Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA

A quantitative molecular technique was developed for rapid analysis of microbial community diversity in various environments. The technique employed PCR in which one of the two primers used was fluorescently labeled at the 5' end and was used to amplify a selected region of bacterial genes encoding 16S rRNA from total community DNA. The PCR product was digested with restriction enzymes, and the fluorescently labeled terminal restriction fragment was precisely measured by using an automated DNA sequencer. Computer-simulated analysis of terminal restriction fragment length polymorphisms (T-RFLP) for 1,002 eubacterial sequences showed that with proper selection of PCR primers and restriction enzymes, 686 sequences could be PCR amplified and classified into 233 unique terminal restriction fragment lengths or "ribotypes." Using T-RFLP, we were able to distinguish all bacterial strains in a model bacterial community, and the pattern was consistent with the predicted outcome. Analysis of complex bacterial communities with T-RFLP revealed high species diversity in activated sludge, bioreactor sludge, aquifer sand, and termite guts; as many as 72 unique ribotypes were found in these communities, with 36 ribotypes observed in the termite guts. The community T-RFLP patterns were numerically analyzed and hierarchically clustered. The pattern derived from termite guts was found to be distinctly different from the patterns derived from the other three communities. Overall, our results demonstrated that T-RFLP is a powerful tool for assessing the diversity of complex bacterial communities and for rapidly comparing the community structure and diversity of different ecosystems.     

Diversity of uncultured microorganisms associated with the seagrass Halophila stipulacea estimated by restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes

Muscle phosphofructokinase deficiency (PFKD) is characterized by exercise intolerance due to the enzymatic block in muscle glycolysis. Glucose infusion increases exertional fatigue in these patients, probably by decreasing the availability of free fatty acids (FFA) and ketones, which play a crucial role in ATP production during exercise in PFKD. This suggests that a lower than normal hepatic glucose production would be appropriate during exercise in PFKD. To investigate glucoregulation in PFKD, we measured glucose turnover and hormonal and metabolic responses to 20 minutes of cycle exercise at near maximal effort in three patients with PFKD and in healthy matched controls studied at the same absolute (A, 15 to 30 Watts) and relative (R, 35 to 80 Watts, matched heart rates) work load as the patients. During exercise, mean glucose production was higher in all patients versus controls (30 +/- 4 versus A: 18 +/- 2 and R: 20 +/- 1 mumol.min-1.kg-1). Mean glucose utilization during exercise was similar in patients and controls working at the same relative work load and higher than in controls at the low work load. Exercise-induced increases in arterialized blood were higher in all patients for glucose, FFA, growth hormone, glucagon, and norepinephrine. Plasma alanine and lactate always decreased during exercise in patients and consistently increased in controls. In conclusion, an enhanced neuroendocrine response and a paradoxically exaggerated mobilization of glucose occurs during exercise in PFKD. The responses are probably initiated by neural feedback elicited by disturbances in local muscle metabolism. The responses promote delivery of oxidizable fat to muscle, but at the expense of accumulation and futile cycling of glucose.     

Higher-level snake phylogeny inferred from mitochondrial DNA sequences of 12S rRNA and 16S rRNA genes

N-Nitrosomethylbenzylamine (NMBA) is a potent esophagus-specific carcinogen that has been utilized extensively in the study of esophageal carcinogenesis in rats. While many studies have focused on the pathogenesis of NMBA-induced esophageal tumors, the tumorigenicity of NMBA itself has not been thoroughly investigated in any single, systematic dose-response study. Therefore, in this study we evaluated NMBA tumorigenicity in rats following various short-term s.c. treatment regimens with the aim of developing an abbreviated treatment protocol which could be used in future studies. To assess the possible correlation of basal cell proliferation with NMBA tumorigenicity, we evaluated the expression of proliferating cell nuclear antigen (PCNA) in both control and NMBA-treated rats. In rats which received a cumulative NMBA dosage of 7.5 mg/kg over the course of 5 weeks, tumor incidence and multiplicity were as follows: 40% with 0.4 +/- 0.3 tumors/rat at week 10; 100% with 2.2 +/- 1.0 tumors/rat at week 20; and 100% with 2.3 +/- 1.0 tumors/rat at week 30. These rats exhibited marked increases in basal cell labeling, with indices that were 1.5- to 1.8-fold higher than controls. NMBA treatment regimens of shorter duration with equivalent or higher cumulative dosages were generally ineffective in producing esophageal tumors, even though significantly elevated levels of basal cell proliferation occurred. Together, these findings indicate that the duration of NMBA treatment is of critical importance in the tumorigenic potential of the carcinogen.     

