Search

We studied the prevalence and clonality of high-level gentamicin-resistant enterococci (HLGRE) in a Dutch university hospital. Of 238 enterococcal strains isolated from blood cultures between 1991 and 1997, 57 were HLGRE. Genomic analysis of these strains revealed 19 different genotypes, two of which were encountered more frequently [type A (12/57), type B (23/57)]. The spread of these types largely explained the rise in HLGRE incidence from 14% in 1991 to 31% in 1997. However, the contribution of unique strains to the total HLGRE burden also increased from 4% to 16%. We conclude that both clonal expansion and the emergence of unique HLGRE have contributed significantly to the increasing incidence of HLGRE. [ABSTRACT FROM AUTHOR]

The activity of quinupristin/dalfopristin was compared with that of other widely used antibiotics against 355 strains isolated from patients with endocarditis. MICs were determined by a standard agar dilution method. Quinupristin/dalfopristin was inhibitory at 1 mg/L for all coagulase-negative staphylococci (n = 36) and for the majority of Staphylococcus aureus strains (n = 87). The activity of quinupristin/dalfopristin against 186 viridans streptococci was somewhat dependent on the species, with MIC50s ranging from 0.5 to 2 mg/L, being least active against Streptococcus bovis and most active against Streptococcus gordonii and Streptococcus mitis. For the staphylococci, quinupristin/dalfopristin was as active against erythromycin-susceptible as erythromycin-resistant strains. Viridans streptococci showed a slight but significant correlation between sensitivity to erythromycin and quinupristin/dalfopristin. It is concluded that quinupristin/dalfopristin has the potential to treat serious infections such as endocarditis caused by Gram-positive cocci. [ABSTRACT FROM PUBLISHER]

Abstract Background Campylobacter jejuni is the predominant cause of antecedent infection in post-infectious neuropathies such as the Guillain-Barré (GBS) and Miller Fisher syndromes (MFS). GBS and MFS are probably induced by molecular mimicry between human gangliosides and bacterial lipo-oligosaccharides (LOS). This study describes a new C. jejuni-specific high-throughput AFLP (htAFLP) approach for detection and identification of DNA polymorphism, in general, and of putative GBS/MFS-markers, in particular. Results We compared 6 different isolates of the "genome strain" NCTC 11168 obtained from different laboratories. HtAFLP analysis generated approximately 3000 markers per stain, 19 of which were polymorphic. The DNA polymorphisms could not be confirmed by PCR-RFLP analysis, suggesting a baseline level of 0.6% AFLP artefacts. Comparison of NCTC 11168 with 4 GBS-associated strains revealed 23 potentially GBS-specific markers, 17 of which were identified by DNA sequencing. A collection of 27 GBS/MFS-associated and 17 enteritis control strains was analyzed with PCR-RFLP tests based on 11 of these markers. We identified 3 markers, located in the LOS biosynthesis genes cj1136, cj1138 and cj1139c, that were significantly associated with GBS (P = 0.024, P = 0.047 and P < 0.001, respectively). HtAFLP analysis of 13 highly clonal South African GBS/MFS-associated and enteritis control strains did not reveal GBS-specific markers. Conclusion This study shows that bacterial GBS markers are limited in number and located in the LOS biosynthesis genes, which corroborates the current consensus that LOS mimicry may be the prime etiologic determinant of GBS. Furthermore, our results demonstrate that htAFLP, with its high reproducibility and resolution, is an effective technique for the detection and subsequent identification of putative bacterial disease markers.

Ganglioside mimicry in the lipopolysaccharide (LPS) fraction of Campylobacter jejuni isolated from Guillain-Barré syndrome (GBS) and Miller Fisher syndrome (MFS) patients was compared with isolates from patients with an uncomplicated enteritis. The antibody response to C. jejuni LPS and gangliosides in neuropathy patients and controls was compared as well. LPS from GBS and MFS-associated isolates more frequently contained ganglioside-like epitopes compared to control isolates. Almost all neuropathy patients showed a strong antibody response against LPS and multiple gangliosides in contrast to enteritis patients. Isolates from GBS patients more frequently had a GM1-like epitope than isolates from MFS patients. GQ1b-like epitopes were present in all MFS-associated isolates and was associated with anti-GQ1b antibody reactivity and the presence of oculomotor symptoms. These results demonstrate that the expression of ganglioside mimics is a risk factor for the development of post-Campylobacter neuropathy. This study provides additional evidence for the hypothesis that the LPS fraction determines the antiganglioside specificity and clinical features in post-Campylobacter neuropathy patients. [ABSTRACT FROM AUTHOR]

