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H Verbrugh

Publications and source records attributed to H Verbrugh.

At least 19 recordsLinked to original sources
PubMed1999

Validation of binary typing for Staphylococcus aureus strains.

Most of the DNA-based methods for genetic typing of Staphylococcus aureus strains generate complex banding patterns. Therefore, we have developed a binary typing procedure involving strain-differentiating DNA probes which were generated on the basis of randomly amplified polymorphic DNA (RAPD) analysis. We present and validate the usefulness of 15 DNA probes, according to generally accepted performance criteria for molecular typing systems. RAPD analysis with multiple primers was performed on 376 S. aureus strains of which 97% were methicillin resistant (MRSA). Among the 1,128 RAPD patterns generated, 66 were selected which identified 124 unique DNA fragments. From these amplicons, only 12% turned out to be useful for isolate-specific binary typing. The nature of the RAPD-generated DNA fragments was investigated by partial DNA sequence analysis. Several homologies with known S. aureus sequences and with genes from other species were discovered; however, 87% of the probe sequences are of previously unknown origin. The locations of most of the DNA probes on the chromosome of S. aureus NCTC 8325 were determined by hybridization. Seven fragments were randomly dispersed along the genome, five were clustered within the 2500- to 2600-kb position of the genome, and the remaining four did not recognize complementary sequences in S. aureus NCTC 8325. A total of 103 S. aureus strains (69% MRSA) were used for the validation of the binary typing technique. The 15 DNA probes provided stable epidemiological markers, both in vitro (type consistency after serial passages on culture media) and in vivo (comparison of sequential isolates recovered from cases of persistent colonization). The discriminatory power of binary typing (D = 0.998) exceeded that of pulsed-field gel electrophoresis (D = 0.966) and RAPD analysis (D = 0.949). Reproducibility, measured by analyzing multiple strains belonging to a multitude of different epidemiological clusters, was comparable to that of other genotyping techniques used. Contribution of the DNA probes to the discriminatory power of the system was analyzed by comparison of dendrograms. This study demonstrates that binary typing is a robust tool for the genetic typing of S. aureus isolates.

Base Sequence↗
PubMed1998

Assessment of resolution and intercenter reproducibility of results of genotyping Staphylococcus aureus by pulsed-field gel electrophoresis of SmaI macrorestriction fragments: a multicenter study.

Twenty well-characterized isolates of methicillin-resistant Staphylococcus aureus were used to study the optimal resolution and interlaboratory reproducibility of pulsed-field gel electrophoresis (PFGE) of DNA macrorestriction fragments. Five identical isolates (one PFGE type), 5 isolates that produced related PFGE subtypes, and 10 isolates with unique PFGE patterns were analyzed blindly in 12 different laboratories by in-house protocols. In several laboratories a standardized PFGE protocol with a commercial kit was applied successfully as well. Eight of the centers correctly identified the genetic homogeneity of the identical isolates by both the in-house and standard protocols. Four of 12 laboratories failed to produce interpretable data by the standardized protocol, due to technical problems (primarily plug preparation). With the five related isolates, five of eight participants identified the same subtype interrelationships with both in-house and standard protocols. However, two participants identified multiple strain types in this group or classified some of the isolates as unrelated isolates rather than as subtypes. The remaining laboratory failed to distinguish differences between some of the related isolates by utilizing both the in-house and standardized protocols. There were large differences in the relative genome lengths of the isolates as calculated on the basis of the gel pictures. By visual inspection, the numbers of restriction fragments and overall banding pattern similarity in the three groups of isolates showed interlaboratory concordance, but centralized computer analysis of data from four laboratories yielded percent similarity values of only 85% for the group of identical isolates. The differences between the data sets obtained with in-house and standardized protocols could be the experimental parameters which differed with respect to the brand of equipment used, imaging software, running time (20 to 48 h), and pulsing conditions. In conclusion, it appears that the standardization of PFGE depends on controlling a variety of experimental intricacies, as is the case with other bacterial typing procedures.

Bacterial Typing Techniques↗
PubMed1998

Persistence of clones of coagulase-negative staphylococci among premature neonates in neonatal intensive care units: two-center study of bacterial genotyping and patient risk factors.