Detection of Helicobacter-like bacteria in porcine gastric biopsy samples by amplification of 16S rRNA, ureB, vacA and cagA genes by PCR

Preliminary evidence for the presence of Helicobacter-like bacteria was sought in 395 porcine gastric samples by a urease test. Of the samples, 37% (146/395) were urease-positive and 82% (82/100) of the Gram-stained urease-positive samples showed large, tightly spiralled organisms. Several methods were applied to culture the organisms but isolation was unsuccessful, contaminant organisms being considered to be one of the major problems. PCR with Helicobacter genus-specific primers for 16S rRNA and ureB genes, and primers for H. pylori vacA and cagA genes were tested with 102 ureasepositive biopsy samples. The PCR results showed some evidence for the presence of the urease and the vacA genes in porcine Helicobacter-like bacteria and raises the possibility of pathogenicity by these organisms.     

Diversity and structure of the methanogenic community in anoxic rice paddy soil microcosms as examined by cultivation and direct 16S rRNA gene sequence retrieval

A dual approach consisting of cultivation and molecular retrieval of partial archaeal 16S rRNA genes was carried out to characterize the diversity and structure of the methanogenic community inhabiting the anoxic bulk soil of flooded rice microcosms. The molecular approach identified four groups of known methanogens. Three environmental sequences clustered with Methanobacterium bryantii and Methanobacterium formicicum, six were closely related but not identical to those of strains of Methanosaeta concilii, two grouped with members of the genus Methanosarcina, and two were related to the methanogenic endosymbiont of Plagiopyla nasuta. The cultivation approach via most-probable-number counts with a subsample of the same soil as an inoculum yielded cell numbers of up to 10(7) per g of dry soil for the H2-CO2-utilizing methanogens and of up to 10(6) for the acetate-utilizing methanogens. Strain VeH52, isolated from the terminal positive dilution on H2-CO2, grouped within the phylogenetic radiation characterized by M. bryantii and M. formicicum and the environmental sequences of the Methanobacterium-like group. A consortium of two distinct methanogens grew in the terminal positive culture on acetate. These two organisms showed absolute 16S rRNA gene identities with environmental sequences of the novel Methanosaeta-like group and the Methanobacterium-like group. Methanosarcina spp. were identified only in the less-dilute levels of the same dilution series on acetate. These data correlate well with acetate concentrations of about 11 microM in the pore water of this rice paddy soil. These concentrations are too low for the growth of known Methanosarcina spp. but are at the acetate utilization threshold of Methanosaeta spp. Thus, our data indicated Methanosaeta spp. and Methanobacterium spp. to be the dominant methanogenic groups in the anoxic rice soil, whereas Methanosarcina spp. appeared to be less abundant.     

Characterization of a defined 2,3,5, 6-tetrachlorobiphenyl-ortho-dechlorinating microbial community by comparative sequence analysis of genes coding for 16S rRNA

Defined microbial communities were developed by combining selective enrichment with molecular monitoring of total community genes coding for 16S rRNAs (16S rDNAs) to identify potential polychlorinated biphenyl (PCB)-dechlorinating anaerobes that ortho dechlorinate 2,3, 5,6-tetrachlorobiphenyl. In enrichment cultures that contained a defined estuarine medium, three fatty acids, and sterile sediment, a Clostridium sp. was predominant in the absence of added PCB, but undescribed species in the delta subgroup of the class Proteobacteria, the low-G+C gram-positive subgroup, the Thermotogales subgroup, and a single species with sequence similarity to the deeply branching species Dehalococcoides ethenogenes were more predominant during active dechlorination of the PCB. Species with high sequence similarities to Methanomicrobiales and Methanosarcinales archaeal subgroups were predominant in both dechlorinating and nondechlorinating enrichment cultures. Deletion of sediment from PCB-dechlorinating enrichment cultures reduced the rate of dechlorination and the diversity of the community. Substitution of sodium acetate for the mixture of three fatty acids increased the rate of dechlorination, further reduced the community diversity, and caused a shift in the predominant species that included restriction fragment length polymorphism patterns not previously detected. Although PCB-dechlorinating cultures were methanogenic, inhibition of methanogenesis and elimination of the archaeal community by addition of bromoethanesulfonic acid only slightly inhibited dechlorination, indicating that the archaea were not required for ortho dechlorination of the congener. Deletion of Clostridium spp. from the community profile by addition of vancomycin only slightly reduced dechlorination. However, addition of sodium molybdate, an inhibitor of sulfate reduction, inhibited dechlorination and deleted selected species from the community profiles of the class Bacteria. With the exception of one 16S rDNA sequence that had the highest sequence similarity to the obligate perchloroethylene-dechlorinating Dehalococcoides, the 16S rDNA sequences associated with PCB ortho dechlorination had high sequence similarities to the delta, low-G+C gram-positive, and Thermotogales subgroups, which all include sulfur-, sulfate-, and/or iron(III)-respiring bacterial species.     

