Biomedical subjects

Alex van Belkum

Publications and source records attributed to Alex van Belkum.

At least 37 records · Page 2Linked to original sources

PubMed2005

The role of nasal carriage in Staphylococcus aureus infections.

Staphylococcus aureus is a frequent cause of infections in both the community and hospital. Worldwide, the increasing resistance of this pathogen to various antibiotics complicates treatment of S aureus infections. Effective measures to prevent S aureus infections are therefore urgently needed. It has been shown that nasal carriers of S aureus have an increased risk of acquiring an infection with this pathogen. The nose is the main ecological niche where S aureus resides in human beings, but the determinants of the carrier state are incompletely understood. Eradication of S aureus from nasal carriers prevents infection in specific patient categories-eg, haemodialysis and general surgery patients. However, recent randomised clinical trials in orthopaedic and non-surgical patients failed to show the efficacy of eliminating S aureus from the nose to prevent subsequent infection. Thus we must elucidate the mechanisms behind S aureus nasal carriage and infection to be able to develop new preventive strategies. We present an overview of the current knowledge of the determinants (both human and bacterial) and risks of S aureus nasal carriage. Studies on the population dynamics of S aureus are also summarised.

Adolescent↗

PubMed2005

Major epidemic clones of Staphylococcus aureus in Nigeria.

Clinical isolates of Staphylococcus aureus (n=276) were collected in 10 different hospitals located in the Southwest (cities of Ibadan and Lagos) and North-Central (city of Jos) parts of Nigeria. Resistance profiling of these strains revealed that the vast majority was still susceptible to methicillin (98.6% MSSA). Pulsed field gel electrophoresis (PFGE) performed for these strains identified 45 different genotypes, nine of which were identified in four or more different hospitals. The major PFGE type (genotype 2) comprised 23% of all isolates. In addition, several other strains were shown to be endemic in individual hospitals. Three out of four multi-resistant MRSA strains that were detected were sequence type 8 (ST8) as determined by array-based multi-locus sequence typing (MLST). PCRs for cataloguing the staphylococcal cassette chromosome (SCCmec) type were negative for two of these, suggesting the existence of a new methicillin-resistance gene complex in the ST8 genetic background. In conclusion, major clones of MSSA circulate in Nigeria, and the MRSA incidence is still low. However, the occurrence of old and new versions of SCCmec in some of the ecologically abundant MSSA strains should be taken as a serious warning since clonal expansion of this type of strains is not unprecedented.

Genotype↗

PubMed2005

Persistent (not intermittent) nasal carriage of Staphylococcus aureus is the determinant of CPD-related infections.

BACKGROUND: We investigated the impact of staphylococcal carriage among patients on continuous peritoneal dialysis (CPD) in a university hospital. METHODS: Patients were screened for Staphylococcus aureus carriage and categorized as persistent, intermittent, or non-S. aureus nasal carriers. Patients were subsequently recultured every 12 weeks for S. aureus and coagulase negative staphylococcal (CoNS) carriage, and followed-up for CPD-related infections and antibiotic resistance. RESULTS: Fifty-two patients were included: 20 peristent, 10 intermittent, and 22 non-S. aureus carriers. Only persistent S. aureus carriage was significantly associated with an increased risk for all CPD-related infections [incidence rate ratio (IRR) 3.52 (95% CI: 2.56-4.85)], exit site infections [IRR 5.59 (95% CI: 3.50-8.92)], and peritonitis [IRR 2.19 (95% CI: 1.39-3.45)], as well as increased antibiotic use [IRR 3.43 (95% CI: 2.50-4.72)], including vancomycin [IRR 2.15 (95%: 2.13-2.16)]. No vancomycin-resistant S. aureus strains were detected. However, eight (2%) out of 407 CoNS strains isolated were vancomycin intermediately susceptible. In all five patients (four persistent and one intermittent carriers) concerned, this was significantly related to a higher antibiotic (including vancomycin) usage [IRR 2.65 (95% CI: 1.82-3.84)]. CONCLUSION: Persistent-but not intermittent-S. aureus nasal carriage is the major determinant of CPD-related infections, and is associated with a significantly higher consumption of antibiotics, including vancomycin. The highly diverse population of CoNS appears to be the prime reservoir of staphylococcal vancomycin resistance. Accurate determination of the S. aureus nasal carriage state of CPD patients is essential to better target intervention strategies to prevent CPD-related infections.

Adult↗

PubMed2005

Testing of the in vitro susceptibilities of Madurella mycetomatis to six antifungal agents by using the Sensititre system in comparison with a viability-based 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5- [(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) assay and a modified NCCLS method.