From 1 January 1995 until 1 January 1996, we studied the molecular epidemiology of blood isolates of coagulase-negative staphylococci (CoNS) in the Neonatal Intensive Care Units (NICUs) of the Sophia Children's Hospital (SCH; Rotterdam, The Netherlands) and the Wilhelmina Children's Hospital (WCH; Utrecht, The Netherlands). The main goal of the present study was to detect putatively endemic clones of CoNS persisting in these NICUs. Pulsed-field gel electrophoresis was used to detect the possible presence of endemic clones of clinical significance. In addition, clinical data of patients in the SCH were analyzed retrospectively to identify risk factors for the acquisition of positive blood cultures. In both centers, endemic CoNS clones were persistently present. Thirty-three percent of the bacterial isolates derived from blood cultures in the SCH belonged to a single genotype. In the WCH, 45% of all bacterial strains belonged to a single clone. These clones were clearly different from each other, which implies that site specificity is involved. Interestingly, we observe that the clonal type in the SCH differed significantly from the incidentally occurring strains with respect to both the average pH and partial CO2 pressure of the patient's blood at the time of bacterial culture. We found that the use of intravascular catheters, low gestational age, and a long hospital stay were important risk factors for the development of a putative CoNS infection. When the antibiotic susceptibility of the bacterial isolates was assessed, a clear correlation between the nature of the antibiotics most frequently used as a first line of defense versus the resistance profile was observed. We conclude that the intensive use of antibiotics in an NICU setting with highly susceptible patients causes selection of multiresistant clones of CoNS which subsequently become endemic.

Anti-Bacterial Agents↗
PubMed1998

Short-sequence DNA repeats in prokaryotic genomes.

Short-sequence DNA repeat (SSR) loci can be identified in all eukaryotic and many prokaryotic genomes. These loci harbor short or long stretches of repeated nucleotide sequence motifs. DNA sequence motifs in a single locus can be identical and/or heterogeneous. SSRs are encountered in many different branches of the prokaryote kingdom. They are found in genes encoding products as diverse as microbial surface components recognizing adhesive matrix molecules and specific bacterial virulence factors such as lipopolysaccharide-modifying enzymes or adhesins. SSRs enable genetic and consequently phenotypic flexibility. SSRs function at various levels of gene expression regulation. Variations in the number of repeat units per locus or changes in the nature of the individual repeat sequences may result from recombination processes or polymerase inadequacy such as slipped-strand mispairing (SSM), either alone or in combination with DNA repair deficiencies. These rather complex phenomena can occur with relative ease, with SSM approaching a frequency of 10(-4) per bacterial cell division and allowing high-frequency genetic switching. Bacteria use this random strategy to adapt their genetic repertoire in response to selective environmental pressure. SSR-mediated variation has important implications for bacterial pathogenesis and evolutionary fitness. Molecular analysis of changes in SSRs allows epidemiological studies on the spread of pathogenic bacteria. The occurrence, evolution and function of SSRs, and the molecular methods used to analyze them are discussed in the context of responsiveness to environmental factors, bacterial pathogenicity, epidemiology, and the availability of full-genome sequences for increasing numbers of microorganisms, especially those that are medically relevant.

Adhesins, Bacterial↗
PubMed1997

Simultaneous persistence of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus in a neonatal ward of a Warsaw hospital.

Fifty-seven methicillin-resistant Staphylococcus aureus (MRSA) isolates from babies (N = 31), carriers amongst health care workers (N = 16; 10% of all staff members) and the environment (N = 10); 39 MSSA isolates, from babies (N = 18), health care workers (N = 5) and environment (N = 16) were analysed. The strains were from the neonatal ward of a teaching hospital in Warsaw and were collected over a period of 16 months (1993/1994). The isolates were characterized by phage-typing, arbitrary-primed polymerase chain reaction (AP PCR), DNA repeat polymorphism within the protein A gene and the resistance pattern to antimicrobial agents. The presence of the mecA gene was determined by PCR. MRSA were classified as heterogeneously resistant to methicillin, susceptible to other antimicrobial agents and, except for three isolates, appeared to be genotypically almost identical. The first example of mupirocin resistant MRSA in Poland was documented. Amongst MSSA isolates, increased variability was seen, however, the persistence of one predominate clone of MSSA was shown. In this particular hospital environment, several different strains of both MRSA and MSSA were capable of maintaining persistent colonization.

Bacteriophage Typing↗
PubMed1997

Genetic homogeneity among methicillin-resistant Staphylococcus aureus strains from Saudi Arabia.