Guillain-Barré syndrome (GBS) and Miller-Fisher syndrome (MFS) are correlated with prior infection by Campylobacter jejuni in up to 40% of cases. Nucleotide sequence-based typing of 25 C. jejuni isolates associated with neuropathy permitted robust comparisons with equivalent data from approximately 800 C. jejuni isolates not associated with neuropathy. A total of 13 genetic lineages and 20 flaA short variable region nucleotide sequences were present among the 25 isolates. A minority of isolates (4 of 25) had the flaA short variable region nucleotide sequences that were previously proposed as a marker for GBS-associated isolates. These 4 isolates probably represented the Penner serotype 19 lineage, which has been proposed to have an association with GBS. [ABSTRACT FROM AUTHOR]

Examines the variable flagella expression among clonal toxin Clostridium difficile strains. Presence of flagella and lack of polymorphism in flagellin genes; Function of flagella as virulence determinant for several microbial species; Detection and identification of toxin and genetic strains.

In the recent years, vibrational spectroscopies (infrared and Raman spectroscopy) have been developed for all sorts of analyses in microbiology. Important features of these methods are the relative ease with which measurements can be performed. Furthermore, in order to obtain infrared or Raman spectra, there is only a limited amount of sample handling involved without the need for expensive chemicals, labels or dyes. Here, we review the potential application of vibrational spectroscopies for the use in medical microbiology. After describing some of the basics of the techniques, considerations on reproducibility and standardisation are presented. Finally, the use of infrared and Raman spectroscopy for the (rapid) identification of medically relevant microorganisms is discussed. It can be concluded that vibrational spectroscopies show high potential as novel methods in medical microbiology. [Copyright &y& Elsevier]

Background: The lead symptom of small fibre neuropathy (SFN) is neuropathic pain. Recent functional magnetic resonance imaging (fMRI) studies have indicated central changes in SFN patients of different etiologies. However, less is known about brain functional connectivity during acute pain processing in idiopathic SFN., Methods: We conducted fMRI with thermal heat pain application (left volar forearm) in 32 idiopathic SFN patients and 31 healthy controls. We performed functional connectivity analyses with right supplementary motor area (SMA), left insula, and left caudate nucleus (CN) as seed regions, respectively. Since pathogenic gain-of-function variants in voltage gated sodium channels (Nav) have been linked to SFN pathophysiology, explorative connectivity analyses were performed in a homogenous subsample of patients carrying rare heterozygous missense variants., Results: For right SMA, we found significantly higher connectivity with the right thalamus in SFN patients compared to controls. This connectivity correlated significantly with intraepidermal nerve fibre density, suggesting a link between peripheral and central pain processing. We found significantly reduced connections between right SMA and right middle frontal gyrus in patients with Nav variants. Likewise, connectivity between left CN and right frontal pole was decreased., Conclusions: Aberrant functional connectivity in SFN is in line with previous research on other chronic pain syndromes. Functional connectivity changes may be linked to SFN, highlighting the need to determine if they result from peripheral changes causing abnormal somatosensory processing. This understanding may be crucial for assessing their impact on painful symptoms and therapy response., Significance Statement: We found increased functional connectivity between SMA and thalamus during painful stimulation in patients with idiopathic SFN. Connectivity correlated significantly with intraepidermal nerve fibre density, suggesting a link between peripheral and central pain processing. Our findings emphasize the importance of investigating functional connectivity changes as a potential feature of SFN., (© 2024 The Author(s). European Journal of Pain published by John Wiley & Sons Ltd on behalf of European Pain Federation ‐ EFIC ®.)

Small-fiber neuropathy (SFN) is defined by degeneration or dysfunction of peripheral sensory nerve endings. Central correlates have been identified on the level of gray matter volume (GMV) and cortical thickness (CT) changes. However, across SFN etiologies knowledge about a common structural brain signature is still lacking. Therefore, we recruited 26 SFN patients and 25 age- and sex-matched healthy controls to conduct voxel-based- and surface-based morphometry. Across all patients, we found reduced GMV in widespread frontal regions, left caudate, insula and superior parietal lobule. Surface-based morphometry analysis revealed reduced CT in the right precentral gyrus of SFN patients. In a region-based approach, patients had reduced GMV in the left caudate. Since pathogenic gain-of-function variants in voltage-gated sodium channels (Nav) have been associated with SFN pathophysiology, we explored brain morphological patterns in a homogenous subsample of patients carrying rare heterozygous missense variants. Whole brain- and region-based approaches revealed GMV reductions in the bilateral caudate for Nav variant carriers. Further research is needed to analyze the specific role of Nav variants for structural brain alterations. Together, we conclude that SFN patients have specific GMV and CT alterations, potentially forming potential new central biomarkers for this condition. Our results might help to better understand underlying or compensatory mechanisms of chronic pain perception in the future. PERSPECTIVE: This study reveals structural brain changes in small-fiber neuropathy (SFN) patients, particularly in frontal regions, caudate, insula, and parietal lobule. Notably, individuals with SFN and specific Nav variants exhibit bilateral caudate abnormalities. These findings may serve as potential central biomarkers for SFN and provide insights into chronic pain perception mechanisms., (Copyright © 2024 United States Association for the Study of Pain, Inc. Published by Elsevier Inc. All rights reserved.)