The in vitro susceptibilities of 36 clinical isolates of Madurella mycetomatis, the prime agent of eumycetoma in Africa, to ketoconazole, itraconazole, fluconazole, voriconazole, amphotericin B, and flucytosine were determined by the Sensititre YeastOne system. This system appeared to be a rapid and easy test, and by use of hyphal suspensions it generated results comparable to those of a modified NCCLS method. After 10 days of incubation, the antifungal activities of ketoconazole (MIC at which 90% of isolates were inhibited [MIC90], 0.125 microg/ml), itraconazole (MIC90, 0.064 microg/ml), and voriconazole (MIC90, 0.125 microg/ml) appeared superior to those of fluconazole (MIC90, 128 microg/ml) and amphotericin B (MIC90, 1 microg/ml), with MICs in the clinically relevant range. All isolates were resistant to flucytosine (all MICs above 64 microg/ml). Based on the relatively broad range of MICs obtained for the antifungal agents, routine testing of M. mycetomatis isolates for susceptibility to antifungal agents seems to be relevant to adequate therapeutic management.

Antifungal Agents↗

PubMed2005

Effect of mupirocin treatment on nasal, pharyngeal, and perineal carriage of Staphylococcus aureus in healthy adults.

Nasal carriage of Staphylococcus aureus is an important risk factor for S. aureus infections. Mupirocin nasal ointment is presently the treatment of choice for decolonizing the anterior nares. However, recent clinical trials show limited benefit from mupirocin prophylaxis in preventing nosocomial S. aureus infections, probably due to (re)colonization from extranasal carriage sites. Therefore, we studied the effectiveness of mupirocin nasal ointment treatment on the dynamics of S. aureus nasal and extranasal carriage. Twenty noncarriers, 26 intermittent carriers, and 16 persistent carriers had nasal, throat, and perineum samples taken 1 day before and 5 weeks after mupirocin treatment (twice daily for 5 days) and assessed for growth of S. aureus. The identities of cultured strains were assessed by restriction fragment length polymorphisms of the coagulase and protein A genes. The overall carriage rate (either nasal, pharyngeal, or perineal carrier or a combination) was significantly reduced after mupirocin treatment from 30 to 17 carriers (P = 0.003). Of the 17 carriers, 10 (60%) were still colonized with their old strain, 6 (35%) were colonized with an exogenous strain, and 1 (5%) was colonized with both. Two noncarriers became carriers after treatment. The acquisition of exogenous strains after mupirocin treatment is a common phenomenon. The finding warrants the use of mupirocin only in proven carriers for decolonization purposes. Mupirocin is effective overall in decolonizing nasal carriers but less effective in decolonizing extranasal sites.

Administration, Intranasal↗

PubMed2005

Host- and tissue-specific pathogenic traits of Staphylococcus aureus.

Comparative genomics were used to assess genetic differences between Staphylococcus aureus strains derived from infected animals versus colonized or infected humans. A total of 77 veterinary isolates were genetically characterized by high-throughput amplified fragment length polymorphism (AFLP). Bacterial genotypes were introduced in a large AFLP database containing similar information for 1,056 human S. aureus strains. All S. aureus strains isolated from animals in close contact with humans (e.g., pet animals) were predominantly classified in one of the five main clusters of the AFLP database (cluster I). In essence, mastitis-associated strains from animals were categorized separately (cluster IVa) and cosegregated with bacteremia-associated strains from humans. Distribution of only 2 out of 10 different virulence genes differed across the clusters. The gene encoding the toxic shock syndrome protein (tst) was more often encountered among veterinary strains (P < 0.0001) and even more in the mastitis-related strains (P<0.0001) compared to human isolate results. The gene encoding the collagen binding protein (cna) was rarely detected among invasive human strains. The virulence potential, as indicated by the number of virulence genes per strain, did not differ significantly between the human- and animal-related strains. Our data show that invasive infections in pets and humans are usually due to S. aureus strains with the same genetic background. Mastitis-associated S. aureus isolated in diverse farm animal species form a distinct genetic cluster, characterized by an overrepresentation of the toxic shock syndrome toxin superantigen-encoding gene.

Adhesins, Bacterial↗

PubMed2005

Identification and characterization of a novel outer membrane protein (OMP J) of Moraxella catarrhalis that exists in two major forms.

