Biomedical subjects

H Verbrugh

Publications and source records attributed to H Verbrugh.

53 records · Page 3Linked to original sources

PubMed1996

Serotyping, ribotyping, PCR-mediated ribosomal 16S-23S spacer analysis and arbitrarily primed PCR for epidemiological studies on Legionella pneumophila.

Fifty clinical and environmental isolates of Legionella pneumophila were typed serologically and by DNA fingerprinting using arbitrarily primed polymerase chain reaction (AP-PCR). Furthermore, variability in and around ribosomal operons was assessed by conventional ribotyping and PCR-mediated amplification of the spacer region separating the 16S and 23S genes. It appears that serotyping suffers from low resolution capabilities, and ribotyping and spacer PCR display intermediate resolving capabilities, whereas AP-PCR is more discriminating. Results from AP-PCR and both forms of ribotyping analysis correlate with epidemiological and environmental data. It is suggested that AP-PCR typing may be the method of choice for rapidly determining clonality among L. pneumophila isolates.

Bacterial Typing Techniques↗

PubMed1996

Elimination of nasal carriage of Staphylococcus aureus in hemodialysis patients.

OBJECTIVE: To study the efficacy of mupirocin for the elimination of nasal carriage of Staphylococcus aureus in hemodialysis patients. DESIGN: The efficacy of mupirocin was studied in a prospectively followed cohort. The effect of this intervention on the rate of S aureus bacteremia was evaluated using a historic control group. SETTING: Patients on the hemodialysis unit of the University Hospital Rotterdam, a tertiary referral center. PATIENTS: The study group consisted of consecutive patients on hemodialysis from February 1, 1992, until November 1, 1993. They were screened by taking nasal cultures monthly during their time on hemodialysis. If S aureus was isolated, treatment with mupirocin nasal ointment was initiated. The control group consisted of patients treated on the same hemodialysis unit from January 1, 1990, until January 1, 1992. RESULTS: The study group consisted of 226 patients, of whom 172 were evaluated to determine the efficacy of mupirocin. Sixty-seven (39%) were identified as nasal carriers. Following the initial treatment, 66 nasal cultures (98.5%) became negative. After 3 months and 6 months, respectively, 63 (94%) and 61 (91%) of the treated carriers had negative cultures. The rate of bacteremia (defined as the number of episodes of S aureus bacteremia per patient-year on hemodialysis) was significantly lower among the 226 patients in the study group (0.04 per patient-year) than among the 273 patients in the control group (0.25 per patient year, P < .001). Development of resistance and adverse effects were not observed. CONCLUSIONS: Mupirocin nasal ointment effectively eliminates nasal carriage of S aureus in patients on hemodialysis. This was associated with a significant reduction of the incidence of S aureus bacteremia, as compared to historic controls.

Administration, Intranasal↗

PubMed1996

Characterization of methicillin-resistant Staphylococcus aureus isolates from Portuguese hospitals by multiple genotyping methods.

One hundred and eighty-three methicillin-resistant Staphylococcus aureus (MRSA) isolates from eight different Portuguese hospitals were genetically typed by random amplification of polymorphic DNA (RAPD) employing different oligonucleotide primers. Fourteen different RAPD genotypes were identified. A subset of the same strains was also characterized by pulsed-field gel electrophoresis (PFGE) and/or hybridization using mecA and Tn554 probes. In the majority of cases, the different genotyping methods have identified the same MRSA clones. However, PFGE combined with the DNA probes was clearly the method providing higher resolution. Most strains that have already been identified by PFGE and DNA probes as members of the widely spread Iberian clone of MRSA generated a common RAPD genotype. The most prevalent Iberian clone was not detected in a collection of MRSA from Poland that was also examined by RAPD. On the other hand, MRSA strains second most frequent in prevalence in the Portuguese and Polish collection appear to be identical by RAPD, indicating extensive geographic spread of this particular clone. No correlation was apparent between epidemic behavior and the number of protein A gene repeats in this particular collection of MRSA strains.

DNA Probes↗

PubMed1996

On the nature and use of randomly amplified DNA from Staphylococcus aureus.