Occurrence of introns in the 16S rRNA genes of members of the genus Thermoproteus

Cervical smears (n = 150) from five departments showing high-grade dyskaryosis were examined by three cytologists. All the smears came from patients with biopsy-proven CIN III. One hundred had been correctly reported (true positives) but 50 had originally been reported as negative and had been found to be positive only on review (false negatives). There were significant differences between the two sets in the characteristics of the dyskaryotic cell population. The false-negative smears tended to have fewer than 200 dyskaryotic cells. The nuclei of the dyskaryotic cells tended to have fine rather than coarse nuclear chromatin. A smear with fewer than 50 dyskaryotic cells is 26 times more likely to be reported as negative than one with more than 200 dyskaryotic cells. The results suggest that there is a type of severely dyskaryotic smear that is inherently likely to be missed on routine screening.     

Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis

Sequence heterogeneities in 16S rRNA genes from individual strains of Paenibacillus polymyxa were detected by sequence-dependent separation of PCR products by temperature gradient gel electrophoresis (TGGE). A fragment of the 16S rRNA genes, comprising variable regions V6 to V8, was used as a target sequence for amplifications. PCR products from P. polymyxa (type strain) emerged as a well-defined pattern of bands in the gradient gel. Six plasmids with different inserts, individually demonstrating the migration characteristics of single bands of the pattern, were obtained by cloning the PCR products. Their sequences were analyzed as a representative sample of the total heterogeneity. An amount of 10 variant nucleotide positions in the fragment of 347 bp was observed, with all substitutions conserving the relevant secondary structures of the V6 and V8 regions in the RNA molecules. Hybridizations with specifically designed probes demonstrated different chromosomal locations of the respective rRNA genes. Amplifications of reverse-transcribed rRNA from ribosome preparations, as well as whole-cell hybridizations, revealed a predominant representation of particular sequences in ribosomes of exponentially growing laboratory cultures. Different strains of P. polymyxa showed not only remarkably differing patterns of PCR products in TGGE analysis but also discriminative whole-cell labeling with the designed oligonucleotide probes, indicating the different representation of individual sequences in active ribosomes. Our results demonstrate the usefulness of TGGE for the structural analysis of heterogeneous rRNA genes together with their expression, stress problems of the generation of meaningful data for 16S rRNA sequences and probe designs, and might have consequences for evolutionary concepts.     

Temperature gradient gel electrophoresis analysis of 16S rRNA from human fecal samples reveals stable and host-specific communities of active bacteria

The diversity of the predominant bacteria in the human gastrointestinal tract was studied by using 16S rRNA-based approaches. PCR amplicons of the V6 to V8 regions of fecal 16S rRNA and ribosomal DNA (rDNA) were analyzed by temperature gradient gel electrophoresis (TGGE). TGGE of fecal 16S rDNA amplicons from 16 individuals showed different profiles, with some bands in common. Fecal samples from two individuals were monitored over time and showed remarkably stable profiles over a period of at least 6 months. TGGE profiles derived from 16S rRNA and rDNA amplicons showed similar banding patterns. However, the intensities of bands with similar mobilities differed in some cases, indicating a different contribution to the total active fraction of the prominent fecal bacteria. Most 16S rRNA amplicons in the TGGE pattern of one subject were identified by cloning and sequence analysis. Forty-five of the 78 clones matched 15 bands, and 33 clones did not match any visible band in the TGGE pattern. Nested PCR of amplified 16S rDNA indicated preferential amplification of a sequence corresponding to 12 of the 33 nonmatching clones with similar mobilities in TGGE. The sequences matching 15 bands in the TGGE pattern showed 91.5 to 98.7% homology to sequences derived from different Clostridium clusters. Most of these were related to strains derived from the human intestine. The results indicate that the combination of cloning and TGGE analysis of 16S rDNA amplicons is a reliable approach to monitoring different microbial communities in feces.     

