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A C Fluit

Publications and source records attributed to A C Fluit.

At least 109 records · Page 6Linked to original sources
PubMed1999

Invasive and noninvasive group A streptococcal isolates with different speA alleles in The Netherlands: genetic relatedness and production of pyrogenic exotoxins A and B.

Streptococcal pyrogenic exotoxin A (SPE-A) and SPE-B have been implicated in the pathogenesis of severe group A streptococcal (GAS) disease. We studied 31 invasive GAS strains including 18 isolates from patients with toxic shock syndrome and 22 noninvasive strains isolated in The Netherlands between 1994 and 1998. These strains were associated with the different allelic variants of the gene encoding SPE-A. We selected endemic strains with speA-positive M and T serotypes: speA2-associated M1T1 and M22-60T12 strains, speA3-associated M3T3 strains, and speA4-associated M6T6 strains. Since speA1-positive isolates were not frequently encountered, we included speA1 strains of different serotypes. The GAS strains were compared genotypically by pulsed-field gel electrophoresis and phenotypically by the in vitro production of SPE-A and SPE-B. All strains within one M and T type appeared to be of clonal origin. Most strains produced SPE-A and SPE-B, but only a minority of the speA4-positive isolates did so. Among our isolates, speA1- and speA3-positive strains produced significantly more SPE-A than speA2- and speA4-carrying strains, while SPE-B production was most pronounced among speA1- and speA2-containing strains. There was a marked degree of variability in the amounts of exotoxins produced in vitro by strains that shared the same genetic profile. We conclude that the differences in the in vitro production of SPE-A and SPE-B between our selected strains with identical M and T types were not related to either genetic heterogeneity or the clinical course of GAS disease in the patient from whom they were isolated.

Alleles↗
PubMed1999

The prevalence of aminoglycoside resistance and corresponding resistance genes in clinical isolates of staphylococci from 19 European hospitals.

Aminoglycosides still play an important role in antistaphylococcal therapies, although emerging resistance amongst staphylococci is widespread. To further our understanding of the prevalence of aminoglycoside resistance in Europe, we tested 699 and 249 consecutive unrelated clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci (CNS), respectively, from the SENTRY Antimicrobial Surveillance Program, for susceptibility to gentamicin, tobramycin, kanamycin and streptomycin, and examined the relationship between susceptibility to these antimicrobials and susceptibility to methicillin. Three hundred and sixty-three staphylococcal isolates demonstrated resistance to at least one of the aminoglycosides tested; all of these isolates were screened for the presence of aac(6')-Ie + aph(2"), ant(4')-Ia and aph(3')-IIIa, the genes encoding the most clinically relevant aminoglycoside-modifying enzymes. S. aureus isolates derived from hospital-acquired pneumonia tended to be more resistant to aminoglycosides and methicillin than isolates from blood or wound infections. In S. aureus, resistance to aminoglycosides was closely associated with methicillin resistance. Susceptibility of S. aureus to gentamicin has decreased by 9% from previous European studies to a current level of 77%, while susceptibility of CNS, currently at 67%, has increased by 21%. Geographical variation occurred, correlating with methicillin resistance, although intra-country variation was considerable. aac(6')-Ie + aph(2"), ant(4')-Ia and aph(3')-IIIa were found throughout Europe in 68%, 48% and 14% respectively of staphylococci resistant to at least one aminoglycoside. aph(3')-IIIa was considerably more common in methicillin-susceptible S. aureus and CNS isolates; the reverse was true for the other two resistance genes. The prevalence of ant(4')-Ia and aph(3')-IIIa genes in aminoglycoside-resistant staphylococci was significantly greater than that reported in previous European studies.

Acetyltransferases↗
PubMed1998

Cloning and characterization of an accessory gene regulator (agr)-like locus from Staphylococcus epidermidis.

