A comparison of multilocus sequence typing and fluorescent fragment-length polymorphism analysis genotyping of clone complex and other strains of Neisseria meningitidis.

Five National Collection of Type Culture (NCTC) strains and 14 isolates of Neisseria meningitidis, representing 13 outbreak isolates from within the UK, were examined by multilocus sequence typing (MLST) for seven house-keeping genes. The results were compared with those of fluorescent amplified fragment-length polymorphism (FAFLP) analysis. Phylogenetic inferences were made from 3284-nucleotide lengths of sequence for the 19 isolates, by distance and parsimony methods. Two clusters of isolates were delineated. The larger, comprising eight isolates--S1, S3, Ironville, P9, ET-37 (M99-241951), P7, P10 and P60--shared 100-99.2% similarity and varied in only 40 nucleotides (approximately 1.22% variation) from the consensus sequence alignment. This cluster could be equated to the ET-37 complex because it had allelic signatures identical to MLST sequence types 11 and 50. These eight isolates were also assigned to one group by FAFLP. The reference ET-5 complex isolate 'ET-5 (NG144/82)' and an isolate (X9) from an outbreak in the north of England were also grouped together by MLST. They shared 99.2% similarity and differed within the aroE and fumC genes by 4 and 17 nucleotides, respectively. Their MLST sequence types were 32 and 661 and, therefore, these two isolates could be equated to the ET-5 complex. They also grouped together by FAFLP. A comparison of the resources required to apply MLST to the 19 isolates examined with those needed to characterise them by FAFLP indicated that FAFLP (a fragment-based genotyping method) is more cost-effective than the partial sequencing approach, MLST.

Fluorescent amplified-fragment length polymorphism genotyping of Neisseria meningitidis identifies clones associated with invasive disease.

Fluorescent amplified-fragment length polymorphism (FAFLP), a genotyping technique with phylogenetic significance, was applied to 123 isolates of Neisseria meningitidis. Nine of these were from an outbreak in a British university; 9 were from a recent outbreak in Pontypridd, Glamorgan; 15 were from sporadic cases of meningococcal disease; 26 were from the National Collection of Type Cultures; 58 were carrier isolates from Ironville, Derbyshire; 1 was a disease isolate from Ironville; and five were representatives of invasive clones of N. meningitidis. FAFLP analysis results were compared with previously published multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) results. FAFLP was able to identify hypervirulent, hyperendemic lineages (invasive clones) of N. meningitidis as well as did MLST. PFGE did not discriminate between two strains from the outbreak that were classified as similar but distinct by FAFLP. The results suggest that high resolution of N. meningitidis for outbreak and other epidemiological analyses is more cost efficient by FAFLP than by sequencing procedures.

The Nocardia salmonicida clade, including descriptions of Nocardia cummidelens sp. nov., Nocardia fluminea sp. nov. and Nocardia soli sp. nov.

Large numbers of strains selectively isolated from soil, water and deteriorating vulcanised natural rubber pipe rings were provisionally assigned to the genus Nocardia. Twenty-eight representative isolates were found to have chemical and morphological properties typical of nocardiae. These organisms formed a monophyletic clade in the 16S rDNA tree together with Nocardia salmonicida. Three of the strains, isolates S1, W30 and R89, were distinguished from one another and from representatives of the validly described species of Nocardia using genotypic and phenotypic data. These organisms were considered to merit species status and were named Nocardia cummidelens sp. nov., Nocardia fluminea sp. nov. and Nocardia soli sp. nov. respectively. Additional comparative studies are needed to resolve the finer taxonomic relationships of the remaining isolates assigned to the Nocardia salmonicida clade and to further unravel the extent of nocardial diversity in artificial and natural ecosystems.

PCR-RFLP analysis of the coagulase gene of Staphylococcus aureus: application to the differentiation of epidemic and sporadic methicillin-resistant strains.