Moraxella catarrhalis is a common commensal of the human respiratory tract that has been associated with a number of disease states, including acute otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults. During studies to investigate the outer membrane proteins of this bacterium, two novel major proteins, of approximately 19 kDa and 16 kDa (named OMP J1 and OMP J2, respectively), were identified. Further analysis indicated that these two proteins possessed almost identical gene sequences, apart from two insertion/deletion events in predicted external loops present within the putative barrel-like structure of the proteins. The development of a PCR screening strategy found a 100% (96/96) incidence for the genes encoding the OMP J1 and OMP J2 proteins within a set of geographically diverse M. catarrhalis isolates, as well as a significant association of OMP J1/OMP J2 with both the genetic lineage and the complement resistance phenotype (Fisher's exact test; P < 0.01). Experiments using two DeltaompJ2 mutants (one complement resistant and the other complement sensitive) indicated that both were less easily cleared from the lungs of mice than were their isogenic wild-type counterparts, with a significant difference in bacterial clearance being observed for the complement-resistant isolate but not for its isogenic DeltaompJ2 mutant (unpaired Student's t test; P < 0.001 and P = 0.32). In this publication, we characterize a novel outer membrane protein of Moraxella catarrhalis which exists in two variant forms associated with particular genetic lineages, and both forms are suggested to contribute to bacterial clearance from the lungs.

Amino Acid Sequence↗

PubMed2005

Clonal spread of a Clostridium difficile strain with a complete set of toxin A, toxin B, and binary toxin genes among Polish patients with Clostridium difficile-associated diarrhea.

Clinically relevant Clostridium difficile strains usually produce toxins A and B. Some C. difficile strains can produce an additional binary toxin. We report clonality among five strains carrying all toxin genes from Polish patients with C. difficile-associated diarrhea. In another strain, possible recombination between binary toxin genes is documented.

Adolescent↗

PubMed2005

Isolation of nontuberculous mycobacteria in Zambia: eight case reports.

The isolation of nontuberculous mycobacteria (NTM) raises the question of their clinical significance, especially in an African setting. We found a high percentage of NTM isolated from various specimens, including ones that are normally sterile, among 213 patients in Zambia. Because tuberculosis can affect all parts of the body, we decided to include patients who had signs and symptoms in any part of the body for more than 2 weeks. Most patients had tractus respiratorius (80%) and tractus digestivus (10%) symptoms. During three consecutive days, sputum was collected and two separate sputum specimens were cultured for mycobacteria. Depending on the clinical picture, pleural effusion, ascites, abscess material, or enlarged lymph nodes were also cultured for mycobacteria. A specimen from one sterile body site was collected from 25 patients (60% human immunodeficiency virus [HIV] positive). NTM were isolated from 8 of these 25 specimens. Mycobacterium lentiflavum was isolated from four patients, and Mycobacterium goodii was isolated from one patient. In order to exclude the possibility of laboratory cross-contamination, a novel amplified fragment length polymorphism DNA typing method for M. lentiflavum was developed. Genetic variation was detected, rendering the likelihood of laboratory contamination unlikely. Clinically relevant infection due to NTM occurs in both HIV-positive and HIV-negative patients in Zambia, and their clinical impact seems to be underestimated. This is the first report of M. lentiflavum and M. goodii infections in Africa.

AIDS-Related Opportunistic Infections↗

PubMed2005

Loss of the mecA gene during storage of methicillin-resistant Staphylococcus aureus strains.

The mecA gene was lost in 36 (14.4%) of 250 methicillin-resistant Staphylococcus aureus isolates after 2 years of storage at -80 degrees C with the Microbank system (Pro-lab Diagnostics, Austin, Tex.). Further analysis of 35 of these isolates confirmed loss of the mecA gene in 32 isolates. This finding has important implications for the management of strain collections.

Bacterial Proteins↗

PubMed2005

Performance of CHROMagar MRSA medium for detection of methicillin-resistant Staphylococcus aureus.

CHROMagar MRSA was evaluated for its ability to identify methicillin-resistant Staphylococcus aureus (MRSA). A well-defined collection consisting of 216 MRSA strains and 241 methicillin-susceptible Staphylococcus aureus isolates was used. The sensitivity of CHROMagar MRSA after 24 h of incubation was 95.4%, increasing to 100% after 48 h. The specificity was already 100% after 24 h.

Chromogenic Compounds↗

PubMed2005

Use of an automated multiple-locus, variable-number tandem repeat-based method for rapid and high-throughput genotyping of Staphylococcus aureus isolates.