The presence of sequences related to the agr of Staphylococcus aureus was demonstrated in Staphylococcus epidermidis by agr-specific PCR, and Southern blot. The agr-like locus of S. epidermidis A086 was cloned and sequenced. An overall homology of 68% was found between the agr locus from S. epidermidis and S. aureus. The agr locus from S. epidermidis was organized similar to those from S. aureus and S. lugdunensis. The putative RNAII molecule contains four open reading frames, agr A, B, C and D. AgrA was a response regulator. AgrB showed homology with transducer and translocase molecules. AgrC is expected to act as a histidine protein kinase in which a leucine zipper is present. AgrD is presumably processed into an autoinducer peptide. The putative RNAIII molecule contained an open reading frame encoding a putative 26 amino acid (aa) polypeptide, which differed in 3 aa from the RNAIII encoded delta-toxin of S. aureus. Kinetic studies showed that the production of this RNAIII was elevated during the post-exponential phase. delta-Toxin activity was demonstrated for 21 of 23 tested S. epidermidis strains. Kinetic studies of the production of delta-toxin showed that the toxin was produced during the post-exponential phase. Sequencing of S. epidermidis A097, which showed a delayed agr-response, revealed a truncated AgrC lacking the histidine kinase domain. These data indicate that an agr-like locus is active in S. epidermidis during the post-exponential phase.

Amino Acid Sequence↗
PubMed1998

Detection of staphylococcal genes directly from cerebrospinal and peritoneal fluid samples using a multiplex polymerase chain reaction.

A multiplex polymerase chain reaction (PCR) that can simultaneously detect eubacterial isolates and the methicillin-susceptibility of staphylococcal isolates from cerebrospinal and peritoneal fluid samples was compared to conventional microbiological methods. Using conventional methods, bacteria were isolated from 8% (29/350) of the cerebrospinal fluid samples and from 5% (3/60) of the peritoneal fluid samples. All isolates except two Staphylococcus epidermidis isolates were also detected using the multiplex PCR. Coagulase-negative staphylococci and Staphylococcus aureus were correctly identified using both methods. The multiplex PCR can rapidly and simultaneously detect eubacteria, and the methicillin susceptibility of staphylococci from samples containing > or = 10(2) cfu/ml of bacteria.

Ascitic Fluid↗
PubMed1998

The prevalence of low- and high-level mupirocin resistance in staphylococci from 19 European hospitals.

The topical agent mupirocin plays a crucial role in strategies designed to control outbreaks of methicillin-resistant Staphylococcus aureus. The extent of high- or low-level mupirocin resistance amongst S. aureus from European hospitals is not known. Six hundred and ninety-nine S. aureus and 249 coagulase-negative staphylococci (CNS) derived from blood, hospital-acquired pneumonia or skin and soft tissue infections from 19 European hospitals were tested for susceptibility to mupirocin and oxacillin. Methicillin sensitivity was found in 72% and 32% of S. aureus and CNS, respectively. High-level mupirocin resistance was detected in 1.6% of S. aureus and 5.6% of CNS isolates, while low-level mupirocin resistance was detected in 2.3% of S. aureus and 7.2% of CNS isolates. Amongst S. aureus, methicillin-resistant isolates were twice as likely to have high- or low-level mupirocin resistance. This difference was less pronounced in CNS. No relationship was found between the site of infection and prevalence of mupirocin resistance. High- and low-level mupirocin resistance was detected amongst staphylococci from 10 and 16 of the hospitals studied, respectively. To maintain the relatively low prevalence of mupirocin resistance in Europe amongst both S. aureus and CNS, the prudent use of mupirocin restricted to defined infection control strategies should be emphasized.

Anti-Bacterial Agents↗
PubMed1998

Class I integrons in Gram-negative isolates from different European hospitals and association with decreased susceptibility to multiple antibiotic compounds.