Various DNA-based methods have been introduced to genetically type Staphylococcus aureus strains but not a single technique is universally applicable. In order to search for DNA probes suitable for differentiating strains, randomly amplified polymorphic DNA patterns were generated for 243 S. aureus strains and a single isolate of Staphylococcus intermedius. All fingerprints were examined for unique amplicons, and the nature of 42 of these DNA fragments was investigated. Partial DNA sequences were determined, and several homologies were discovered with known S. aureus sequences (plasmid pSH6 DNA with insertion sequences, agrA and agrB sequences, hld genes, the gene for 23S rRNA, the lysyl tRNA synthetase gene, and the threonyl tRNA synthetase gene) and with genes from other species (Haemophilus influenzae bexA and Bacillus subtilis spoF and ctrA). Thirty fragments were of previously unknown origin. In Southern blots containing Eco RI-digested DNA from S. aureus strains and the S. intermedius strain, nine probes demonstrated the capacity to differentiate strains on the basis of the presence or absence of the sequence element in the staphylococcal genome involved. The remainder of the probes displayed restriction fragment length polymorphisms (n = 12), hybridized in a homogeneously positive fashion (n = 13) or hybridized only with their source strains (n = 8) (four of the latter were specific to S. intermedius). Three of the nine strain-specific probes were overlapping, and two of the others were found to display a high level of inconsistency among epidemiologically related strains. Thus, five strain-specific probes remained that, in a 5-digit typing system, accurately distinguished epidemiologically related and unrelated strains of S. aureus. We conclude that application of strain-specific DNA probes, selected on the basis of differing randomly amplified polymorphic DNA patterns, promises to become a technically simple, robust, and reproducible tool that may significantly facilitate the study of the epidemiology of S. aureus infections.

Bacterial Typing Techniques↗

PubMed1996

Comparative typing of Pseudomonas aeruginosa by random amplification of polymorphic DNA or pulsed-field gel electrophoresis of DNA macrorestriction fragments.

Eighty-seven strains of Pseudomonas aeruginosa were typed by random amplification of polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) of macrorestriction fragments. Stains were clustered on the basis of interpretative criteria as presented previously for the PFGE analysis. Clusters of strains were also defined on the basis of epidemiological data and subsequently reanalyzed by RAPD. It was found that in an RAPD assay employing the enterobacterial repetitive intergenic consensus sequence ERIC2 as a primer, single band differences can be ignored; in this case, clonally related strains could be grouped as effectively and reliably as with PFGE. These data could be corroborated by the use of other primer species. However, some primers either showed reduced resolution or, in contrast, identified DNA polymorphisms beyond epidemiologically and PFGE-defined limits. Apparently, different primers define different windows of genetic variation. It is suggested that criteria for interpretation of the ERIC2 PCR fingerprints can be simple and straightforward: when single band differences are ignored, RAPD-determined grouping of P. aeruginosa is congruent with that obtained by PFGE. Consequently, this implies that RAPD can be used with trust as a first screen in epidemiological characterization of P. aeruginosa. The ability to measure the rate of molecular evolution of the P. aeruginosa genome clearly depends on the choice of restriction enzyme or primer when RAPD or PFGE, respectively, is applied for the detection of DNA polymorphisms.

Bacterial Typing Techniques↗

PubMed1996

Molecular epidemiology of apparent outbreak of invasive aspergillosis in a hematology ward.

During a 2-month period, five patients suffering from invasive infections caused by Aspergillus flavus or Aspergillus fumigatus were identified in the Hematology Department of the University Hospital Dijkzigt (Rotterdam, The Netherlands). To study the epidemiological aspects of invasive aspergillosis, strains from these patients and from the hospital environment, isolated during extensive microbiological screening, were subjected to genotyping. A novel DNA extraction technique, involving freezing, grinding, and direct lysis in guanidium isothiocyanate-containing buffers of mycelial material, was applied. DNA isolation was followed by typing by random amplification of polymorphic DNA (RAPD) analysis. This showed that strains isolated from all patients infected with the same fungal species were genotypically distinct, thus providing evidence against the possibility of an ongoing, single-source nosocomial outbreak. Strains could also be differentiated from strains of geographically diverse origins. However, an A. flavus strain from one of the patients was also frequently encountered in the hospital environment. As all environmental strains were collected after this patient had been diagnosed with invasive disease, the epidemiological value of this observation could not be ascertained. Intensive investigations showed no single source of A. flavus or other aspergilli. RAPD genotyping proved that the outbreak of invasive aspergillosis in the hematology ward consisted of a series of unrelated events and was not due to a common source within the hospital. RAPD fingerprinting of aspergilli may greatly facilitate future investigations of the epidemiology of invasive disease caused by these pathogens.