Fast and reliable genotyping methods that allow real-time epidemiological surveillance would be instrumental to monitoring of the spread of methicillin-resistant Staphylococcus aureus. We describe an automated variable-number tandem repeat-based method for the rapid genotyping of Staphylococcus aureus. Multiplex PCR amplifications with eight primer pairs that target gene regions with variable numbers of tandem repeats were resolved by microcapillary electrophoresis and automatically assessed by cluster analysis. This genotyping technique was evaluated for its discriminatory power and reproducibility with clinical isolates of various origins, including a panel of control strains previously characterized by several typing methods and collections from either long-term carriers or defined nosocomial outbreaks. All steps of this new procedure were developed to ensure a rapid turnaround time and moderate cost. The results obtained suggest that this rapid approach is a valuable tool for the genotyping of S. aureus isolates in real time.

Automation↗

PubMed2005

Genotyping of Madurella mycetomatis by selective amplification of restriction fragments (amplified fragment length polymorphism) and subtype correlation with geographical origin and lesion size.

One of the causative organisms of mycetoma is the fungus Madurella mycetomatis. Previously, extensive molecular typing studies identified Sudanese isolates of this fungus as clonal, but polymorphic genetic markers have not yet been identified. Here, we report on the selective amplification of restriction fragment (AFLP) analysis of 37 Sudanese clinical isolates of M. mycetomatis. Of 93 AFLP fragments generated, 25 were polymorphic, and 12 of these 25 polymorphic fragments were found in a large fraction of the strains. Comparative analysis resulted into a tree, composed of two main (clusters I and II) and one minor cluster (cluster III). Seventy-five percent of the strains found in cluster I originated from central Sudan, while the origin of the strains in cluster II was more heterogeneous. Furthermore, the strains found in cluster I were generally obtained from lesions larger than those from which the strains found in cluster II were obtained (chi-square test for trend, P = 0.03). Among the 12 more commonly found polymorphisms, 4 showed sequence homology with known genes. Marker A7 was homologous to an endo-1,4-beta-glucanase from Aspergillus oryzae, 97% identical markers A12 and B3 matched a hypothetical protein from Gibberella zeae, and marker B4 was homologous to casein kinase I from Danio rerio. The last marker seemed to be associated with strains originating from central Sudan (P = 0.001). This is the first report on a genotypic study where genetic markers which may be used to study pathogenicity in M. mycetomatis were obtained.

Antifungal Agents↗

PubMed2005

Fluoroquinolone-resistant Escherichia coli, Indonesia.

In a recent, population-based survey of 3,996 persons in Indonesia, fluoroquinolone (FQ)-resistant Escherichia coli was prevalent in the fecal flora of 6% of patients at hospital admission and 23% of patients at discharge, but not among healthy relatives or patients visiting primary healthcare centers (2%). Molecular typing showed extensive genetic diversity with only limited clonality among isolates. This finding suggests that independent selection of resistant mutants occurs frequently. FQ-resistant isolates exhibited a higher rate of spontaneous mutation, but sparser virulence profiles, than FQ-susceptible isolates from the same population. The resistant isolates belonged predominantly to phylogenetic groups A (57%) and B1 (22%) but also to the moderately virulent group D (20%). Hypervirulent strains from the B2 cluster were underrepresented (1%). Because FQ-resistant E. coli can cause disease, especially nosocomial infections in immunocompromised patients, spread of such strains must be stopped.

Anti-Infective Agents↗

PubMed2004

A novel plasmid (pEMCJH03) isolated from moraxella catarrhalis possibly useful as a cloning and expression vector within this species.

A preliminary screening study of six Moraxella catarrhalis isolates from primary school children in the Netherlands identified a small 3.5 kb plasmid (pEMCJH03), containing four open reading frames, which encoded three mobilizing and one replicase protein. Insertion of a kanamycin containing transposon (yielding pEMCJH04) allowed selection and isolation of the plasmid in Escherichia coli. Natural transformation of pEMCJH04 into M. catarrhalis was successful for 25% (3/12) of non-isogenic isolates, with no link between (lack of) transformability and genetic lineage or (lack of) transformability and complement phenotype, though the transformation efficiency was found to be rather low at approximately 615CFU/microg (range=60-1040CFU/microg ). This is only the second publication detailing a plasmid isolated from this important respiratory pathogen, and the ability to clone and express foreign proteins in M. catarrhalis using pEMCJH04 could help in the development of a vaccine expression vector, as well as providing a useful tool for studying promoter activity and in complementation studies of gene knockout mutants.

Bacterial Proteins↗

PubMed2004

Predicting the Staphylococcus aureus nasal carrier state: derivation and validation of a "culture rule".