Class I integrons are associated with carriage of genes encoding resistance to antibiotics. Expression of inserted resistance genes within these structures can be poor and, as such, the clinical relevance in terms of the effect of integron carriage on susceptibility has not been investigated. Of 163 unrelated Gram-negative isolates randomly selected from the intensive care and surgical units of 14 different hospitals in nine European countries, 43.0% (70/163) of isolates were shown to be integron-positive, with inserted gene cassettes of various sizes. Integrons were detected in isolates from all hospitals with no particular geographical variations. Integron-positive isolates were statistically more likely to be resistant to aminoglycoside, quinolone and beta8-lactam compounds, including third-generation cephalosporins and monobactams, than integron-negative isolates. Integron-positive isolates were also more likely to be multi-resistant than integron-negative isolates. This association implicates integrons in multi-drug resistance either directly through carriage of specific resistance genes, or indirectly by virtue of linkage to other resistance determinants such as extended-spectrum beta-lactamase genes. As such their widespread presence is a cause for concern. There was no association between the presence of integrons and susceptibility to cefepime, amikacin and the carbapenems, to which at least 97% of isolates were fully susceptible.

Anti-Bacterial Agents↗
PubMed1998

The effect of reserpine, an inhibitor of multidrug efflux pumps, on the in-vitro activities of ciprofloxacin, sparfloxacin and moxifloxacin against clinical isolates of Staphylococcus aureus.

In Staphylococcus aureus, in addition to mutations in the grl and gyr gene loci, multidrug efflux pumps like NorA contribute to decreased fluoroquinolone susceptibility. Efflux pumps can be inhibited by the plant alkaloid reserpine, which, at 20 mg/L, reduced sparfloxacin, moxifloxacin and ciprofloxacin IC50s and MICs by up to four-fold in 11, 21 and 48 of the 102 unrelated clinical isolates tested, respectively. The effect was less pronounced with the hydrophobic drugs sparfloxacin and moxifloxacin than with the hydrophilic drug ciprofloxacin and was stable in all 25 clonally related isolates tested.

Anti-Infective Agents↗
PubMed1998

The effect of culture conditions on the in-vitro adherence of methicillin-resistant Staphylococcus aureus.

Methicillin-resistant isolates of Staphylococcus aureus (MRSA) were divided on the basis of their epidemiologic behaviour into two subgroups, epidemic MRSA (EMRSA) and sporadic MRSA (SMRSA) strains. An existing adherence assay was modified to determine differences in adherence properties between these two groups of MRSA, and the influence of culture conditions on the adherence of SMRSA and EMRSA strains to plastic, human collagen I (HuCol I) and pharyngeal carcinoma Detroit 562 cells (D562) was determined. In-vitro parameters, such as culture medium, growth temperature and growth phase of the bacterium, influenced the adherence of MRSA strains to plastic significantly. Even more pronounced differences in adherence due to changes in growth conditions and growth phase of the bacteria were found for the adherence of MRSA strains to HuCol I. Growth phase had a significant effect on the adherence of MRSA strains to the pharyngeal carcinoma cells D562. However, the study did not find conditions which made it possible to distinguish EMRSA from SMRSA strains. These data show that extrapolation of in-vitro data concerning adherence of MRSA strains to in-vivo conditions should be treated with caution.

Bacterial Adhesion↗
PubMed1995

Molecular identification of bacteria by fluorescence-based PCR-single-strand conformation polymorphism analysis of the 16S rRNA gene.

PCR-single-strand conformation polymorphism (PCR-SSCP) analysis is a rapid and convenient technique for the detection of mutations and allelic variants. We have adapted this technique for the identification of bacteria by PCR with fluorescein-labeled primers chosen from the conserved regions of the 16S rRNA gene flanking a variable region. The PCR product was denatured, separated on a nondenaturing gel, and detected by an automated DNA sequencer. The mobility of the single-stranded DNA is sequence dependent and allows the identification of a broad panel of bacteria. A single nucleotide difference in the amplified region was sufficient to obtain different PCR-SSCP patterns. The simultaneous amplification of multiple polymorphic regions by multiplex PCR with subsequent multiplex SSCP increased the discriminatory power of PCR-SSCP. A broad range of gram-negative and gram-positive bacteria were tested by PCR-SSCP, including, e.g., Escherichia coli, Enterobacter spp., Klebsiella spp., Haemophilus spp., Neisseria spp., Staphylococcus spp, Streptococcus spp., Enterococcus spp., and Bacillus spp. In total, a panel of 178 strains of bacteria representing 51 species in 21 genera was examined. Although a limited number of strains from each species were tested, the strains tested gave species-specific patterns, with only one exception: Shigella species were indistinguishable from E. coli. PCR is a sensitive technique; as few as 10 CFU of E. coli was sufficient to produce PCR-SSCP patterns suitable for identification. The whole fluorescence PCR-SSCP procedure takes approximately 8 h for the detection and identification of low numbers of bacteria.2+ fluorescence PCR-SSCP seems to be a promising method for the differentiation of a broad range of pathogens found in usually sterile clinical sites, such as blood and cerebrospinal fluid.