Preventing cross-infection with epidemic strains of methicillin-resistant Staphylococcus aureus (MRSA) requires effective control measures. These call for simple, rapid, discriminatory and reproducible methods for typing this pathogen. In this study 140 isolates/strains from 105 hospitals in England and Wales, representing 72 diverse phage types, were analysed by bacteriophage typing and PCR coagulase (coa) gene restriction fragment length polymorphism (RFLP). Isolates gave a coa gene PCR product that was either 660 base pairs (bp), 603 bp or 547 pb in size. The PCR products were digested with Alu I and Cfo I, and the fragments separated by gel electrophoresis. Eight coa gene RFLP patterns, numbered 1 to 8, were observed. Pattern 3 was most common (N = 25 isolates), followed by patterns 2 and 5 (18 isolates each), pattern 1 (14 isolates), pattern 4 (11 isolates), pattern 7 (10 isolates), pattern 8 (eight isolates) and pattern 6 (six isolates). Isolates of the same phage type often gave different coa gene RFLP patterns, and the patterns within the epidemic types EMRSA-03, EMRSA-15 and EMRSA-16 were heterogeneous. Thus, representatives of EMRSA-03 were subtyped to coa RFLP patterns 1 and 2, those of EMRSA-05 to coa RFLP patterns 1, 2, 7 and 8, and those for EMRSA-16 to coa RFLP patterns 2, 3, 4, 5 and 6. The range of patterns within single phage types of S. aureus could help to discriminate between isolates/strains, and in a hierarchical approach coa gene RFLP could occupy an intermediate position between phage typing and pulsed-field gel electrophoresis (PFGE).

Use of fluorescent amplified fragment length polymorphism (fAELP) to characterise methicillin-resistant Staphylococcus aureus.

The new PCR-based genotyping technique, fluorescent amplified fragment length polymorphism (fAFLP), was compared for discriminatory power and reproducibility with standard phenotypic methods, a coagulase gene (coa) restriction fragment length polymorphism (RFLP) method and pulsed-field gel electrophoresis (PFGE), in typing 34 isolates and four reference strains of methicillin-resistant Staphylococcus aureus (MRSA). The fAFLP showed from 40 to 75 fragments, 50 to 450 base pairs (bp) in size. Based on replicate studies, the isolates were judged indistinguishable when their fAFLP pattern was >93.7% similar. Only two of the isolates were indistinguishable by this criterion. Thirty-one MRSA fell into four major fAFLP groups (1, 2, 3 and 4) at the level of >79.9% similarity. Three other isolates and an EMRSA-16 strain fell outside these major groups. Within both fAFLP groups 1 and 2, two subgroups, A and B, could be identified at approximately 82.0% similarity. While most isolates within group 1 could also be separated by their phenotypic and coagulase gene (coa) RFLP pattern, all the isolates within fAFLP groups 2A and 2B were identical on the basis of these characters. The MRSA within fAFLP groups 3 and 4 were heterogeneous by their phenotypic characteristics and coa gene RFLP patterns. fAFLP was reproducible and distinguished between MRSA isolates that appeared identical by other methods. It is likely to contribute to the epidemiological analysis of outbreaks of MRSA infection.

Analysis of methicillin-resistant Staphylococcus aureus by IS1181 profiling.

Variation in the genomic location and copy number of the insertion element IS1181 in methicillin-resistant Staphylococcus aureus (MRSA) was investigated. Sixty-three isolates representing the Jevons type strain (NCTC 10442), phage-propagating strains, and epidemic strains were examined. A PCR amplicon of the insertion element was used to probe genomic restriction endonuclease digests. HindIII genomic digests gave 25 distinct IS1181 patterns, while EcoRI digests gave 20 patterns. EMRSA-01, -02, -04, -06, -07, -09, -10, -11, -13 and -14 contained the element but could not be subtyped by profiling it. EMRSA-16 did not contain IS1181, consistent with a unique evolutionary origin for this major UK epidemic strain. Marked heterogeneity was observed among isolates of EMRSA-03. Each EMRSA-03 strain examined gave a unique pattern, thereby allowing subtyping of an important epidemic phage type for the purposes of hospital cross-infection control.

