Efficient DNA fingerprinting of Clostridium botulinum types A, B, E, and F by amplified fragment length polymorphism analysis

Amplified fragment length polymorphism (AFLP) analysis was applied to characterize 33 group I and 37 group II Clostridium botulinum strains. Four restriction enzyme and 30 primer combinations were screened to tailor the AFLP technique for optimal characterization of C. botulinum. The enzyme combination HindIII and HpyCH4IV, with primers having one selective nucleotide apiece (Hind-C and Hpy-A), was selected. AFLP clearly differentiated between C. botulinum groups I and II; group-specific clusters showed <10% similarity between proteolytic and nonproteolytic C. botulinum strains. In addition, group-specific fragments were detected in both groups. All strains studied were typeable by AFLP, and a total of 42 AFLP types were identified. Extensive diversity was observed among strains of C. botulinum type E, whereas group I had lower genetic biodiversity. These results indicate that AFLP is a fast, highly discriminating, and reproducible DNA fingerprinting method with excellent typeability, which, in addition to its suitability for typing at strain level, can be used for C. botulinum group identification.     

Genetic Diversity of the Flagellin Genes of Clostridium botulinum Groups I and II

Botulinum neurotoxins (BoNTs) are produced by phenotypically and genetically different Clostridium species, including Clostridium botulinum and some strains of Clostridium baratii (serotype F) and Clostridium butyricum (serotype E). BoNT-producing clostridia responsible for human botulism encompass strains of group I (secreting proteases, producing toxin serotype A, B, or F, and growing optimally at 37°C) and group II (nonproteolytic, producing toxin serotype E, B, or F, and growing optimally at 30°C). Here we report the development of real-time PCR assays for genotyping C. botulinum strains of groups I and II based on flaVR (variable region sequence of flaA) sequences and the flaB gene. Real-time PCR typing of regions flaVR1 to flaVR10 and flaB was optimized and validated with 62 historical and Canadian C. botulinum strains that had been previously typed. Analysis of 210 isolates of European origin allowed the identification of four new C. botulinum flaVR types (flaVR11 to flaVR14) and one new flaVR type specific to C. butyricum type E (flaVR15). The genetic diversity of the flaVR among C. botulinum strains investigated in the present study reveals the clustering of flaVR types into 5 major subgroups. Subgroups 1, 3, and 4 contain proteolytic Clostridium botulinum, subgroup 2 is made up of nonproteolytic C. botulinum only, and subgroup 5 is specific to C. butyricum type E. The genetic variability of the flagellin genes carried by C. botulinum and the possible association of flaVR types with certain geographical areas make gene profiling of flaVR and flaB promising in molecular surveillance and epidemiology of C. botulinum.     

Intraspecies Genomic Groups in Enterococcus faecium and Their Correlation with Origin and Pathogenicity

Seventy-eight Enterococcus faecium strains from various sources were characterized by random amplified polymorphic DNA (RAPD)-PCR, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE) analysis of SmaI restriction patterns. Two main genomic groups (I and II) were obtained in both RAPD-PCR and AFLP analyses. DNA-DNA hybridization values between representative strains of both groups demonstrated a mean DNA-DNA reassociation level of 71%. PFGE analysis revealed high genetic strain diversity within the two genomic groups. Only group I contained strains originating from human clinical samples or strains that were vancomycin-resistant or beta-hemolytic. No differentiating phenotypic features between groups I and II were found using the rapid ID 32 STREP system. The two groups could be further subdivided into, respectively, four and three subclusters in both RAPD-PCR and AFLP analyses, and a high correlation was seen between the subclusters generated by these two methods. Subclusters of group I were to some extent correlated with origin, pathogenicity, and bacteriocinogeny of the strains. Host specificity of E. faecium strains was not confirmed.     