Ninety-four strains of methicillin-resistant Staphylococcus aureus (MRSA) were collected from patients nursed in several hospitals in Saudi Arabia, before they were referred to King Faisal Specialist Hospital and Research Centre for tertiary care. The hospitals were from geographically diverse regions and as such the entirety of Saudi Arabia was covered. All strains were genetically typed by random amplification of polymorphic DNA (RAPD) analysis using three different primers and a representative subset of the strains was analyzed with pulsed-field gel electrophoresis (PFGE) as well. It was concluded that 87 out of 94 (93%) belong to a single clonally related lineage of MRSA. In the other 7 cases, the DNA banding patterns were shown to differ only slightly from those determined for the clonal type. PFGE analysis confirmed the homogeneity of the collection of strains. When the RAPD and PFGE fingerprints obtained for the Saudi clone were compared to those generated for a collection of MRSA with a more diverse geographical background, it was shown that the clonal type from Saudi Arabia was not identical to any of these MRSA strains. Our data provide another example of the capacity of certain MRSA clones to expand through entire nations and establish themselves permanently among large number of hospitals and, consequently, even larger numbers of patients.

DNA, Bacterial↗
PubMed1997

Coagulase and protein A polymorphisms do not contribute to persistence of nasal colonisation by Staphylococcus aureus.

The nasal carriage rate of Staphylococcus aureus was examined in a longitudinal study of 31 healthy Danish volunteers. Each person was classified as persistent (>8 positive cultures from 10 examinations), an intermittent carrier (50-80% positive cultures) or an ocassional carrier (positive cultures on 10-40% of ocassions only). One hundred and twenty strains from these persons were subjected to phage typing and random amplification of polymorphic DNA (RAPD) analysis. Phage and RAPD typing were in close agreement. RAPD confirmed the spread of a particular S. aureus clone (phage type 95) throughout Denmark. However, no common genotype or phenotype characteristics of S. aureus that could separate persistent from intermittent or incidental colonisers were identified. The immunoglobulin binding protein A and the prothrombin binding coagulase protein are both putative S. aureus virulence or defence factors. Analysis of polymorphisms in the variable repeat regions in the genes for these proteins showed no correlation between the number of repeat units and, consequently, the protein structure with the ability of strains to persist in the human nasal mucosa. The amount of protein A, detectable by its IgG binding activity, appeared not to be correlated to persistence of carriage. Thus protein A and coagulase gene polymorphisms do not seem to play a significant role in the propensity of S. aureus to colonise human nasal epithelium. Furthermore, based on the genetic heterogeneity encountered among the S. aureus strains it is suggested that within the current study population, no single clonal lineage of S. aureus has increased capability to colonise the human nasal epithelium.

Adult↗
PubMed1997

Nasal carriage of Staphylococcus aureus: epidemiology, underlying mechanisms, and associated risks.

Staphylococcus aureus has long been recognized as an important pathogen in human disease. Due to an increasing number of infections caused by methicillin-resistant S. aureus (MRSA) strains, therapy has become problematic. Therefore, prevention of staphylococcal infections has become more important. Carriage of S. aureus appears to play a key role in the epidemiology and pathogenesis of infection. The ecological niches of S. aureus are the anterior nares. In healthy subjects, over time, three patterns of carriage can be distinguished: about 20% of people are persistent carriers, 60% are intermittent carriers, and approximately 20% almost never carry S. aureus. The molecular basis of the carrier state remains to be elucidated. In patients who repeatedly puncture the skin (e.g., hemodialysis or continuous ambulatory peritoneal dialysis [CAPD] patients and intravenous drug addicts) and patients with human immunodeficiency virus (HIV) infection, increased carriage rates are found. Carriage has been identified as an important risk factor for infection in patients undergoing surgery, those on hemodialysis or CAPD, those with HIV infection and AIDS, those with intravascular devices, and those colonized with MRSA. Elimination of carriage has been found to reduce the infection rates in surgical patients and those on hemodialysis and CAPD. Elimination of carriage appears to be an attractive preventive strategy in patients at risk. Further studies are needed to optimize this strategy and to define the groups at risk.

Bacterial Adhesion↗
PubMed1997

Variable number of tandem repeats in clinical strains of Haemophilus influenzae.