Adult↗

PubMed1996

Novel BOX repeat PCR assay for high-resolution typing of Streptococcus pneumoniae strains.

Typing data obtained by specifically targeting a single, high-stringency PCR at the pneumococcal BOX repeat element for 28 strains of Streptococcus pneumoniae completely corroborated the resolutions attained by five genotypic procedures as described by Hermans et al. (P.W.M. Hermans, M. Sluijter, T. Hoogenboezem, H. Heersma, A. van Belkum, and R. de Groot, J. Clin. Microbiol. 33:1606-1612, 1995). All pairs of strains, except one, derived from both the cerebrospinal fluid and blood of the same individual were shown to be identical. Moreover, other, epidemiologically unrelated isolates were demonstrated to be unique. Considering the combined data from the five typing techniques applied previously as the "gold standard," the single BOX PCR test demonstrated excellent resolving powers while maintaining epidemiological linkage.

Bacterial Typing Techniques↗

PubMed1996

Comparison of five tests for identification of Staphylococcus aureus from clinical samples.

Five different laboratory tests for the identification of Staphylococcus aureus were compared. Analyses of 271 presumptive S. aureus strains, supplemented with 59 well-defined methicillin-resistant S. aureus (MRSA) isolates, were performed. Only the Staphaurex Plus (Murex Diagnostics, Dartford, United Kingdom) and the Pastorex Staphplus (Sanofi, Marnes-La-Coquette, France) tests displayed 100% sensitivity. The observed difference with the free-coagulase test (Bacto coagulase plasma; Difco, Detroit, Mich.), a bound-coagulase (clumping factor) test, and the former Staphaurex test (Murex Diagnostics) was caused mainly by the inability of these three tests to identify some MRSA strains correctly. Among Polish MRSA isolates included in the analysis, a group of free-coagulase-negative S. aureus strains was detected. Genetic typing by random amplification of polymorphic DNA revealed that the strains showing aberrant behavior when the different test results were compared belonged to limited number of S. aureus clones.

Bacterial Typing Techniques↗

PubMed1996

Monitoring persistence of coagulase-negative staphylococci in a hematology department using phenotypic and genotypic strategies.

OBJECTIVE: To determine persistence of coagulase-negative staphylococci (CNS) on a hematology-oncology ward and to determine the value of phenotypic and genotypic procedures for establishing clonality among CNS isolates. DESIGN: Strains of CNS isolated from bacteremic patients (n = 139) were typed by biochemical reactivity, antibiotic susceptibility, DNA macrorestriction analysis by pulsed-field gel electrophoresis (PFGE), and arbitrary primed polymerase chain reaction (AP PCR). Coagulase-negative staphylococci were subgrouped in a random collection (n = 20) used for the evaluation of the typing procedures and a collection of 119 CNS isolates from hematologic patients displaying multiple bacteremic episodes. RESULTS: Analysis of the reference collection demonstrated the usefulness of the DNA typing procedures, indicating that AP PCR and PFGE can be used for epidemiologic typing of CNS in a concordant fashion. Certain strains appeared to be permanent colonizers of the hematology ward or ward-related personnel. In individual patients, persistent colonization by a single type was demonstrated. However, a number of patients also experienced bacteremic episodes caused by CNS belonging to different types. CONCLUSION: We conclude that monitoring of CNS infections on a hematology ward by various genotypic techniques provides insight into nosocomial epidemiology and elucidates the complexity of the infections taking place. DNA typing is preferred over phenotypic procedures and can identify persistent CNS strains in a given location.

Bacteremia↗

PubMed1995

Investigation into the repeated recovery of coagulase-negative staphylococci from blood taken at the end of cardiopulmonary by-pass.

Forty-six strains of coagulase-negative staphylococci (CNS) were typed by biochemical reactivity, antibiotic susceptibility pattern, macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) and arbitrary primed polymerase chain reaction (AP PCR). Twenty-four strains were obtained in 1993 from blood cultures of as many patients coupled to a heart-lung machine during cardiac surgery. Since over 30% of the latter belonged to a single type, it was concluded that during the year of analysis a single clone of CNS persisted in this hospital setting. Subsequent epidemiological surveillance putatively identified four possible carriers among surgical personnel. For this reason, 22 strains collected from the hands and nose of two cardiac surgeons and two perfusionists were also tested; none were identical to the persistent clone. Thus either the operation equipment was colonized longitudinally or the causative CNS had disappeared from the suspect individuals' flora. Longitudinal monitoring of CNS infections by various techniques gives a valuable insight into nosocomial epidemiology and elucidates the complexity of the CNS colonization.