Genomic Analysis of Clostridium botulinum Group II by Pulsed-Field Gel Electrophoresis

Pulsed-field gel electrophoresis (PFGE) was optimized for genomic analyses of Clostridium botulinum (nonproteolytic) group II. DNA degradation problems caused by extracellular DNases were overcome by fixation of cells with formaldehyde prior to isolation. A rapid (4-h) in situ DNA isolation method was also assessed and gave indistinguishable results. Genomic DNA from 21 strains of various geographical and temporal origins was digested with 15 rare-cutting restriction enzymes. Of these, ApaI, MluI, NruI, SmaI, and XhoI gave the most revealing PFGE patterns, enabling strain differentiation. Twenty strains yielded PFGE patterns containing 13 pulsotypes. From summation of MluI, SmaI, and XhoI restriction fragments, the genome size of C. botulinum group II was estimated to be 3.6 to 4.1 Mb (mean ± standard deviation = 3,890 ± 170 kb). The results substantiate that after problems due to DNases are overcome, PFGE analysis will be a reproducible and highly discriminating epidemiological method for studying C. botulinum group II at the molecular level.     

Diversity of Proteolytic Clostridium botulinum Strains, Determined by a Pulsed-Field Gel Electrophoresis Approach

Pulsed-field gel electrophoresis (PFGE) was applied to the study of the similarity of 55 strains of proteolytic Clostridium botulinum (C. botulinum group I) types A, AB, B, and F. Rare-cutting restriction enzymes ApaI, AscI, MluI, NruI, PmeI, RsrII, SacII, SmaI, and XhoI were tested for their suitability for the cleavage of DNA of five proteolytic C. botulinum strains. Of these enzymes, SacII, followed by SmaI and XhoI, produced the most convenient number of fragments for genetic typing and were selected for analysis of the 55 strains. The proteolytic C. botulinum species was found to be heterogeneous. In the majority of cases, PFGE enabled discrimination between individual strains of proteolytic C. botulinum types A and B. The different toxin types were discriminated at an 86% similarity level with both SacII and SmaI and at an 83% similarity level with XhoI. Despite the high heterogeneity, three clusters at a 95% similarity level consisting of more than three strains of different origin were noted. The strains of types A and B showed higher diversity than the type F organisms which formed a single cluster. According to this survey, PFGE is to be considered a useful tool for molecular epidemiological analysis of proteolytic C. botulinum types A and B. However, epidemiological conclusions based on PFGE data only should be made with discretion, since highly similar PFGE patterns were noticed, especially within the type B strains.     

Molecular Gene Profiling of Clostridium botulinum Group III and Its Detection in Naturally Contaminated Samples Originating from Various European Countries

We report the development of real-time PCR assays for genotyping Clostridium botulinum group III targeting the newly defined C. novyi sensu lato group; the nontoxic nonhemagglutinin (NTNH)-encoding gene ntnh; the botulinum neurotoxin (BoNT)-encoding genes bont/C, bont/C/D, bont/D, and bont/D/C; and the flagellin (fliC) gene. The genetic diversity of fliC among C. botulinum group III strains resulted in the definition of five major subgroups named fliC-I to fliC-V. Investigation of fliC subtypes in 560 samples, with various European origins, showed that fliC-I was predominant and found exclusively in samples contaminated by C. botulinum type C/D, fliC-II was rarely detected, no sample was recorded as fliC-III or fliC-V, and only C. botulinum type D/C samples tested positive for fliC-IV. The lack of genetic diversity of the flagellin gene of C. botulinum type C/D would support a clonal spread of type C/D strains in different geographical areas. fliC-I to fliC-III are genetically related (87% to 92% sequence identity), whereas fliC-IV from C. botulinum type D/C is more genetically distant from the other fliC types (with only 50% sequence identity). These findings suggest fliC-I to fliC-III have evolved in a common environment and support a different genetic evolution for fliC-IV. A combination of the C. novyi sensu lato, ntnh, bont, and fliC PCR assays developed in this study allowed better characterization of C. botulinum group III and showed the group to be less genetically diverse than C. botulinum groups I and II, supporting a slow genetic evolution of the strains belonging to C. botulinum group III.     