Bacteria↗
PubMed1995

Phenotypic characterization of epidemic versus sporadic strains of methicillin-resistant Staphylococcus aureus.

Forty strains of methicillin-resistant Staphylococcus aureus (MRSA) were divided on the basis of their epidemiologic behavior into two subgroups, sporadic MRSA (SMRSA) and epidemic MRSA (EMRSA) strains. The strains were examined for binding of 125I-labelled fibronectin, vitronectin, collagen, Fc fragments of immunoglobulin G, and fibrinogen. A significant difference between EMRSA and SMRSA strains was found for binding of 125I-labelled fibrinogen and for Fc fragments of immunoglobulin G, (P < 0.05). No significant difference in the binding of 125I-labelled fibronectin and collagen was found between EMRSA and SMRSA strains. The binding of 125I-labelled vitronectin to MRSA strains was found to be aspecific. Capsular serotypes of the strains were determined with monoclonal antibodies against capsular types 5 and 8. Strains could be divided into the following four groups: types 5, 8, and 5/8 and nontypeable. More nontypeable strains were found in the EMRSA group (66.6%). Significantly more EMRSA strains (79%) than SMRSA strains (44%) produced alpha-toxin (P < 0.025). Logistic regression analysis using a combination of the parameters 125I-labelled immunoglobulin G binding, capsular type, and alpha-toxin production predicted the epidemic character with a sensitivity of 83% and a specificity of 75%.

Bacterial Toxins↗
PubMed1994

Multivalent binding of toxin A from Clostridium difficile to carbohydrate receptors.

A specific monoclonal antibody against toxin A from Clostridium difficile was generated that did not show thermolabile binding. Nonspecific murine monoclonal antibodies bound toxin A at 4 degrees C, but less effectively at 37 degrees C. Nonspecific human monoclonal antibodies did not bind to toxin A at 4 degrees C. Cytotoxic properties of purified toxin A were not inhibited by Bandeiraea simplicifolia lectin. This points to a carbohydrate moiety on the cell surface and a multivalent nonspecific carbohydrate binding ligand on toxin A.

Animals↗
PubMed1994

Reactivity of monoclonal antibodies to Pseudomonas aeruginosa isolates from hospitalized adults and patients with cystic fibrosis.

To assess the potential therapeutic use of three human monoclonal antibodies (MAbs) and one murine MAb, we tested the reactivity of these MAbs toward 100 clinical isolates of Pseudomonas aeruginosa. Seventy-five isolates were from hospitalized patients, while 25 were recovered from patients with documented cystic fibrosis. Nine isolates showed autoagglutination. The three human MAbs and one murine MAb exhibited agglutination with 81% of the P. aeruginosa isolates obtained from hospitalized patients and with 72% of the isolates from patients with cystic fibrosis. Eight of the autoagglutinating isolates were reactive in whole-cell ELISAs with the MAbs. The isolates recovered from patients with cystic fibrosis were reactive mainly with a MAb that is directed to the outer core of lipopolysaccharide (LPS); they showed hardly any reaction with O antigen-specific MAbs or typing sera, a finding indicating that these isolates lacked the O antigen and that the outer core of the LPS was still present. The reactivity of the MAb specific for the outer core of LPS toward P. aeruginosa isolates from patients with cystic fibrosis has potential value in eradicating P. aeruginosa from these patients.

Adult↗
PubMed1994

Toxins A and B of Clostridium difficile.