Cardiopulmonary Bypass↗

PubMed1995

PCR ribotyping and arbitrarily primed PCR for typing strains of Clostridium difficile from a Polish maternity hospital.

Detection of the source of Clostridium difficile strains is of importance for the control of the nosocomial spread of this microorganism. For this purpose, vaginal and rectal swabs from 183 mothers, duplicate fecal samples (taken on days 1 and 4 after birth) from 183 neonates, and 94 environmental samples were cultured for C. difficile. The microorganism was never detected in the meconium obtained on day 1 after birth. On the other hand, an incidence of 17% C. difficile positivity was noted in the fecal samples obtained on day 4 after birth. Forty-two percent of the 31 colonized neonates had been delivered with complications. The bacteria were never encountered in the rectal swabs of the mothers, and C. difficile was identified in only one vaginal swab. In contrast, 13% of the environmental samples were positive for C. difficile. No major difference was encountered between patient and environmental isolates with respect to toxigenicity (58 to 65% toxigenic isolates). All strains were subsequently typed by PCR amplification of the 16S-23S ribosomal intergenic spacer regions and by arbitrarily primed PCR (AP-PCR) with different primers and combinations thereof. All environmental isolates and 11 of 31 neonatal strains were of a single type. The vaginal strain was unique, and among the maternity ward- and neonate-related isolates, only two additional AP-PCR types were identified. When a collection of C. difficile strains from patients hospitalized in other institutions and suffering from antibiotic-associated diarrhea or pseudomembranous colitis was analyzed in a similar manner, it appeared that the strain from the maternity ward was unique. The other strain commonly encountered among the neonates was also identified frequently among the isolates from patients with antibiotic-associated diarrhea or pseudomembranous colitis, indicating its general occurrence. On the basis of both epidemiological studies and PCR-mediated genotyping, it was shown that the environment and not the birth canal is the major source of C. difficile acquisition by neonates in this maternity hospital setting. Furthermore, AP-PCR appears to be a fast and useful method for epidemiologically relevant typing of C. difficile isolates.

Bacterial Typing Techniques↗

PubMed1995

Molecular nosocomial epidemiology: high speed typing of microbial pathogens by arbitrary primed polymerase chain reaction assays.

Arbitrary primed polymerase chain reaction (AP PCR) assays are suited for the discrimination of isolates of all clinically relevant bacterial species. In a hospital setting, this type of DNA amplification test can be used for the timely detection of ongoing nosocomial outbreaks. For rapid screening of isolates of many medically important bacterial species, including Acinetobacter baumannii, Staphylococcus aureus, Serratia marcescens, Xanthomonas maltophilia and others, a single AP PCR assay can be used as a primary typing screen for genetic relatedness. In combination with epidemiological data, AP PCR testing is particularly useful for identifying true outbreaks caused by a single strain.

Acinetobacter↗

PubMed1982

Serum-induced lysis of Pseudomonas aeruginosa.

The sensitivity of 12 Pseudomonas aeruginosa strains (5 mucoid and 7 non-mucoid strains) to serum and the interaction of these strains with the complement system was studied. Five strains (4 mucoid and 1 non-mucoid strains) were lysed in 20% normal serum as measured by the release of radiolabelled material from 3H-adenine labelled bacteria. Three of these strains were also lysed in MgEGTA chelated serum. All strains activated complement via the classical pathway, and six strains were able to activate the alternative complement pathway as well. Slime production did not interfere with bacteriolysis and complement consumption.

Bacteriolysis↗

PubMedDate not supplied

Genetic diversification of methicillin-resistant Staphylococcus aureus as a function of prolonged geographic dissemination and as measured by binary typing and other genotyping methods.

The aim of the present study was to determine the extent of genome evolution among methicillin-resistant Staghylococcus aureus (MRSA) strains. Three different collections of strains were analysed, comprising locally, nationally and internationally disseminated genotypes. Various genotyping assays displaying different levels of resolution were used. Geographically and temporally diverse MRSA strains comprised the international group. MRSA strains recovered during an outbreak in a New York City hospital and Portuguese MRSA isolates, all resembling the so-called Iberian clone, were included in the local and national collections, respectively. Genotypes were determined by genome scanning typing techniques and procedures which analyse specific DNA elements only. The outbreak strains showed subclonal variation, whereas the Portuguese isolates displayed an increased number of genotypes. Among the epidemiologically unrelated MRSA strains, the different genotyping techniques revealed a wide heterogeneity of types. Different typing techniques appeared to show different levels of resolution, which could be correlated with the extent of geographic spread; the more pronounced the spread, the higher the degree of genome evolution. Binary typing and randomly amplified polymorphic DNA analysis are the typing methods of choice for determining (non)identity among strains that have a recent common ancestor and have undergone yet limited dissemination.