Comparative Genomic Hybridization Analysis of Two Predominant Nordic Group I (Proteolytic) Clostridium botulinum Type B Clusters

Comparative genomic hybridization analysis of 32 Nordic group I Clostridium botulinum type B strains isolated from various sources revealed two homogeneous clusters, clusters BI and BII. The type B strains differed from reference strain ATCC 3502 by 413 coding sequence (CDS) probes, sharing 88% of all the ATCC 3502 genes represented on the microarray. The two Nordic type B clusters differed from each other by their response to 145 CDS probes related mainly to transport and binding, adaptive mechanisms, fatty acid biosynthesis, the cell membranes, bacteriophages, and transposon-related elements. The most prominent differences between the two clusters were related to resistance to toxic compounds frequently found in the environment, such as arsenic and cadmium, reflecting different adaptive responses in the evolution of the two clusters. Other relatively variable CDS groups were related to surface structures and the gram-positive cell wall, suggesting that the two clusters possess different antigenic properties. All the type B strains carried CDSs putatively related to capsule formation, which may play a role in adaptation to different environmental and clinical niches. Sequencing showed that representative strains of the two type B clusters both carried subtype B2 neurotoxin genes. As many of the type B strains studied have been isolated from foods or associated with botulism, it is expected that the two group I C. botulinum type B clusters present a public health hazard in Nordic countries. Knowing the genetic and physiological markers of these clusters will assist in targeting control measures against these pathogens.Clostridium botulinum produces a potent neurotoxin during its growth. The toxin causes a potentially lethal paralytic disease, botulism, in humans and animals. The classical food-borne botulism follows the consumption of toxin-containing food or drink, while infant and adult intestinal botulism results from in vivo spore germination, outgrowth, and toxin production in the gut. Apart from attenuated intestinal microbial population, other factors affecting the colonization of C. botulinum in the intestinal forms of botulism are not known.Based on their physiology and genetic background, C. botulinum strains are divided into groups I to IV (13). Strains of groups I and II are associated with human disease. Group I strains produce neurotoxin serotypes A, B, and/or F, while the group II strains produce type B, E, or F toxin. Physiologically, groups I and II differ markedly from each other as well as from groups III and IV. Genomic analysis of group I and II C. botulinum strains by 16S rrn sequencing (13), ribotyping (10), and amplified fragment length polymorphism (11, 15, 16) is consistent with the divergent physiologies of the two groups (18).Nordic C. botulinum group I strains show a remarkable homogeneity (15, 20, 21, 23). In a large pulsed-field gel electrophoresis (PFGE) analysis, the majority of group I strains isolated from various sources from Finland, Norway, and Denmark formed type B neurotoxin and clustered into two large groups, with the members of each group sharing identical or nearly identical restriction patterns (20, 23). Many of these strains were recovered from honey for human consumption (23), and one strain was related to an infant botulism case (22). Apart from a recent study showing that strains of the two type B clusters, further referred to as clusters BI and BII, differ in their abilities to grow at extreme temperatures (12), the physiological, epidemiological, and genetic markers of the two clusters are not known. An understanding of such traits will assist in designing control measures against these potential food- and environment-borne pathogens.The availability of group I C. botulinum genome sequences has enabled the construction of whole-genome DNA microarrays and a comprehensive genomic analysis of C. botulinum strains (26, 27). In this paper, we describe a comparative genomic hybridization (CGH) analysis of 32 Nordic group I C. botulinum type B cluster BI or BII strains with a DNA microarray based on the protein-coding sequences (CDS) in the ATCC 3502 genome. Strains within each cluster showed no substantial variation. Furthermore, strains belonging to the two clusters differed by their responses to 145 CDS probes, suggesting differential resistance to toxic compounds and a relatively large antigenic variability. Sequencing of botB in a representative cluster BI strain and a representative cluster BII strain revealed subtype B2 neurotoxin genes in both strains.     

Purification,properties, and metabolic roles of NAD+-glutamate dehydrogenase in Clostridium botulinum 113B

Cell-free extracts of proteolytic strains of Clostridium botulinum types A, B and F (group I) were found to have unusually high specific activities of NAD⁺-dependent L-glutamate dehydrogenase (NAD-GDH). In comparison, nonproteolytic strains of types B, E and F (group II) had low specific activities. The enzyme was purified 131-fold from C. botulinum 113B to a final specific activity of >1,092 molxmin^-1xmg protein^-1. The enzyme is a hexamer of a polypeptide of Mr=42,500, and the native molecular weight is 250,800. The apparent K _m values for substrates were 5.3 mM for glutamate and 0.028 mM for NAD⁺ in the deamination reaction, and 7.2 mM for -ketoglutarate, 243 mM for NH ₄ ⁺ and 0.028 mM for NADH in the reverse reaction. NADP⁺ did not serve as a hydrogen acceptor for the enzyme. Activity in the animation direction was inhibited by fumarate, oxalacetate, aspartate, glutamate and glutamine. The results suggest that GDH is important in group I (proteolytic) C. botulinum to generate -ketoglutarate as a substrate for transamination reactions. We have also found that the high activity decreases significantly when cells are exposed to sodium chloride. Therefore GDH probably has several important physiological roles in group I proteolytic C. botulinum.     