The toxins produced by Clostridium difficile share several functional properties with other bacterial toxins, like the heat-labile enterotoxin of Escherichia coli and cholera toxin. However, functional and structural differences also exist. Like cholera toxin, their main target is the disruption of the microfilaments in the cell. However, since these effects are not reversible, as found with cholera toxin, additional mechanisms add to the cytotoxic potential of these toxins. Unlike most bacterial toxins, which are built from two structurally and functionally different small polypeptide chains, the functional and binding properties of the toxins of C. difficile are confined within one large polypeptide chain, making them the largest bacterial toxins known so far.

Bacterial Proteins↗
PubMed1994

Rapid identification of bacteria by PCR-single-strand conformation polymorphism.

A new molecular biological approach for the identification of bacteria is described. This approach employs PCR of bacterial cell lysates with conserved primers located in the 16S rRNA sequence flanking a variable region, and analysis of the amplified product was based on the principle of single-strand conformation polymorphism (SSCP). The PCR product was denatured and separated on a nondenaturing polyacrylamide gel. SSCP patterns were detected by silver staining the nucleic acids. The mobility of the single-stranded DNA is sequence dependent and could be used to identify the unknown bacteria. Feasibility of the technique was demonstrated for a broad panel of gram-negative and gram-positive bacteria. We tested over 100 strains of bacteria representing 15 genera and 40 species. With the use of only two primer sets, P11P-P13P and ER10-ER11, we were capable to discriminate the tested species at the genus and species levels. Species-specific patterns were obtained for, e.g., Clostridium spp., Listeria spp., Pseudomonas spp., and Enterobacter spp. PCR-SSCP is a sensitive technique; e.g., the sensitivity obtained for Escherichia coli cells was 30 CFU. This technique is a simple and rapid method for the detection and identification of a wide spectrum of bacteria by whole-cell-based PCR amplification with the use of conserved primers and identification by nondenaturing gel electrophoresis.

Bacteria↗
PubMed1994

Rapid detection of toxigenic Clostridium difficile in fecal samples by magnetic immuno PCR assay.

Rapid detection of toxigenic Clostridium difficile in fecal samples was accomplished with the magnetic immuno PCR assay (MIPA). Elaborate DNA extraction techniques were unnecessary. First, we generated a mouse monoclonal antibody (MAb) reactive with only C. difficile, Clostridium sordellii, and Clostridium bifermentans. Then, magnetic beads were coated with the MAb, incubated with fecal samples to allow binding with C. difficile, extracted from the stool with a magnet, and processed in the PCR with primers specific for the toxin B gene. After optimizing MIPA by raising the number of PCR cycles from 35 to 40 and adding Chelex 100 to the PCR mixture, we found a sensitivity of 96.7%, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 94.1% when compared with the culture of cytotoxic C. difficile from fecal samples. MIPA is a rapid, easy, and sensitive PCR method for demonstrating the presence of toxigenic C. difficile in stool samples and avoids the disadvantage of elaborate extraction of DNA from fecal samples.

Antibodies, Monoclonal↗
PubMed1994

Clinical significance of Clostridium difficile and its toxins in faeces of immunocompromised children.

In this study, clinical and laboratory findings were tested for correlation with the presence of Clostridium difficile. The toxigenicity of the isolated strains and the toxins were determined in faecal samples of immunocompromised children admitted to a single room for protective isolation. Using the toxin assay as the gold standard, the culture sensitivity of toxigenic C difficile was 94.1%, the specificity 93.8%, the positive predictive value 62.8%, and the negative predictive value 99.3%. Correction for stools with a positive culture of toxigenic C difficile preceding detection of toxin, resulted in a positive prediction value of 78.4%. A statistically significant association was found between a positive faecal toxin assay and fever, and between a positive culture of toxigenic C difficile and abdominal pain: 42% of the patients with positive toxin assays had fever versus 21% with negative toxin assays, and 66% of the patients with a positive culture for toxigenic C difficile had abdominal pain, versus 22% with negative cultures. Further analysis of the cultures and toxin assays showed no statistically significant association with diarrhoea, fever, white blood cell count, C reactive protein concentrations, or abdominal pain. Based on these findings, it is suggested that immunocompromised children should be treated when toxigenic C difficile is cultured or when toxin is detected in stool samples.

Abdominal Pain↗