Rett syndrome (RTT; OMIM 312750) is an X-linked dominant neurodevelopmental disorder leading to cognitive and motor impairment, epilepsy, and autonomic dysfunction in females. Since the discovery that RTT is caused by mutations in MECP2, large retrospective genotype-phenotype correlation studies have been performed. A number of general genotype-phenotype relationships were confirmed and specific disorder profiles were described. Nevertheless, conflicting results are still under discussion, partly due to the variability in classification of mutations, assessment tools, and structure of the data sets. The aim of this study was to investigate relationships between genotype and specific clinical data collected by the same experienced physician in a well-documented RTT cohort, and evaluate its prognostic value in counseling young parents with a newly diagnosed RTT girl regarding her future outcome. The Maastricht-Leuven Rett Syndrome Database is a register of 137 molecularly confirmed clinical RTT cases, containing both molecular and clinical data on examination and follow up by the same experienced physician. Although the general genotype-phenotype relationships were confirmed, the clinical severity was still found to be very variable. We therefore recommend caution in using genotype-phenotype data in the prognosis of outcome for children in Rett syndrome. Early diagnosis, early intervention, and preventive management are imperative for better outcomes and better quality of daily life for RTT females and their families., (Copyright © 2011 Wiley Periodicals, Inc.)

Molecular mimicry of Campylobacter jejuni lipooligosaccharides (LOS) by gangliosides in peripheral nerve tissue probably triggers the Guillain-Barré syndrome due to the induction of cross-reactive antibodies. PCR-restriction fragment length polymorphism analysis of C. jejuni genes involved in the biosynthesis of LOS demonstrated that specific genes were associated with the expression of ganglioside mimics and the development of neuropathy.

Background: Campylobacter jejuni is the predominant cause of antecedent infection in post-infectious neuropathies such as the Guillain-Barré (GBS) and Miller Fisher syndromes (MFS). GBS and MFS are probably induced by molecular mimicry between human gangliosides and bacterial lipo-oligosaccharides (LOS). This study describes a new C. jejuni-specific high-throughput AFLP (htAFLP) approach for detection and identification of DNA polymorphism, in general, and of putative GBS/MFS-markers, in particular., Results: We compared 6 different isolates of the "genome strain" NCTC 11168 obtained from different laboratories. HtAFLP analysis generated approximately 3000 markers per stain, 19 of which were polymorphic. The DNA polymorphisms could not be confirmed by PCR-RFLP analysis, suggesting a baseline level of 0.6% AFLP artefacts. Comparison of NCTC 11168 with 4 GBS-associated strains revealed 23 potentially GBS-specific markers, 17 of which were identified by DNA sequencing. A collection of 27 GBS/MFS-associated and 17 enteritis control strains was analyzed with PCR-RFLP tests based on 11 of these markers. We identified 3 markers, located in the LOS biosynthesis genes cj1136, cj1138 and cj1139c, that were significantly associated with GBS (P = 0.024, P = 0.047 and P < 0.001, respectively). HtAFLP analysis of 13 highly clonal South African GBS/MFS-associated and enteritis control strains did not reveal GBS-specific markers., Conclusion: This study shows that bacterial GBS markers are limited in number and located in the LOS biosynthesis genes, which corroborates the current consensus that LOS mimicry may be the prime etiologic determinant of GBS. Furthermore, our results demonstrate that htAFLP, with its high reproducibility and resolution, is an effective technique for the detection and subsequent identification of putative bacterial disease markers.