Genomic Relatedness within Five Common Finnish Campylobacter jejuni Pulsed-Field Gel Electrophoresis Genotypes Studied by Amplified Fragment Length Polymorphism Analysis, Ribotyping, and Serotyping

Thirty-five Finnish Campylobacter jejuni strains with five SmaI/SacII pulsed-field gel electrophoresis (PFGE) genotypes selected among human and chicken isolates from 1997 and 1998 were used for comparison of their PFGE patterns, amplified fragment length polymorphism (AFLP) patterns, HaeIII ribotypes, and heat-stable (HS) serotypes. The discriminatory power of PFGE, AFLP, and ribotyping with HaeIII were shown to be at the same level for this selected set of strains, and these methods assigned the strains into the same groups. The PFGE and AFLP patterns within a genotype were highly similar, indicating genetic relatedness. The same HS serotypes were distributed among different genotypes, and different serotypes were identified within one genotype. HS serotype 12 was only associated with the combined genotype G1 (PFGE-AFLP-ribotype). These studies using polyphasic genotyping methods suggested that common Finnish C. jejuni genotypes form genetic lineages which colonize both humans and chickens.     

Whole-Genome Single-Nucleotide-Polymorphism Analysis for Discrimination of Clostridium botulinum Group I Strains

Clostridium botulinum is a genetically diverse Gram-positive bacterium producing extremely potent neurotoxins (botulinum neurotoxins A through G [BoNT/A-G]). The complete genome sequences of three strains harboring only the BoNT/A1 nucleotide sequence are publicly available. Although these strains contain a toxin cluster (HA⁺ OrfX⁻) associated with hemagglutinin genes, little is known about the genomes of subtype A1 strains (termed HA⁻ OrfX⁺) that lack hemagglutinin genes in the toxin gene cluster. We sequenced the genomes of three BoNT/A1-producing C. botulinum strains: two strains with the HA⁺ OrfX⁻ cluster (69A and 32A) and one strain with the HA⁻ OrfX⁺ cluster (CDC297). Whole-genome phylogenic single-nucleotide-polymorphism (SNP) analysis of these strains along with other publicly available C. botulinum group I strains revealed five distinct lineages. Strains 69A and 32A clustered with the C. botulinum type A1 Hall group, and strain CDC297 clustered with the C. botulinum type Ba4 strain 657. This study reports the use of whole-genome SNP sequence analysis for discrimination of C. botulinum group I strains and demonstrates the utility of this analysis in quickly differentiating C. botulinum strains harboring identical toxin gene subtypes. This analysis further supports previous work showing that strains CDC297 and 657 likely evolved from a common ancestor and independently acquired separate BoNT/A1 toxin gene clusters at distinct genomic locations.     

Cloning, Nucleotide Sequence, and Expression of the Gene Encoding the Bacteriocin Boticin B from Clostridium botulinum Strain 213B

Boticin B is a heat-stable bacteriocin produced by Clostridium botulinum strain 213B that has inhibitory activity against various strains of C. botulinum and related clostridia. The gene encoding the bacteriocin was localized to a 3.0-kb HindIII fragment of an 18.8-kb plasmid, cloned, and sequenced. DNA sequencing revealed the boticin B structural gene, btcB, to be an open reading frame encoding 50 amino acids. A C. botulinum strain 62A transconjugant containing the HindIII fragment inserted into a clostridial shuttle vector expressed boticin B, although at much lower levels than those observed in C. botulinum 213B. To our knowledge, this is the first demonstration and characterization of a bacteriocin from toxigenic group I C. botulinum.     