Molecular typing of Staphylococcus aureus based on PCR restriction fragment length polymorphism and DNA sequence analysis of the coagulase gene.

A typing procedure for Staphylococcus aureus was developed based on improved PCR amplification of the coagulase gene and restriction fragment length polymorphism (RFLP) analysis of the product. All coagulase-positive staphylococci produced a single PCR amplification product of either 875, 660, 603, or 547 bp. Those strains of epidemic methicillin-resistant S. aureus 16 (EMRSA-16) studied all gave a product of 547 bp. PCR products were digested with AluI and CfoI, and the fragments were separated by gel electrophoresis. Ten distinct RFLP patterns were found among 85 isolates of methicillin-resistant S. aureus (MRSA) and 10 propagating strains (PS) of methicillin-sensitive S. aureus (MSSA) examined. RFLP patterns 1, 2, and 3 were specific to strains of EMRSA-3, -15, and -16, respectively. By contrast, RFLP patterns 4 and 5 were seen with a heterogeneous collection of strains, together with drug-resistant forms of S. aureus isolated in Europe and four propagating strains used for the international phage set. RFLP pattern 6 was given by the Airedale isolate and PS 95. RFLP pattern 7 encompassed EMRSA-2 (isolate 331), PS 94, and PS 96. An isolate from Germany gave RFLP pattern 8. Eight strains of MSSA gave patterns similar to those of methicillin-resistant strains (RFLP patterns 3, 4, 5, 6, and 7), but two, PS 42E and PS 71, gave unique RFLP patterns 9 and 10, respectively. The coagulase gene PCR products for 24 isolates of MRSA and two isolates of MSSA were sequenced for both strands. The sequences were aligned, and evolutionary lineages were inferred based on pairwise distances between isolates.

PCR-enzyme-linked immunosorbent assay and sequencing as an alternative to serology for M-antigen typing of Streptococcus pyogenes.

A rapid PCR-enzyme-linked immunosorbent assay for identification of 10 important emm gene types of Streptococcus pyogenes was developed. The emm genotypes of a coded panel of strains of known M serotype were determined, and in 144 of 149 cases (97%) the results were congruous. Strains of types that were not included in the panel of capture probes were emm genotyped by sequencing.

Virulence regulon polymorphism in group A streptococci revealed by long PCR and implications for epidemiological and evolutionary studies.

A method based on long PCR amplification and restriction endonuclease analysis of the virulence regulon was developed for a rapid (2 days), simple differentiation of group A streptococci. The PCR product size varied from 12.3 kb for serotypes M1 (NCTC 8198) and M12 (NCTC 10085) to 7.8 kb for serotype M6 (NCTC 8302). The fragment patterns formed on HaeIII digestion of the products were unique and this allowed the differentiation of each of the M-type strains (M1, M3, M4, M5, M6, M11, M12, M28, M76 and M78) studied. Contemporary M1 isolates all gave the same fragment pattern but differed from the prototype strain (NCTC 8198) in not having a 1.25-kb fragment. Isolates of serotypes M1 and M3 each had similar patterns, an indication of their clonality and global dispersion. In contrast, more than one restriction fragment length polymorphism (RFLP) pattern was detected among clinical isolates of serotypes M5, M6, M12, M4, M(R)28 and M78. Two strains that were M-protein non-typable by serological means were provisionally classified as M6 by comparisons of HaeIII long PCR fragment patterns.

Heterogeneity of human intestinal spirochaetes demonstrated by one-dimensional polyacrylamide gel electrophoresis of proteins visualised by (35)S-methionine labelling and Coomassie blue staining.

The relatedness of strains of a human intestinal spirochaete was investigated by comparison of electrophoretic protein profiles produced by Coomassie Blue staining of proteins separated by polyacrylamide gel electrophoresis (PAGE) of lysed organisms and by examination of autoradiographs following PAGE of lysed (35)S-methionine-labelled organisms. A wide diversity of strains was revealed by both techniques but clustering of strains was different by the two methods. These findings support the view that the human intestinal spirochaetes comprise a group of bacteria of considerable heterogeneity.