Campylobacter jejuni is the predominant cause of antecedent infection in Guillain-Barré syndrome (GBS) or Miller Fisher syndrome (MFS). C. jejuni probably triggers GBS or MFS through molecular mimicry between bacterial sialylated lipo-oligosaccharides (LOS) and gangliosides in peripheral nerve tissue. We investigated whether co-infections with multiple C. jejuni strains occur in GBS or MFS patients and we further characterized these strains. PFGE analysis of 83 C. jejuni isolates from single primary colonies from stool cultures of 13 patients with GBS or MFS revealed co-infection with two different strains in one patient (8%). We showed that only strain GB5.1 contained an LOS biosynthesis gene locus that is associated with neuropathy. The patient serum strongly reacted with the LOS of strain GB5.1 and not with the LOS of strain GB5.2. Mass spectrometry revealed that both strains expressed a non-sialylated outer core structure in their LOS. The patient serum contained anti-asialo-GM2 antibodies that cross-reacted with the LOS of strain GB5.1. This study demonstrates that co-infection with multiple C. jejuni strains occurs in GBS patients. Consequently, not all C. jejuni strains isolated from the faeces of a GBS patient are involved in the pathogenesis of GBS per se. Furthermore, this is the first report in which cross-reactivity of antibodies to asialo-GM2 and to the LOS of a C. jejuni strain from a GBS patient has been demonstrated. This finding suggests that molecular mimicry with non-sialylated structures may also be involved in the pathogenesis of GBS.

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.

A steady increase in the incidence of Guillain-Barré syndrome (GBS) with a seasonal preponderance, almost exclusively related to Campylobacter jejuni, and a rise in the incidence of laboratory-confirmed Campylobacter enteritis have been reported from Curaçao, Netherlands Antilles. We therefore investigated possible risk factors associated with diarrhea due to epidemic C. jejuni. Typing by pulsed-field gel electrophoresis identified four epidemic clones which accounted for almost 60% of the infections. One hundred six cases were included in a case-control study. Infections with epidemic clones were more frequently observed in specific districts in Willemstad, the capital of Curaçao. One of these clones caused infections during the rainy season only and was associated with the presence of a deep well around the house. Two out of three GBS-related C. jejuni isolates belonged to an epidemic clone. The observations presented point toward water as a possible source of Campylobacter infections.

Campylobacter jejuni isolates (n = 234) associated with gastroenteritis and the Guillain-Barré syndrome (GBS) in the island of Curaçao, Netherlands Antilles, and collected from March 1999 to March 2000 were investigated by a range of molecular typing techniques. Data obtained by pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) analysis, multilocus sequence typing (MLST), automated ribotyping, and sequence analysis of the short variable region of the flagellin gene (flaA) were analyzed separately and in combination. Similar groupings were obtained by all methods, with the data obtained by MLST and AFLP analysis exhibiting the highest degree of congruency. MLST identified 29 sequence types, which were assigned to 10 major clonal complexes. PFGE, AFLP analysis, and ribotyping identified 10, 9, and 8 of these clonal groups, respectively; however, these three techniques permitted subdivision of the clonal groups into more different types. Members of seven clonal groups comprising 107 isolates were obtained from November 1999 to February 2000, and no distinguishing characteristics were identified for two GBS-associated strains. The sequence type 41 (ST-41), ST-508, and ST-657 clonal complexes and their corresponding AFLP types have been rare or absent in the Campylobacter data sets described to date. We conclude that several clonal complexes of C. jejuni are associated with human disease in Curaçao, and some of these have not been reported elsewhere. Furthermore, given the observation that C. jejuni-associated diseases appear to be more severe from November to February, it can be speculated that this may be due to the presence of virulent clones with a limited span of circulation.

Analysis of both the antibiotic resistance and the virulence characteristics of anaerobic human microbial pathogens is important in order to improve our understanding of a number of clinically significant infectious diseases, including Clostridium difficile-associated diarrhea (CDAD). We determined the presence of the clindamycin resistance-associated gene ermB and the ribotype of 33 C. difficile strains isolated from Polish patients suffering from CDAD. While all strains produced cytotoxin B (TcdB), enterotoxin A (TcdA) was produced by a subset of 15 strains only. The results showed that a single ermB-positive, TcdA(-)B(+) C. difficile strain with ribotype A has disseminated widely in the two Warsaw hospitals under investigation. Although different strains with the same phenotype were detected, the genotype A strain appeared to be the only one with a clear epidemic character. Apparently, enhanced local spread of CDAD-causing C. difficile may be restricted to a limited number of bacterial genotypes only.

