MAB clone appeared to be strongly associated with the presence of the efflux-type gene tet(A), but the fact that some members lack this gene or have acquired an additional tet gene [i.e. tet(M)] suggests that the tet gene carriage in the pan-European clone III population may have diversified in time and space. In contrast, all clone III strains shared the previously described aminoglycoside resistotype encoded by the aminoglycoside-modifying genes aphA6 and the class 1 integron-associated aadB, which may point to the fact that these genes probably are more stably inherited in MAB clone III compared to tet genes.

Acinetobacter Infections↗

PubMed2005

Species-level identification of isolates of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex by sequence analysis of the 16S-23S rRNA gene spacer region.

The species Acinetobacter calcoaceticus, A. baumannii, genomic species 3, and genomic species 13TU included in the Acinetobacter calcoaceticus-Acinetobacter baumannii complex are genetically highly related and difficult to distinguish phenotypically. Except for A. calcoaceticus, they are all important nosocomial species. In the present study, the usefulness of the 16S-23S rRNA gene intergenic spacer (ITS) sequence for the differentiation of (genomic) species in the A. calcoaceticus-A. baumannii complex was evaluated. The ITSs of 11 reference strains of the complex and 17 strains of other (genomic) species of Acinetobacter were sequenced. The ITS lengths (607 to 638 bp) and sequences were highly conserved for strains within the A. calcoaceticus-A. baumannii complex. Intraspecies ITS sequence similarities ranged from 0.99 to 1.0, whereas interspecies similarities varied from 0.86 to 0.92. By using these criteria, 79 clinical isolates identified as A. calcoaceticus (18 isolates) or A. baumannii (61 isolates) with the API 20 NE system (bioMerieux Vitek, Marcy l'Etoile, France) were identified as A. baumannii (46 isolates), genomic species 3 (19 isolates), and genomic species 13TU (11 isolates) by ITS sequencing. An identification rate of 96.2% (76 of 79 isolates) was obtained by using ITS sequence analysis for identification of isolates in the A. calcoaceticus-A. baumannii complex, and the accuracy of the method was confirmed for a subset of strains by amplified rRNA gene restriction analysis and genomic DNA analysis by AFLP analysis by using libraries of profiles of reference strains. In conclusion, ITS sequence-based identification is reliable and provides a promising tool for elucidation of the clinical significance of the different species of the A. calcoaceticus-A. baumannii complex.

Acinetobacter baumannii↗

PubMed2005

Standardization and interlaboratory reproducibility assessment of pulsed-field gel electrophoresis-generated fingerprints of Acinetobacter baumannii.

A standard procedure for pulsed-field gel electrophoresis (PFGE) of macrorestriction fragments of Acinetobacter baumannii was set up and validated for its interlaboratory reproducibility and its potential for use in the construction of an Internet-based database for international monitoring of epidemic strains. The PFGE fingerprints of strains were generated at three different laboratories with ApaI as the restriction enzyme and by a rigorously standardized procedure. The results were analyzed at the respective laboratories and also centrally at a national reference institute. In the first phase of the study, 20 A. baumannii strains, including 3 isolates each from three well-characterized hospital outbreaks and 11 sporadic strains, were distributed blindly to the participating laboratories. The local groupings of the isolates in each participating laboratory were identical and allowed the identification of the epidemiologically related isolates as belonging to three clusters and identified all unrelated strains as distinct. Central pattern analysis by using the band-based Dice coefficient and the unweighted pair group method with mathematical averaging as the clustering algorithm showed 95% matching of the outbreak strains processed at each local laboratory and 87% matching of the corresponding strains if they were processed at different laboratories. In the second phase of the study, 30 A. baumannii isolates representing 10 hospital outbreaks from different parts of Europe (3 isolates per outbreak) were blindly distributed to the three laboratories, so that each laboratory investigated 10 epidemiologically independent outbreak isolates. Central computer-assisted cluster analysis correctly identified the isolates according to their corresponding outbreak at an 87% clustering threshold. In conclusion, the standard procedure enabled us to generate PFGE fingerprints of epidemiologically related A. baumannii strains at different locations with sufficient interlaboratory reproducibility to set up an electronic database to monitor the geographic spread of epidemic strains.

Acinetobacter Infections↗

PubMed2004

Distribution of tetracycline resistance genes in genotypically related and unrelated multiresistant Acinetobacter baumannii strains from different European hospitals.