Identification and analysis of PCB dechlorinating anaerobic enrichments by amplification: accuracy of community structure based on restriction analysis and partial sequencing of 16S rRNA genes

The composition of polychlorinated biphenyl (PCB) dechlorinating mixed communities was analysed by restriction fragment length polymorphism of PCR amplified rDNAs (ARDRA) and partial sequencing of 16S rRNA genes amplified from PCB degrading enrichments. Restriction analysis confirms that the 16S rRNA genes amplified from PCB dechlorinating communities vary depending on the PCB congener dechlorinated. Comparison of 16S rRNA sequences to published ribosomal databases indicates that the two most abundant Operational Taxonomic Units (OTUs) appear to be species of the genus Clostridium. The amount that the amplification procedure contributed to this result was determined by varying the amplification procedure and by creating an artificial template mixture. Varying the amount of template by sixfold in the amplification did not affect the distribution of OTUs but the number of OTUs observed decreased with decreasing template concentration. Comparison of products amplified from mixtures of 16S rDNA clones indicates that the more abundant Clostridium OTU did not amplify more efficiently than those of less abundant OTUs. Hybridization to a probe designed to detect the most abundant OTUs indicates that two other OTUs are closely related to this Clostridium species.     

Hierarchical analysis of variation in the mitochondrial 16S rRNA gene among Hymenoptera

Nucleotide sequences from a 434-bp region of the 16S rRNA gene were analyzed for 65 taxa of Hymenoptera (ants, bees, wasps, parasitoid wasps, sawflies) to examine the patterns of variation within the gene fragment and the taxonomic levels for which it shows maximum utility in phylogeny estimation. A hierarchical approach was adopted in the study through comparison of levels of sequence variation among taxa at different taxonomic levels. As previously reported for many holometabolous insects, the 16S data reported here for Hymenoptera are highly AT-rich and exhibit strong site-to-site variation in substitution rate. More precise estimates of the shape parameter (alpha) of the gamma distribution and the proportion of invariant sites were obtained in this study by employing a reference phylogeny and utilizing maximum-likelihood estimation. The effectiveness of this approach to recovering expected phylogenies of selected hymenopteran taxa has been tested against the use of maximum parsimony. This study finds that the 16S gene is most informative for phylogenetic analysis at two different levels: among closely related species or populations, and among tribes, subfamilies, and families. Maximization of the phylogenetic signal extracted from the 16S gene at higher taxonomic levels may require consideration of the base composition bias and the site-to-site rate variation in a maximum-likelihood framework.     

In vitro amplification of the 16S rRNA genes from Mycoplasma bovirhinis, Mycoplasma alkalescens and Mycoplasma bovigenitalium by PCR

Polymerase chain reaction (PCR) systems were developed for detection of 3 pathogenic bovine mycoplasmas, Mycoplasma alkalescens (Mak), M. bovigenitalium (Mbg) and M. bovirhinis (Mbr). The primers were selected from the sequences of the 16S rRNA from each of the mycoplasmas. Neither the Mbg-PCR system nor the Mbr-PCR system showed cross-amplification among 24 bovine, caprine-ovine and human mycoplasma species (including Acholeplasma and Ureaplasma species) tested. The Mak-PCR system showed cross-amplification with M. canadens ATCC29418T (T = type strain). The sensitivity of each PCR system to detect the proper mycoplasma was 10(3) colony forming units (CFU) when a pure culture was tested, and 2 x 10(3) CFU when the mycoplasma culture was spiked into a calf nasal swab sample. The target mycoplasmas in a clinical nasal swab sample that could be detected by the direct plate method could also be detected by these PCR systems.     