Background: The aim of our research was to investigate the role of enterotoxin- producing anaerobic bacteria other than Clostridium difficile in the etiology of antibiotic-associated diarrhea. This article presents data related to C. perfringens., Material/methods: Stool samples taken from 158 patients with suspected antibiotic-associated diarrhea were specifically cultured for Clostridium difficile, Bacteroides fragilis and Clostridium perfringens. In order to associate the presence of virulence factors in the bacterial isolates thus collected with disease features, all strains were genetically and phenotypically analyzed for toxin production. All isolated C. perfringens strains were cultured in Ellner sporulation-promoting medium., Results: In 21 of the 158 patients (13%) C. perfringens could be cultivated from the fecal specimen. None of the strains produced enterotoxin, and consequently the cpe gene was not detected by PCR in any of these strains. C. perfringens and C. difficile were cultivated from the same stool samples in 4 cases. Interestingly, in one case toxin A-negative/toxin B positive C. difficile and non-enterotoxigenic C. perfringens were co-cultured. After application of a heat shock (100 degrees C at 30 min.) only two C. perfringens strains producing thermoresistant spores were detected. Pulsed field gel electrophoresis (PFGE) demonstrated genetic heterogenicity among the C. perfringens strains, suggesting that these bacteria were already presented upon hospital admission., Conclusions: It seems unlikely that nosocomial transfer has taken place. The relatively low incidence suggests that C. perfringens is not a major primary cause of antibiotic-associated diarrhea.

Ganglioside mimicry in the lipopolysaccharide (LPS) fraction of Campylobacter jejuni isolated from Guillain-Barré syndrome (GBS) and Miller Fisher syndrome (MFS) patients was compared with isolates from patients with an uncomplicated enteritis. The antibody response to C. jejuni LPS and gangliosides in neuropathy patients and controls was compared as well. LPS from GBS and MFS-associated isolates more frequently contained ganglioside-like epitopes compared to control isolates. Almost all neuropathy patients showed a strong antibody response against LPS and multiple gangliosides in contrast to enteritis patients. Isolates from GBS patients more frequently had a GM1-like epitope than isolates from MFS patients. GQ1b-like epitopes were present in all MFS-associated isolates and was associated with anti-GQ1b antibody reactivity and the presence of oculomotor symptoms. These results demonstrate that the expression of ganglioside mimics is a risk factor for the development of post-Campylobacter neuropathy. This study provides additional evidence for the hypothesis that the LPS fraction determines the antiganglioside specificity and clinical features in post-Campylobacter neuropathy patients.

Guillain-Barré syndrome (GBS) and Miller-Fisher syndrome (MFS) are correlated with prior infection by Campylobacter jejuni in up to 40% of cases. Nucleotide sequence-based typing of 25 C. jejuni isolates associated with neuropathy permitted robust comparisons with equivalent data from approximately 800 C. jejuni isolates not associated with neuropathy. A total of 13 genetic lineages and 20 flaA short variable region nucleotide sequences were present among the 25 isolates. A minority of isolates (4 of 25) had the flaA short variable region nucleotide sequences that were previously proposed as a marker for GBS-associated isolates. These 4 isolates probably represented the Penner serotype 19 lineage, which has been proposed to have an association with GBS.

Ertapenem (MK-0826, L-749,345) is a 1-beta-methyl carbapenem with a long serum half-life. Its in vitro activity was determined by broth microdilution against 3,478 bacteria from 12 centers in Europe and Australia, with imipenem, cefepime, ceftriaxone, and piperacillin-tazobactam used as comparators. Ertapenem was the most active agent tested against members of the family Enterobacteriaceae, with MICs at which 90% of isolates are inhibited (MIC(90)s) of < or =1 microg/ml for all species. Ertapenem also was more active than imipenem against fastidious gram-negative bacteria and Moraxella spp.; on the other hand, ertapenem was slightly less active than imipenem against streptococci, methicillin-susceptible staphylococci, and anaerobes, but its MIC(90)s for these groups remained < or =0.5 microg/ml. Acinetobacter spp. and Pseudomonas aeruginosa were also much less susceptible to ertapenem than imipenem, and most Enterococcus faecalis strains were resistant. Ertapenem resistance, based on a provisional NCCLS MIC breakpoint of > or =16 microg/ml, was seen in only 3 of 1,611 strains of the family Enterobacteriaceae tested, all of them Enterobacter aerogenes. Resistance was also seen in 2 of 135 anaerobes, comprising 1 Bacteroides fragilis strain and 1 Clostridium difficile strain. Ertapenem breakpoints for streptococci have not been established, but an unofficial susceptibility breakpoint of < or =2 microg/ml was adopted for clinical trials to generate corresponding clinical response data for isolates for which MICs were as high as 2 microg/ml. Of 234 Streptococcus pneumoniae strains tested, 2 required ertapenem MICs of 2 microg/ml and one required an MIC of 4 microg/ml, among 67 non-Streptococcus pyogenes, non-Streptococcus pneumoniae streptococci, single isolates required ertapenem MICs of 2 and 16 microg/ml. These streptococci also had diminished susceptibilities to other beta-lactams, including imipenem as well as ertapenem. The Etest and disk diffusion gave susceptibility test results in good agreement with those of the broth microdilution method for ertapenem.