An algorithm capable of identifying short repeat motifs was developed and used to screen the whole genome sequence available for Haemophilus influenzae, since some of these repeats have been shown to affect bacterial virulence. Various di- to hexanucleotide repeats were identified, confirming and extending previous findings on the existence of variable-number-of-tandem-repeat loci (VNTRs). Repeats with units of 7 or 8 nucleotides were not encountered. For all of the 3- to 6-nucleotide repeats in the H. influenzae chromosome, PCR tests capable of detecting allelic polymorphisms were designed. Fourteen of 18 of the potential VNTRs were indeed highly polymorphic when different strains were screened. Two of the potential VNTRs appeared to be short and homogeneous in length; another one may be specific for the H. influenzae Rd strain only. One of the primer sets generated fingerprint-type DNA banding patterns. The various repeat types differed with respect to intrinsic stability as well. It was noted for separate colonies derived from a single clinical specimen or strains passaged for several weeks on chocolate agar plates that the lengths of the VNTRs did not change. When several strains from different patients infected during an outbreak of lung disease were analyzed, increased but limited variation was encountered in all VNTR sites analyzed. One of the 5-nucleotide VNTRs proved to be hypervariable. This variability may reflect the molecular basis of a mechanism used by H. influenzae bacteria to successfully colonize and infect different human individuals.

Algorithms↗
PubMed1997

Vaginal carriage of enterotoxigenic Bacteroides fragilis in pregnant women.

Bacteroides fragilis is an anaerobic bacterial species that is involved in gynecological infections and pathology. The incidence of vaginal carriage is largely unknown, and in order to study this, 120 pregnant women attending a general hospital for delivery were examined. Cultures were positive for eight of these women (6.6%). Interestingly, potential clonal relatedness could be demonstrated among several of the nonenterotoxigenic B. fragilis strains. Among the strains, only one produced metalloprotease enterotoxin. The presence of the gene for the metalloprotease, giving rise to the pathogenic effect on cultured eukaryotic HT29/C1 cells, was confirmed by a newly designed specific PCR assay. The enterotoxigenic B. fragilis (ETBF) strain was analyzed with the help of arbitrarily primed PCR (AP-PCR) and PCR-mediated ribotyping. The ETBF strain was shown to be genetically different compared to several other strains obtained from diverse sources. Our data indicate a relatively high vaginal B. fragilis carriage rate among pregnant women in Warsaw, Poland. Although neither ETBF nor B. fragilis colonization presented a clinical problem, the possible genetic relatedness among the colonizing B. fragilis strains indicates the need for additional research in the field of ETBF transmission and molecular epidemiology.

Bacteroides Infections↗
PubMed1997

Dissemination of a single clone of methicillin-resistant Staphylococcus aureus among Turkish hospitals.

A collection of 39 methicillin-resistant Staphylococcus aureus (MRSA) stains derived from six different hospitals in Ankara and one hospital in Barsa, Turkey, were analyzed by multiple genotyping. In agreement with the other genotyping assays, pulsed-field gel electrophoresis of DNA macrorestriction fragments identified genetic homogeneity among all MRSA isolates studies. It is concluded that a major clone of MRSA has spread through a large part of Turkey, causing longitudinally persistent colonization in all of the institutions surveyed.

Cross Infection↗
PubMed1997

Outbreak of amoxicillin-resistant Haemophilus influenzae type b: variable number of tandem repeats as novel molecular markers.

An outbreak caused by amoxicillin-resistant Haemophilus influenzae type b was noted among patients suffering from chronic obstructive pulmonary disease. Since infections were clustered in time and place, an ongoing outbreak was suspected. The spread of the strain and the course of the outbreak could be followed by random amplification of polymorphic DNA (RAPD) analysis of the different bacterial isolates. In addition, studies were aimed at the determination of length polymorphism in regions of repetitive DNA. By PCR-mediated amplification of variable number of tandem repeat regions (VNTRs), additional insight into the genome composition of the epidemic strain was gained. Our results show that VNTRs comprising repeat units that are 3, 5, or 6 nucleotides in length provided stable genetic markers that can be used for molecular typing of H. influenzae type b. VNTRs built from tetranucleotide units, however, appear to be hypervariable and not suited for epidemiological studies. The observed variability in this latter class of VNTRs might be reminiscent of the bacterium's capacity to deal with unfavorable host factors.

Amoxicillin↗
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↗