Amplified Fragment Length Polymorphism Analysis of Campylobacter jejuni Strains Isolated from Chickens and from Patients with Gastroenteritis or Guillain-Barré or Miller Fisher Syndrome

The high-resolution genotyping method of amplified fragment length polymorphism (AFLP) analysis was used to study the genetic relationships between Campylobacter jejuni strains infecting chickens (n = 54) and those causing gastroenteritis in humans (n = 53). In addition, C. jejuni strains associated with the development of Guillain-Barré syndrome (GBS) (n = 14) and Miller Fisher syndrome (MFS) (n = 4), two related acute paralytic syndromes in human, were included. Strains were isolated between 1989 and 1998 in The Netherlands. The AFLP banding patterns were analyzed with correlation-based and band-based similarity coefficients and UPGMA (unweighted pair group method using average linkages) cluster analysis. All C. jejuni strains showed highly heterogeneous fingerprints, and no fingerprints exclusive for chicken strains or for human strains were obtained. All strains were separated in two distinct genetic groups. In group A the percentage of human strains was significantly higher and may be an indication that genotypes of this group are more frequently associated with human diseases. We conclude that C. jejuni from chickens cannot be distinguished from human strains and that GBS or MFS related strains do not belong to a distinct genetic group.     

Conjugative Botulinum Neurotoxin-Encoding Plasmids in Clostridium botulinum

Background

Clostridium botulinum produces seven distinct serotypes of botulinum neurotoxins (BoNTs). The genes encoding different subtype neurotoxins of serotypes A, B, F and several dual neurotoxin-producing strains have been shown to reside on plasmids, suggesting that intra- and interspecies transfer of BoNT-encoding plasmids may occur. The objective of the present study was to determine whether these C. botulinum BoNT-encoding plasmids are conjugative.

Methodology/Principal Findings

C. botulinum BoNT-encoding plasmids pBotCDC-A3 (strain CDC-A3), pCLJ (strain 657Ba) and pCLL (strain Eklund 17B) were tagged with the erythromycin resistance marker (Erm) using the ClosTron mutagenesis system by inserting a group II intron into the neurotoxin genes carried on these plasmids. Transfer of the tagged plasmids from the donor strains CDC-A3, 657Ba and Eklund 17B to tetracycline-resistant recipient C. botulinum strains was evaluated in mating experiments. Erythromycin and tetracycline resistant transconjugants were isolated from donor∶recipient mating pairs tested. Transfer of the plasmids to the transconjugants was confirmed by pulsed-field gel electrophoresis (PFGE) and Southern hybridizations. Transfer required cell-to-cell contact and was DNase resistant. This indicates that transfer of these plasmids occurs via a conjugation mechanism.

Conclusions/Significance

This is the first evidence supporting conjugal transfer of native botulinum neurotoxin-encoding plasmids in C. botulinum, and provides a probable mechanism for the lateral distribution of BoNT-encoding plasmids to other C. botulinum strains. The potential transfer of C. botulinum BoNT-encoding plasmids to other bacterial hosts in the environment or within the human intestine is of great concern for human pathogenicity and necessitates further characterization of these plasmids.     

Genetic Characterization and Comparison of Clostridium botulinum Isolates from Botulism Cases in Japan between 2006 and 2011

Genetic characterization was performed for 10 group I Clostridium botulinum strains isolated from botulism cases in Japan between 2006 and 2011. Of these, 1 was type A, 2 were type B, and 7 were type A(B) {carrying a silent bont/B [bont/(B)] gene} serotype strains, based on botulinum neurotoxin (BoNT) production. The type A strain harbored the subtype A1 BoNT gene (bont/A1), which is associated with the ha gene cluster. The type B strains carried bont/B5 or bont/B6 subtype genes. The type A(B) strains carried bont/A1 identical to that of type A(B) strain NCTC2916. However, bont/(B) genes in these strains showed single-nucleotide polymorphisms (SNPs) among strains. SNPs at 2 nucleotide positions of bont/(B) enabled classification of the type A(B) strains into 3 groups. Pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem-repeat analysis (MLVA) also provided consistent separation results. In addition, the type A(B) strains were separated into 2 lineages based on their plasmid profiles. One lineage carried a small plasmid (5.9 kb), and another harbored 21-kb plasmids. To obtain more detailed genetic information about the 10 strains, we sequenced their genomes and compared them with 13 group I C. botulinum genomes in a database using whole-genome SNP analysis. This analysis provided high-resolution strain discrimination and enabled us to generate a refined phylogenetic tree that provides effective traceability of botulism cases, as well as bioterrorism materials. In the phylogenetic tree, the subtype B6 strains, Okayama2011 and Osaka05, were distantly separated from the other strains, indicating genomic divergence of subtype B6 strains among group I strains.     