We evaluated the accuracy of the VITEK 2 fully automated system to detect and identify glycopeptide-resistant enterococci (GRE) compared to a reference agar dilution method. The sensitivity of vancomycin susceptibility testing with VITEK 2 for the detection of vanA, vanB, and vanC1 strains was 100%. The sensitivity of vancomycin susceptibility testing of vanC2 strains was 77%. The sensitivity of teicoplanin susceptibility testing of vanA strains was 90%. Of 80 vanC enterococci, 78 (98%) were correctly identified by VITEK 2 as Enterococcus gallinarum/Enterococcus casseliflavus. Since the identification and susceptibility data are produced within 3 and 8 h, respectively, VITEK 2 appears a fast and reliable method for detection of GRE in microbiology laboratories.

Genetic typing of vancomycin-resistant enterococci (VRE) can be performed using a variety of methods, but comparative analyses of the quality of these methods are still relatively scarce. We here compare random amplification of polymorphic DNA (RAPD) analysis with pulsed field gel electrophoresis (PFGE) of DNA macrorestriction fragments as examples of two of the recent and well-accepted molecular typing methods. For the latter method, empirical guidelines for the interpretation of the DNA fingerprints have been proposed in the international literature. Based on our experimental analyses, we define similar criteria for RAPD fingerprinting. A collection of 100 strains of VRE, comprising Enterococcus faecium, Enterococcus faecalis, Enterococcus avium, Enterococcus gallinarum and Enterococcus casseliflavus, was assembled. Fifty isolates were Dutch, another 50 were isolated in the UK. Strains were selected on the basis of previously determined putative identity, close relatedness or uniqueness. The strains were analysed using well-standardised RAPD and PFGE protocols. Resulting fingerprints were interpreted with computerised methods involving band positioning and we show that typing of VRE by PFGE and RAPD generates highly congruent DNA fingerprint clustering. When the proposed international criteria for interpretation of PFGE fingerprints were applied in our case, 86% PFGE homology as discriminating value between close relatedness and uniqueness, a 75% homology cut-off for the comparison of the RAPD-generated DNA fingerprints revealed essentially identical strain clusters. As a spin-off it is revealed that strains from the different species can be efficiently discriminated, that strains from the UK and The Netherlands form separate clusters and that strains from veterinary origin can be identified separately as well.

Amplified-fragment length polymorphism (AFLP) analysis was used to investigate the genetic relationships among 255 vancomycin-resistant Enterococcus faecium (VREF) strains isolated from hospitalized patients, nonhospitalized persons, and various animal sources. Four major AFLP genogroups (A-D) were discriminated. The strains of each taxon shared >/=65% of the restriction fragments. Most isolates recovered from nonhospitalized persons (75%) were grouped together with all pig isolates in genogroup A. Most isolates from hospitalized patients (84%), a subset of veal calf isolates (25%), and all isolates from cats and dogs clustered in genogroup C. Most isolates from chickens (97%) and turkeys (86%) were grouped in genogroup B, whereas most veal calf isolates (70%) clustered in genogroup D. Therefore, VREF strains are predominantly host-specific, and strains isolated from hospitalized patients are genetically different from the prevailing VREF strains present in the fecal flora of nonhospitalized persons.

Objective: To determine the prevalence and determinants of fecal carriage of vancomycin-resistant enterococci (VRE) in intensive care unit (ICU), hematology-oncology, and hemodialysis patients in The Netherlands., Design: Descriptive, multicenter study, with yearly 1-week point-prevalence assessments between 1995 and 1998., Population: All patients hospitalized on the testing days in ICUs and hematology-oncology wards in nine hospitals in The Netherlands were included., Methods: Rectal swabs obtained from 1,112 patients were screened for enterococci in a selective broth and subcultured on selective media with and without 6 mg/L vancomycin. Resistance genotypes were determined by polymerase chain reaction. Further characterization of VRE strains was done by pulsed-field gel electrophoresis (PFGE). We studied possible determinants of VRE colonization with a logistic regression analysis model. Determinants analyzed included gender, age, and log-transformed length of prior hospital stay., Results: The results showed that 614 (55%) of 1,112 patients were colonized with vancomycin-sensitive enterococci, and 15 (1.4%) of 1,112 carried VRE. No increase in VRE colonization was observed from 1995 to 1998. Eleven strains were identified as Enterococcus faecium and four as Enterococcus faecalis. All E faecium and one E faecalis carried the vanA gene; the other E faecalis strains harbored the vanB gene. PFGE revealed that three vanB VRE isolated from patients hospitalized in one single ICU were related, suggesting nosocomial transmission. Though higher age seemed associated with VRE colonization, exclusion of patients with the nosocomial strain from the regression analysis decreased this relation to nonsignificant. Duration of hospital stay was not associated with VRE colonization., Conclusion: VRE colonization in Dutch hospitals is an infrequent phenomenon. Although nosocomial spread occurs, most observed cases were unrelated, which suggests the possibility of VRE acquisition from outside the hospital. Prolonged hospital stay, age, and gender proved unrelated to VRE colonization.