Adhesins, Bacterial↗

PubMedDate not supplied

Occurrence and structure-function relationship of pentameric short sequence repeats in microbial genomes.

It is suggested that genomes found in any form of cellular life contain potentially size-variable repetitive DNA moieties. In eukaryotes, large proportions of the multi-chromosomal genome consist of various classes of repetitive DNA. Also in archaeal genomes, repetitive DNA is encountered and, as is the case for the eukaryotes as well, little or no function is at present attributable to most of it. For prokaryotes, elegant experiments have highlighted so-called slipped strand nucleotide mispairing (SSM) as a basic and causal mechanism, giving rise to repeat unit number variation at a distinct locus. Illegitimate base pairing in regions of repetitive DNA during replication, in association with defective DNA repair and enhanced nuclease susceptibility of replication intermediates, in the end gives rise to deletion or addition of repeat units. Prokaryotic short sequence repeats (SSRs) harbour arrays of short repeat units, between one and approximately 20 nucleotides in length. SSRs are involved in various mechanisms of microbial gene expression regulation. Promoter strength can be affected by altering the spacing between important structural domains as can the integrity of open reading frames. In the present communication the literature on microbial SSRs harbouring repeat units that are five nucleotides in length will be briefly reviewed. Examples of these SSRs with discrete functionality are encountered in bacterial species such as Haemophilus influenzae, Neisseria gonorrhoeae, and Pasteurella haemolytica. In addition, several of the currently known bacterial and archaeal whole genome sequences were scanned for the presence of novel examples of potential five-nucleotide SSRs (and others) in order to gather additional knowledge on the propensity and putative functions of this type of potential genetic switch.

Base Sequence↗

PubMedDate not supplied

Rapid emergence of ciprofloxacin-resistant enterobacteriaceae containing multiple gentamicin resistance-associated integrons in a Dutch hospital.

In a hematology unit in the Netherlands, the incidence of ciprofloxacin-resistant Enterobacter cloacae and Escherichia coli increased from from 1996 to 1999. Clonal spread of single genotypes of both ciprofloxacin-resistant E. coli and Enterobacter cloacae from patient to patient was documented by pulsed-field gel electrophoresis and random amplification of polymorphic DNA. In addition, genetically heterogeneous strains were isolated regularly. Integrons associated with gentamicin resistance were detected in Enterobacter cloacae and E. coli strains. Integron-containing E. coli were detected in all hematology wards. In contrast, in Enterobacter cloacae strains two integron types were encountered only in the isolates from one ward. Although in all patients identical antibiotic regimens were used for selective decontamination, we documented clear differences with respect to the nosocomial emergence of ciprofloxacin-resistant bacterial strains and gentamicin resistance-associated integrons.

Anti-Bacterial Agents↗

PubMedDate not supplied

A noncomparative study of cefprozil at two dose levels in the treatment of acute uncomplicated bacterial sinusitis.

The efficacy and safety of cefprozil at two dose levels were evaluated in 110 patients with acute uncomplicated bacterial sinusitis in an uncontrolled, noncomparative, Phase II trial. Ninety patients received 250 mg of cefprozil (low-dose group) and 20 patients received 500 mg of cefprozil (high-dose group) every 12 hours for ten days. Evaluable patients had symptoms consistent with acute sinusitis, pathogens isolated at pretreatment susceptible to cefprozil, and a radiograph positive for sinusitis within 48 hours before treatment. A satisfactory clinical response was achieved in 34 of 39 evaluable patients (87%) in the low-dose group and in all 16 evaluable patients (100%) in the high-dose group. Pathogens were eradicated in 35 of 39 patients (90%) in the low-dose group and in 15 of 16 patients (94%) in the high-dose group. A total of 140 of 155 pathogens (90%) isolated pretreatment were susceptible to cefprozil. Six patients (7%) in the low-dose group and one patient (5%) in the high-dose group reported at least one adverse clinical event.

Adolescent↗