The presence of tetracycline (TET) resistance genes was investigated in 49 genotypically related and unrelated multidrug-resistant Acinetobacter baumannii (MDRAB) strains from European hospitals including representatives of pan-European clones I and II. Except for one strain, all MDRAB strains displayed resistance to tetracycline (MIC range of 16 to > 512 microg/ml) but were susceptible (MIC < 4 microg/ml) or exhibited intermediate resistance (MIC of 4-8 microg/ml) to minocycline (MIN). In 37 strains, either tet(A) or tet(B) was detected and one of these strains possessed both tet(A) and tet(M). In addition, all MDRAB strains contained the aspecific efflux gene adeB irrespectively of whether they harbored tet genes or not. Repetitive DNA element (rep)-PCR fingerprinting using the (GTG)5 primer [(GTG)5-PCR] revealed that strains previously assigned to pan-European clones I and II were grouped into two separate clusters. In addition, these clusters also contained strains that had not been typed previously, indicating that (GTG)5-PCR is a valuable method for recognizing putative new members of MDRAB clones. Most, but not all, members of clones I and II were linked to the presence of either tet(A) or tet(B) and displayed different levels of TET resistance with MIC values of 32 to > 512 microg/ml and > 512 microg/ml, respectively. Of these two genes only tet(B) encodes an efflux of both TET and MIN, which was reflected by the relatively high MIC values for MIN (4 microg/ml) shown by the majority of the tet(B)-carrying clone II strains as opposed to the low MIC values for MIN (< 1 microg/ml) displayed by most tet(A)-containing clone I strains. Collectively, our phenotypic and genotypic resistance data support the therapeutic evaluation of second-generation tetracyclines like MIN as promising agents for treating MDRAB infections.

Acinetobacter Infections↗

PubMed2004

Identification of a new geographically widespread multiresistant Acinetobacter baumannii clone from European hospitals.

The aim of the study was to investigate the genetic diversity of Acinetobacter baumannii clinical strains that had previously been allocated to three major groups based on automated ribotyping. Forty-seven isolates from European hospitals and one isolate from a South African hospital, geographically representative of the three ribogroups (ribogroups 1, 2 and 3 with 10, 23 and 15 isolates, respectively), were analysed using the highly discriminatory fingerprinting methods AFLP and pulsed-field gel electrophoresis (PFGE). Based on AFLP data, the isolates clustered into three main groups, each corresponding to one ribogroup. Inclusion of reference strains of the previously described clones I and II, responsible for outbreaks in northwestern European hospitals, showed that ribogroups 1 and 2 correspond to clones I and II, respectively, whereas ribogroup 3 apparently represents a new clone. This clone III was found in France, The Netherlands, Italy and Spain. Clones I and II were not limited to northwestern European countries, as they were also recovered from Spain, South Africa, Poland and Italy (clone I) and from Spain, Portugal, South Africa, France, Greece and Turkey (clone II). Combined AFLP and PFGE data showed intraclonal diversity and led to the distinction of 23 different genotypes. Three genotypes, two of them belonging to clone II and one to clone III, were found in different hospitals and may correspond to subsets of isolates with a more recent clonal relationship, which emphasizes the epidemic potential of these organisms.

Acinetobacter Infections↗

PubMed2004

Long-term predominance of two pan-European clones among multi-resistant Acinetobacter baumannii strains in the Czech Republic.

In a recent study, a large proportion of multi-drug-resistant (MDR) Acinetobacter baumannii strains that were isolated from hospitalized patients in the Czech Republic was found to belong to two major groups (A and B). These groups appeared to be similar to epidemic clones I and II, respectively, which were identified previously among outbreak strains from north-western European hospitals. The aim of the present study was to assess in detail the genetic relatedness of Czech A. baumannii strains and those of epidemic clones I and II by using ribotyping with HindIII and HincII and by AFLP fingerprinting. The study collection included 70 MDR strains that were isolated in 30 Czech hospitals in 1991-2001, 15 susceptible Czech strains from 1991 to 1996 and 13 reference strains of clones I and II from 1982 to 1990. One major HindIII/HincIII ribotype (R1-1) was observed in 38 MDR Czech strains and eight reference strains of clone I, whereas another major ribotype (R2-2) was observed in 11 MDR Czech strains and in three reference strains of clone II. A selection of 59 Czech strains (representative of all ribotypes) and the 13 reference strains were investigated by AFLP fingerprinting. At a clustering level of 83%, two large clusters could be distinguished: cluster 1 included all reference strains of clone I and 25 MDR Czech strains, whilst cluster 2 contained all reference strains of clone II and 11 MDR Czech strains. There was a clear correlation between the groupings by AFLP analysis and by ribotyping, as all strains with ribotype R1-1 and four strains with slightly different ribotypes were found in AFLP cluster 1, whereas all strains with ribotype R2-2 and seven strains with similar ribotypes were in AFLP cluster 2. Thus, 41 and 21 MDR Czech strains could be classified as belonging to clones I and II, respectively. The remaining eight MDR and 15 susceptible strains were highly heterogeneous and were distinct from clones I and II by both AFLP fingerprinting and ribotyping. These results indicate that the two predominant groups observed among MDR Czech A. baumannii strains from the 1990s are genetically congruent with the north-western European epidemic clones that were found in the 1980s. Recognition of these clinically relevant, widespread clones is important in infection prevention and control; they are also interesting subjects to study genetic mechanisms that give rise to their antibiotic resistance and epidemic behaviour.

Acinetobacter Infections↗

PubMed2004

Diversity of aminoglycoside-resistance genes and their association with class 1 integrons among strains of pan-European Acinetobacter baumannii clones.