Differentiation of the Gene Clusters Encoding Botulinum Neurotoxin Type A Complexes in Clostridium botulinum Type A, Ab, and A(B) Strains

We describe a strategy to identify the clusters of genes encoding components of the botulinum toxin type A (boNT/A) complexes in 57 strains of Clostridium botulinum types A, Ab, and A(B) isolated in Italy and in the United States from different sources. Specifically, we combined the results of PCR for detecting the ha33 and/or p47 genes with those of boNT/A PCR-restriction fragment length polymorphism analysis. Three different type A toxin gene clusters were revealed; type A1 was predominant among the strains from the United States, whereas type A2 predominated among the Italian strains, suggesting a geographic distinction between strains. By contrast, no relationship between the toxin gene clusters and the clinical or food source of strains was evident. In two C. botulinum type A isolates from the United States, we recognized a third type A toxin gene cluster (designated type A3) which was similar to that previously described only for C. botulinum type A(B) and Ab strains. Total genomic DNA from the strains was subjected to pulsed-filed gel electrophoresis and randomly amplified polymorphic DNA analyses, and the results were consistent with the boNT/A gene clusters obtained.     

Rapid, Quantitative PCR Monitoring of Growth of Clostridium botulinum Type E in Modified-Atmosphere-Packaged Fish

A rapid, quantitative PCR assay (TaqMan assay) which quantifies Clostridium botulinum type E by amplifying a 280-bp sequence from the botulinum neurotoxin type E (BoNT/E) gene is described. With this method, which uses the hydrolysis of an internal fluoregenic probe and monitors in real time the increase in the intensity of fluorescence during PCR by using the ABI Prism 7700 sequence detection system, it was possible to perform accurate and reproducible quantification of the C. botulinum type E toxin gene. The sensitivity and specificity of the assay were verified by using 6 strains of C. botulinum type E and 18 genera of 42 non-C. botulinum type E strains, including strains of C. botulinum types A, B, C, D, F, and G. In both pure cultures and modified-atmosphere-packaged fish samples (jack mackerel), the increase in amounts of C. botulinum DNA could be monitored (the quantifiable range was 10² to 10⁸ CFU/ml or g) much earlier than toxin could be detected by mouse assay. The method was applied to a variety of seafood samples with a DNA extraction protocol using guanidine isothiocyanate. Overall, an efficient recovery of C. botulinum cells was obtained from all of the samples tested. These results suggested that quantification of BoNT/E DNA by the rapid, quantitative PCR method was a good method for the sensitive assessment of botulinal risk in the seafood samples tested.     

Group-Specific Monitoring of Phenol Hydroxylase Genes for a Functional Assessment of Phenol-Stimulated Trichloroethylene Bioremediation

The sequences of the largest subunit of bacterial multicomponent phenol hydroxylases (LmPHs) were compared. It was found that LmPHs formed three phylogenetic groups, I, II, and III, corresponding to three previously reported kinetic groups, low-K_s (the half-saturation constant in Haldane's equation for trichloroethylene [TCE]), moderate-K_s, and high-K_s groups. Consensus sequences and specific amino acid residues for each group of LmPH were found, which facilitated the design of universal and group-specific PCR primers. PCR-mediated approaches using these primers were applied to analyze phenol/TCE-degrading populations in TCE-contaminated aquifer soil. It was found that the aquifer soil harbored diverse genotypes of LmPH, and the group-specific primers successfully amplified LmPH fragments affiliated with each of the three groups. Analyses of phenol-degrading bacteria isolated from the aquifer soil confirmed the correlation between genotype and phenotype. Competitive PCR assays were used to quantify LmPHs belonging to each group during the enrichment of phenol/TCE-degrading bacteria from the aquifer soil. We found that an enrichment culture established by batch phenol feeding expressed low TCE-degrading activity at a TCE concentration relevant to the contaminated aquifer (e.g., 0.5 mg liter⁻¹); group II and III LmPHs were predominant in this batch enrichment. In contrast, group I LmPHs overgrew an enrichment culture when phenol was fed continuously. This enrichment expressed unexpectedly high TCE-degrading activity that was comparable to the activity expressed by a pure culture of Methylosinus trichosporium OB3b. These results demonstrate the utility of the group-specific monitoring of LmPH genes in phenol-stimulated TCE bioremediation. It is also suggested that phenol biostimulation could become a powerful TCE bioremediation strategy when bacteria possessing group I LmPHs are selectively stimulated.     