Physical mapping and characterization of 5S rRNA genes in Douglas-fir

A critical mass of Internet users will soon enable wide diffusion of electronic communication within medical practice. E-mail between physicians and patients offers important opportunities for better communication. Linking patients and physicians through e-mail may increase the involvement of patients in supervising and documenting their own health care, processes that may activate patients and contribute to improved health. These new linkages may have profound implications for the patient-physician relationship. Although the federal government proposes regulation of telemedicine technologies and medical software, communications technologies are evolving under less scrutiny. Unless these technologies are implemented with substantial forethought, they may disturb delicate balances in the patient-physician relationship, widen social disparities in health outcomes, and create barriers to access to health care. This paper seeks to identify the promise and pitfalls of electronic patient-physician communication before such technology becomes widely distributed. A research agenda is proposed that would provide data that are useful for careful shaping of the communications infrastructure. The paper addresses the need to 1) define appropriate use of the various modes of patient-physician communication, 2) ensure the security and confidentiality of patient information, 3) create user interfaces that guide patients in effective use of the technology, 4) proactively assess medicolegal liability, and 5) ensure access to the technology by a multicultural, multilingual population with varying degrees of literacy.     

Detection of kanamycin-resistant Mycobacterium tuberculosis by identifying mutations in the 16S rRNA gene

In Mycobacterium smegmatis and a limited number of Mycobacterium tuberculosis strains, the involvement of alterations of the 16S rRNA gene (rrs) in resistance to kanamycin has been shown. To investigate the extent to which mutations in a specific region of the rrs gene and the kanamycin-resistant phenotype in clinically isolated M. tuberculosis strains were correlated, 43 kanamycin-resistant strains (MICs, > or =200 microg/ml), 71 kanamycin-susceptible strains, and 4 type strains were examined. The 300-bp DNA fragments carrying the rrs gene and the intervening sequence between the rrs gene and 23S rRNA (rrl) gene fragments were amplified by PCR and were subjected to PCR-based direct sequencing. By comparing the nucleotide sequences, substitutions were found in 29 of 43 (67.4%) kanamycin-resistant clinical isolates at positions 1400, 1401, and 1483 but in none of the 71 sensitive isolates or the 4 type strains. The most frequent substitution, from A to G, occurred at position 1400. A substitution from C to T at position 1401 was found once. Two clinical isolates carried the double mutation from C to A at position 1401 and from G to T at position 1483. In addition, we found that these mutants can be distinguished from wild-type strains by digestion with the restriction endonucleases TaiI and Tsp45I. Furthermore, we found that the genotypes of kanamycin-resistant strains can be discriminated from each other by digestion with a restriction endonuclease, BstUI or DdeI.     

Differentiation of Listeria monocytogenes and Listeria innocua by 16S rRNA genes and intraspecies discrimination of Listeria monocytogenes strains by random amplified polymorphic DNA polymorphisms

Differences in the 16S rRNA genes (16S rDNA) which can be used to discriminate Listeria monocytogenes from Listeria innocua have been detected. The 16S rDNA were amplified by polymerase chain reaction with a set of oligonucleotide primers which flank a 1.5-kb fragment. Sequence differences were observed in the V2 region of the 16S rDNA both between L. monocytogenes Scott A and L. innocua and between different L. monocytogenes serotypes. Although L. monocytogenes SLCC2371 had the same V2 region sequence as L. innocua, the two species were different within the V9 region at nucleotides 1259 and 1292, in agreement with previous studies (R.-F. Wang, W.-W. Cao, and M.G. Johnson, Appl. Environ. Microbiol. 57:3666-3670, 1991). Intraspecies discrimination of L. monocytogenes strains was achieved by using the patterns generated by random amplified polymorphic DNA primers. Although some distinction can be made within the L. monocytogenes species by their 16S rDNA sequence, a far greater discrimination within species could be made by generating random amplified polymorphic DNA patterns from chromosomal DNA. By using a number of 10-bp primers, unique patterns for each isolate which in all cases examined differentiate between various L. monocytogenes serotypes, even though they may have the same 16S rRNA sequences, could be generated.     