BACKGROUND: To study determinants and risks of Staphylococcus aureus nasal carriage, adequate differentiation between the different S. aureus carrier states is obligatory. We set out to develop a "culture rule" capable of differentiating between persistent and intermittent or noncarriers that uses a minimum of nasal swab cultures. METHODS: In 51 healthy volunteers (derivation cohort), 12 quantitative nasal cultures were performed to establish S. aureus nasal carriage states. Persons with 11 or 12 cultures positive for S. aureus were classified as persistent carriers, and those with negative results of all cultures were classified as noncarriers. All other persons were classified as intermittent carriers. By means of logistic regression and receiver operating characteristic (ROC) curves, a culture rule was derived. This culture rule was subsequently validated in 106 participants of an ongoing study in 3882 elderly persons, again with the use of 12 quantitative nasal cultures. RESULTS: In both cohorts, the positive predictive value of 2 consecutive positive culture results for persistent carriage was 79%. The model best differentiating between persistent and intermittent or noncarriers used the number of positive culture results combined with the amount of S. aureus in these cultures. By using the outcome of 2 cultures, the areas under the ROC curves were 0.981 (95% confidence interval [CI], 0.949-1.0) for the derivation cohort and 0.936 (95% CI, 0.881-0.990) for the validation cohort. CONCLUSIONS: Combining qualitative and quantitative results of 2 nasal swab cultures accurately predicted the persistent S. aureus carriage state with a reliability of 93.6%. Thus, this culture rule can be used in studies of determinants and risks of S. aureus nasal carriage.

Adult↗

PubMed2004

Molecular epidemiology of Staphylococcus aureus colonization in a burn center.

The aim of this study was to investigate carriage of Staphylococcus aureus by patients and health care workers (HCW) and to define the genetic relationship of S. aureus strains isolated from burn wounds. At admission, 19/55 (34.5%) patients carried S. aureus in their nose and/or throat. Of this group, 95% subsequently colonized their burn wounds with S. aureus. Molecular analysis showed that in 78% of these cases the burn-wound colonizing strain was identical to the strain carried at admission. Importantly, 23/36 (64%) patients who did not carry S. aureus at admission also developed burn-wound colonization. In this group, three dominant genotypes were identified as colonizing strains of burn wounds. These clones represented also the majority (59%) of S. aureus strains cultured from the nose and/or throat of health care workers and patients. If patients were admitted to one of the Intensive Care rooms burn wounds of non-carriers were not colonized with S. aureus as long as they remained in such isolation. Only patients who carried S. aureus at admission developed burn-wound colonization with that genotype they carried in the nose or throat. Both carriage in patients and health care workers and auto-infection play a crucial role in (cross-) colonization events.

Adult↗

PubMed2004

A new high-throughput AFLP approach for identification of new genetic polymorphism in the genome of the clonal microorganism Mycobacterium tuberculosis.

We have here applied high-throughput amplified fragment length polymorphism (htAFLP) analysis to strains belonging to the five classical species of the Mycobacterium tuberculosis complex. Using 20 strains, three enzyme combinations and eight selective amplification primer pairs, 24 AFLP reactions were performed per strain. Overall, this resulted in 480 DNA fingerprints and more than 1200 htAFLP-amplified PCR fragments were visualised per strain. The cumulative dendrogram correctly clustered strains from the various species, albeit within a distance of 6.5% for most of them. The single isolate of Mycobacterium canettii presented separately at 19% distance. All over, 169 fragments (14%) appeared to be polymorphic. Sixty-eight were specific for M. canetti and forty-five for Mycobacterium bovis. For the 10 different M. tuberculosis strains included in the present analysis, 56 polymorphic markers were identified. Upon sequencing 20 of these marker regions and comparisons with the H37Rv genome sequence, 25% appeared to share homology to members of the antigenically variable PE/PPE surface protein encoding gene family confirming previous findings on the genetic heterogeneity within these genes. In addition, homologues for phage genes and insertion element-encoded genes were detected. Forty-five percent of the sequences derived from ORFs with a currently unknown function, which was corroborated by genome sequence comparison for the clinical M. tuberculosis CD 1551 isolate. Sequence variation in M. tuberculosis was assessed in more detail for a subset of these loci by newly designed PCR restriction fragment length polymorphism (RFLP) tests and direct sequencing. Fourteen novel PCR RFLP tests were developed and twelve novel single nucleotide polymorphisms (SNPs) were identified, all suited for epidemiological analysis of M. tuberculosis. The tests allowed for identification of the major Mycobacterium species and M. tuberculosis variants and clones.

Cluster Analysis↗