Intraspecies genomic groups in Enterococcus faecium and their correlation with origin and pathogenicity

Seventy-eight Enterococcus faecium strains from various sources were characterized by random amplified polymorphic DNA (RAPD)-PCR, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE) analysis of SmaI restriction patterns. Two main genomic groups (I and II) were obtained in both RAPD-PCR and AFLP analyses. DNA-DNA hybridization values between representative strains of both groups demonstrated a mean DNA-DNA reassociation level of 71%. PFGE analysis revealed high genetic strain diversity within the two genomic groups. Only group I contained strains originating from human clinical samples or strains that were vancomycin-resistant or beta-hemolytic. No differentiating phenotypic features between groups I and II were found using the rapid ID 32 STREP system. The two groups could be further subdivided into, respectively, four and three subclusters in both RAPD-PCR and AFLP analyses, and a high correlation was seen between the subclusters generated by these two methods. Subclusters of group I were to some extent correlated with origin, pathogenicity, and bacteriocinogeny of the strains. Host specificity of E. faecium strains was not confirmed.     

Multiplex PCR Assay for Detection and Identification of Clostridium botulinum Types A, B, E, and F in Food and Fecal Material

Botulism is diagnosed by detecting botulinum neurotoxin and Clostridium botulinum cells in the patient and in suspected food samples. In this study, a multiplex PCR assay for the detection of Clostridium botulinum types A, B, E, and F in food and fecal material was developed. The method employs four new primer pairs with equal melting temperatures, each being specific to botulinum neurotoxin gene type A, B, E, or F, and enables a simultaneous detection of the four serotypes. A total of 43 C. botulinum strains and 18 strains of other bacterial species were tested. DNA amplification fragments of 782 bp for C. botulinum type A alone, 205 bp for type B alone, 389 bp for type E alone, and 543 bp for type F alone were obtained. Other bacterial species, including C. sporogenes and the nontoxigenic nonproteolytic C. botulinum-like organisms, did not yield a PCR product. Sensitivity of the PCR for types A, E, and F was 10² cells and for type B was 10 cells per reaction mixture. With a two-step enrichment, the detection limit in food and fecal samples varied from 10⁻² spore/g for types A, B, and F to 10⁻¹ spore/g of sample material for type E. Of 72 natural food samples investigated, two were shown to contain C. botulinum type A, two contained type B, and one contained type E. The assay is sensitive and specific and provides a marked improvement in the PCR diagnostics of C. botulinum.     

Molecular characterization of ochratoxin A producing strains of the genus Penicillium

Sixty-six strains classified as P. verrucosum based on morphological criteria were characterized by molecular methods like RAPD, AFLP and ITS sequencing. Two groups could be identified by RAPD and AFLP analyses. The two RAPD as well as the two AFLP groups were completely coincidental. Strains in the two groups differed in their ability to produce ochratoxin A, with group I containing mainly high producing strains, and group II containing moderate to non-producing strains. The strains from group I originate from foods, such as cheeses and meat products, while the strains from group II originate from plants. The ribosomal ITS1-5.8S-ITS2 sequences were similar, except for two single nucleotide exchanges in several strains of each group. A chemotaxonomical analysis of some of the strains identified differences between the groups in secondary metabolite production. Strains from group I possessed the chemotype of P. nordicum and strains from group II that of P. verrucosum. The differences at the RAPD and AFLP level, which parallel the chemotypic differences, are consistent with the recent reclassification of ochratoxin A producing penicillia to be either P. verrucosum or P. nordicum. The homolgy between the ITS sequences however indicates phylogenetic relationship between the two species.