The purpose of the present study was to investigate the diversity of the genes encoding aminoglycoside-modifying enzymes and their association with class 1 integrons in three pan-European clones of Acinetobacter baumannii. The study collection included 106 multidrug-resistant strains previously allocated to clone I (n = 56), clone II (n = 36) and clone III (n = 6) and a heterogeneous group of other strains (n = 8), using AFLP fingerprinting and ribotyping. The strains were from hospitals of the Czech Republic (n = 70; collected 1991-2001) and 12 other European countries (n = 36; 1982-1998). Using PCR, at least one of the following aminoglycoside-resistance genes was detected in 101 (95 %) strains: aphA1 (n = 76), aacC1 (n = 68), aadA1 (n = 68), aphA6 (n = 55), aadB (n = 31), aacC2 (n = 7) and aacA4 (n = 3). A combination of two to five different resistance genes was observed in 89 strains (84 %), with a total of 12 different combinations. PCR mapping revealed that aacC1, aadA1 and aacA4 were each associated with a class 1 integron, as was the case with aadB for six strains of clone III. Six different class 1 integron variable regions were detected in 78 strains (74 %), with two predominant regions (2.5 and 3.0 kb) in two sets of 34 strains each. The 3.0 kb region contained five gene cassettes (aacC1, orfX, orfX, orfX', aadA1) and differed from the 2.5 bp region only by one additional orfX cassette. These two integron regions were confined to clones I and II and were found in strains isolated in seven countries between 1982 and 2001. The clone III strains were homogeneous both in resistance genes and in integron variable regions, whereas clones I and II showed a remarkable intraclonal diversity of these properties, with no clear-cut difference between the two clones. Yet, within the Czech clone I and II strains, the diversity of resistance genes and integron structures was limited as compared to those from other countries. The occurrence of identical resistance genes, gene combinations and class 1 integrons associated with these genes in clonally distinct strains indicates that horizontal gene transfer plays a major role in the dissemination of aminoglycoside resistance in A. baumannii.

Acinetobacter baumannii↗

PubMed2004

The synthetic N-terminal peptide of human lactoferrin, hLF(1-11), is highly effective against experimental infection caused by multidrug-resistant Acinetobacter baumannii.

The lactoferrin-derived peptide hLF(1-11), but not its control peptide, was highly effective against five multidrug-resistant Acinetobacter baumannii strains in vitro (3 to 4 log reduction) and against four of these strains in an experimental infection in mice (2 to 3 log reduction). Therefore, this peptide is a promising candidate as a novel agent against infections with multidrug-resistant A. baumannii.

Acinetobacter Infections↗

PubMed2004

Comparison of Acinetobacter baumannii isolates from United Kingdom hospitals with predominant Northern European genotypes by amplified-fragment length polymorphism analysis.

Acinetobacter baumannii isolates collected between 1999 and 2001 from 46 United Kingdom hospitals were compared with previously identified northern European genotypes by amplified-fragment length polymorphism (AFLP) analysis. Two predominant northern European genotypes associated with outbreaks in the mid-1980s had been superseded by new outbreak-associated genotypes.

Acinetobacter Infections↗

PubMed2004

Characterization of toxin A-negative, toxin B-positive Clostridium difficile isolates from outbreaks in different countries by amplified fragment length polymorphism and PCR ribotyping.

Clinical Clostridium difficile isolates of patients with diarrhea or pseudomembranous colitis usually produce both toxin A and toxin B, but an increasing number of reports mention infections due to toxin A-negative, toxin B-positive (A(-)/B(+)) strains. Thirty-nine clinical toxin A(-)/B(+) isolates, and 12 other unrelated isolates were obtained from Canada, the United States, Poland, the United Kingdom, France, Japan, and The Netherlands. The isolates were investigated by high-resolution genetic fingerprinting by use of amplified fragment length polymorphism (AFLP) and two well-described PCR ribotyping methods. Furthermore, the toxin profile and clindamycin resistance were determined. Reference strains of C. difficile representing 30 known serogroups were also included in the analysis. AFLP discriminated 29 types among the reference strains, whereas the two PCR ribotyping methods distinguished 25 and 26 types. The discriminatory power of AFLP and PCR ribotyping among 12 different unrelated isolates was similar. Typing of 39 toxin A(-)/B(+) isolates revealed 2 AFLP types and 2 and 3 PCR ribotypes. Of 39 toxin A(-)/B(+) isolates, 37 had PCR ribotype 017/20 and AFLP type 20 (95%). A deletion of 1.8 kb was seen in 38 isolates, and 1 isolate had a deletion of approximately 1.7 kb in the tcdA gene, which encodes toxin A. Clindamycin resistance encoded by the erm(B) gene was found in 33 of 39 toxin A(-)/B(+) isolates, and in 2 of the 12 unrelated isolates (P < 0.001, chi-square test). We conclude that clindamycin-resistant C. difficile toxin A(-)/B(+) strain (PCR ribotype 017/20, AFLP type 20, serogroup F) has a clonal worldwide spread.

Bacterial Proteins↗