Campylobacter jejuni has been identified as the predominant cause of antecedent infection in Guillain-Barré syndrome (GBS) and Miller Fisher syndrome (MFS). The risk of developing GBS or MFS may be higher after infection with specific C. jejuni types. To investigate the putative clonality, 18 GBS- or MFS-related C. jejuni strains from The Netherlands and Belgium and 17 control strains were analyzed by serotyping (Penner and Lior), restriction fragment length polymorphism analysis of PCR products of the flaA gene, amplified fragment length polymorphism analysis, pulsed-field gel electrophoresis, and randomly amplified polymorphic DNA analysis. Serotyping revealed 10 different O serotypes and 7 different Lior serotypes, thereby indicating a lack of serotype clustering. Two new O serotypes, O:35 and O:13/65, not previously associated with GBS or MFS were found. Serotype O:19 was encountered in 2 of 18 strains, and none was of serotype O:41. The results of all genotypic methods also demonstrated substantial heterogeneity. No clustering of GBS- or MFS-related strains occurred and no molecular marker capable of separating pathogenic GBS or MFS from non-GBS- or non-MFS-related enteritis strains could be identified in this study. Sialic-acid-containing lipopolysaccharides (LPS) are thought to be involved in the triggering of GBS or MFS through molecular mimicry with gangliosides in human peripheral nerves. Therefore, further characterization of GBS- or MFS-related C. jejuni should target the genes involved in the synthesis of LPS and the incorporation of sialic acid.

We report on a detailed study on the molecular diversity and evolutionary relationships of Tn1546-like elements in vancomycin-resistant enterococci (VRE) from humans and animals. Restriction fragment length polymorphism (RFLP) analysis of the VanA transposon of 97 VRE revealed seven different Tn1546 types. Subsequent sequencing of the complete VanA transposons of 13 VRE isolates representing the seven RFLP types followed by sequencing of the identified polymorphic regions in 84 other VanA transposons resulted in the identification of 22 different Tn1546 derivatives. Differences between the Tn1546 types included point mutations in orf1, vanS, vanA, vanX, and vanY. Moreover, insertions of an IS1216V-IS3-like element in orf1, of IS1251 in the vanS-vanH intergenic region, and of IS1216V in the vanX-vanY intergenic region were found. The presence of insertion sequence elements was often associated with deletions in Tn1546. Identical Tn1546 types were found among isolates from humans and farm animals in The Netherlands, suggesting the sharing of a common vancomycin resistance gene pool. Application of the genetic analysis of Tn1546 to VRE isolates causing infections in Hospitals in Oxford, United Kingdom, and Chicago, Ill., suggested the possibility of the horizontal transmission of the vancomycin resistance transposon. The genetic diversity in Tn1546 combined with epidemiological data suggest that the DNA polymorphism among Tn1546 variants can successfully be exploited for the tracing of the routes of transmission of vancomycin resistance genes.

Vancomycin-resistant enterococci (VRE) pose an emerging health risk, but little is known about the precise epidemiology of the genes coding for vancomycin resistance. To determine whether the bacterial flora of consumer poultry serves as a gene reservoir, the level of contamination of poultry products with VRE was determined. VRE were genotyped by pulsed-field gel electrophoresis (PFGE), and transposon structure mapping was done by PCR. The vanX-vanY intergenic regions of several strains were further analyzed by sequencing. A total of 242 of 305 (79%) poultry products were found to be contaminated with VRE. Of these VRE, 142 (59%) were high-level-vancomycin-resistant Enterococcus faecium strains (VREF). PFGE revealed extensive VREF heterogeneity. Two genotypes were found nationwide on multiple occasions: type A (22 of 142 VREF [15%]) and type B (14 of 142 VREF [10%]). No PFGE-deduced genetic overlap was found when VREF from humans were compared with VREF from poultry. Two vanA transposon types were identified among poultry strains. In 59 of 142 (42%) of the poultry VREF, the size of the intergenic region between vanX and vanY was approximately 1,300 bp. This transposon type was not found in human VREF. In contrast, all human strains and 83 of 142 (58%) of the poultry VREF contained an intergenic region 543 bp in size. Sequencing of this 543-bp intergenic vanX-vanY region demonstrated full sequence conservation. Though preliminary, these data suggest that dissemination of the resistance genes carried on transposable elements may be of greater importance than clonal dissemination of resistant strains. This observation is important for developing strategies to control the spread of glycopeptide resistance.