Molecular evolution of Mycoplasma capricolum subsp. capripneumoniae strains, based on polymorphisms in the 16S rRNA genes

Mycoplasma capricolum subsp. capripneumoniae belongs to the so-called Mycoplasma mycoides cluster and is the causal agent of contagious caprine pleuropneumonia (CCPP). All members of the M. mycoides cluster have two rRNA operons. The sequences of the 16S rRNA genes of both rRNA operons from 20 strains of M. capricolum subsp. capripneumoniae of different geographical origins in Africa and Asia were determined. Nucleotide differences which were present in only one of the two operons (polymorphisms) were detected in 24 positions. The polymorphisms were not randomly distributed in the 16S rRNA genes, and some of them were found in regions of low evolutionary variability. Interestingly, 11 polymorphisms were found in all the M. capricolum subsp. capripneumoniae strains, thus defining a putative ancestor. A sequence length difference between the 16S rRNA genes in a poly(A) region and 12 additional polymorphisms were found in only one or some of the strains. A phylogenetic tree was constructed by comparative analysis of the polymorphisms, and this tree revealed two distinct lines of descent. The nucleotide substitution rate of strains within line II was up to 50% higher than within line I. A tree was also constructed from individual operonal 16S rRNA sequences, and the sequences of the two operons were found to form two distinct clades. The topologies of both clades were strikingly similar, which supports the use of 16S rRNA sequence data from homologous operons for phylogenetic studies. The strain-specific polymorphism patterns of the 16S rRNA genes of M. capricolum subsp. capripneumoniae may be used as epidemiological markers for CCPP.     

Strategy for the detection of Helicobacter species by amplification of 16S rRNA genes and identification of H. felis in a human gastric biopsy

The aim of the present work was to develop polymerase chain reactions (PCRs) based on the conserved nucleotide sequence of the 16S rRNA gene for detection of bacteria of the Helicobacter genus in human antral biopsy samples. The assay for Helicobacter spp was developed by amplifying a 399-bp 16S rRNA gene sequence specific to the genus Helicobacter. The identity of the amplicon was confirmed by hybridization with an internal probe and by restriction by endonuclease VspI showing two expected fragments of 295 and 104 base pairs. A total of 65 dyspeptic patients from France and New Caledonia were screened for Helicobacter spp infection through the use of the following diagnostic assays on biopsy specimens collected through endoscopy: direct detection of bacteria in histological sections by Giemsa and Warthin Starry staining, urease test and bacterial isolation, PCR for Helicobacter pylori ureC/glmM gene, and PCR targeted to 16S rRNA genes. The 16S rRNA gene PCR assay was able to detect down to 680 bacterial cells, as assessed by agarose gel electrophoresis, and down to 4 bacterial cells by hybridization of amplicon with the internal probe. The 16S rRNA PCR test was 100% specific and sensitive; results obtained with this test were in agreement with the visualization of bacteria by histology. Urease test and culture were 86.4% and 22.7% sensitive, and 96.5 and 100% specific, respectively. The H. pylori ureC/glmM gene-based PCR was 100% specific and only 95.4% sensitive, since one biopsy from a Melanesian patient contained a Helicobacter strain other than H. pylori. For this Melanesian patient, a branch-specific PCR targeting the epsilon branch of Proteobacteria was used to amplify a 967-bp amplicon. This amplicon was sequenced and matched with the H. felis sequence. This was confirmed using an H. felis-specific urease PCR test.     

Nearly identical 16S rRNA sequences recovered from lakes in North America and Europe indicate the existence of clades of globally distributed freshwater bacteria

Negative pressure transients (NPT) recorded in a single closing event of mechanical valves in the mitral position in an in vitro setup are compared with data recorded in the left atrium in vivo with the valves implanted in the mitral position in an animal model. The loading at valve closure (dP/dtCL) computed from the in vivo ventricular pressure recording (ranging from 700 to 2300 mm Hg/s) agreed with the magnitudes predicted in our earlier in vitro experiments (750-3000 mm Hg/s). The NPT signals and the corresponding power spectral density plots from the in vivo data were in qualitative agreement with those recorded in vitro. The NPT magnitudes were found to be below the vapor pressure for blood in mechanical valves with rigid occluders suggesting a potential for the valve to cavitate in vivo. Our in vivo results also suggest that the valves with flexible occluders are less likely to cavitate. The correlation of the in vitro and in vivo data also suggests that the flexibility of valve housing used in the in vitro studies is not an important factor in the dynamics of mechanical valve closure in vivo.