A collection of genetically unrelated vancomycin-resistant enterococci (VRE) including 50 vanA, 15 vanB, 50 vanC1, and 30 vanC2 VRE were used to evaluate the accuracy of eight currently available susceptibility test methods (agar dilution, disk diffusion, E-test, agar screen plate, Vitek GPS-TA and GPS-101, and MicroScan overnight and rapid panels). vanA VRE were detected by all methods. vanB VRE were often not detected by Vitek GPS-TA and MicroScan rapid (sensitivities, 47 and 53%, respectively), though the new Vitek GPS-101 was found to be a significant improvement. E-test and the agar screen were the only two methods detecting all VRE, including the vanC1/C2 VRE.

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.

In order to determine the prevalence of vancomycin-resistant enterococci (VRE) in The Netherlands, 624 hospitalized patients from intensive care units or hemato-oncology wards in nine hospitals and 200 patients living in the community were screened for VRE colonization. Enterococci were found in 49% of the hospitalized patients and in 80% of the patients living in the community. Of these strains, 43 and 32%, respectively, were Enterococcus faecium. VRE were isolated from 12 of 624 (2%) and 4 of 200 (2%) hospitalized patients and patients living in the community, respectively. PCR analysis of these 16 strains and 11 additional clinical VRE isolates from one of the participating hospitals revealed 24 vanA gene-containing, 1 vanB gene-containing, and 2 vanC1 gene-containing strains. All strains were cross-resistant to avoparcin but were sensitive to the novel glycopeptide antibiotic LY333328. Genotyping of the strains by arbitrarily primed PCR and pulsed-field gel electrophoresis revealed a high degree of genetic heterogeneity. This underscores a lack of hospital-driven endemicity of VRE clones. It is suggested that the VRE in hospitalized patients have originated from unknown sources in the community.

The in vitro activity of trovafloxacin (CP-99,219), a new fluoroquinolone, was compared with the in vitro activities of other commonly used quinolones and other antimicrobial agents against 445 gram-positive microorganisms isolated between 1986 and 1995 from patients with endocarditis and those with other bloodstream infections. The MICs at which 90% of the isolates are inhibited (MIC90) of trovafloxacin for methicillin-susceptible staphylococci, viridans group streptococci, and enterococci were 0.06, 0.25, and 0.5 mg/liter, respectively. The MIC90 of trovafloxacin for vancomycin-resistant enterococci as well as for methicillin-resistant Staphylococcus aureus and methicillin-susceptible and ciprofloxacin-resistant S. aureus, isolated from sources other than blood, was 1 mg/liter. For the quinolones the rank order of activity was trovafloxacin > sparfloxacin > ciprofloxacin = ofloxacin > pefloxacin. Depending on the species tested, trovafloxacin was 4- to 64-fold more active than ciprofloxacin. Further experimental and in vivo studies are warranted to evaluate the efficacy of trovafloxacin in the treatment of bacterial endocarditis and other infections caused by gram-positive organisms.

In order to gain insight into the epidemiology of Campylobacter lari infection in The Netherlands due to the consumption of raw mussels and oysters, batches of these shellfish were screened for the presence of Campylobacter spp. during a 6 month period in 1993-1994. Apparently, 41 out of 59 batches of mussels and 11 out of 41 batches of oysters were colonized with Campylobacter spp. A subset of the isolates was further characterized by additional phenotypic tests, numerical analysis of electrophoretic protein patterns, and genotyping by random amplification of polymorphic DNA (RAPD). Protein electrophoretic analysis of 39 Campylobacter spp. cultured from 24 batches of mussels and oysters, revealed that all isolates, except two, were C. lari. Two strains with an aberrant protein pattern were identified as C. coli and C. hyointestinalis, respectively. Nalidixic acid susceptible campylobacters (NASC) and urease-positive thermophylic campylobacters (UPTC) did not form separate clusters and should be considered biovars only. Several strains were both urease positive and nalidixic acid susceptible, which represents a new biovar within C. lari. The results of RAPD demonstrated the presence of 3 distinct genetic variants, implying that even within a single batch of shell fish, relatively extensive DNA polymorphisms can be found. It is therefore apparent that this complex group of C. lari is characterized by a high degree of genetic diversity, implying the presence of a heterogenous population of C. lari in crustacean organisms living in marine waters in a restricted area in The Netherlands.