Intergenic 16S rRNA gene (rDNA)-23S rDNA sequence length polymorphisms in members of the family Legionellaceae.

A method based on PCR amplification of the 16S rRNA gene (rDNA)-23S rDNA intergenic regions was developed for the identification of species within the family Legionellaceae. The sizes of the PCR products varied from 1,353 to 350 bp. Strains of Legionella pneumophila were characterized as having products of approximately 900 and 530 bp, and L. birminghamensis had products of 1,390, 960, and 380 bp. Of the 38 species of legionellae examined, only 7 were indistinguishable (L. erythra from L. rubrilucens, L. anisa or L. cherrii from L. tucsonensis, and L. quateirensis from L. shakespearei). Two environmental isolates were identified as L. pneumophila. Strain LLAP-3, which was a symbiont of amoebae, could not be associated with any Legionella sp. studied.

Phylogeny of human intestinal spirochaetes inferred from 16S rDNA sequence comparisons.

The sequence of 1383 nucleotides of the DNA encoding 16S rDNA was determined for strains of human intestinal spirochaetes, comprising an unnamed isolate and "Brachyspira aalborgi" NCTC 11492. A phylogenetic tree was inferred from aligned sequence comparisons between the intestinal spirochaetes, representatives of the Spirochaetales and Escherichia coli. The type strain of Brachyspira aalborgi, though related to the Serpulina spp. at approx. 96.5% sequence similarity was distinct and separated from the unnamed human intestinal isolate, HIS Oman, N26. The latter formed a separated and novel lineage that bisected the Spirochaetales.

The use of 16S rDNA sequence analysis to investigate the phylogeny of Leptospiraceae and related spirochaetes.

The 16S rDNA sequences from 15 Leptospiraceae were determined by automated PCR-directed cycle sequencing. Nucleotide comparisons, including those from published sequences for Leptospira canicola Moulton and Serpulina spp., were used to construct phylogenetic trees. Serpulina hyodysenteriae and S. innocens were related to each other but were distinct from the Leptospiraceae comprising Leptospira parva incertae sedis (Turneria parva H), Leptonema illini and Leptospira spp. The pathogenic and the saprophytic leptospires were distinct and separated from each other. Leptospira inadai occupied an intermediate position between the two forms. The pathogens formed three groups. Group I was represented by L. interrogans sensu stricto and L. kirschneri, Group II by L. weilii, L. borgpetersenii and L. santarosai, and Group III comprised L. noguchii and L. meyeri. The saprophytic species, L. wolbachii and L. biflexa sensu stricto shared about 99% sequence similarity. The freshwater isolates were distinct from the marine isolate L. biflexa sensu lato ancona Ancona Porto.

Characterization of Leptospiraceae by 16S DNA restriction fragment length polymorphisms.

Chromosomal DNA from 37 Leptospiracae representing genetic species, groups and reference strains together with five leptospire isolates and Escherichia coli were digested with the restriction endonucleases BamHI, ClaI and EcoRI. The Southern blots were hybridized with a biotinylated E. coli 1.5 kb 16S rDNA probe and gave 36 reproducible and unique patterns. With the exception of the type strain (Leptospira interrogans serovar icterohaemorrhagiae RGA) and neotype strain (serovar icterohaemorrhagiae Ictero no. 1) all the species and taxa examined could be differentiated from each other on the basis of their BamHI, ClaI and EcoRI restriction fragment length polymorphisms (RFLP). L. interrogans and L. borgpetersenii reference strains were heterogeneous, whereas Leptonema illini and L. parva incertae sedis were distinct and separate. Strains representing L. biflexa sensu lato presented divergent RFLP patterns. A porcine isolate was identified to be L. interrogans pomona Pomona.

Chromosomal rearrangement and serovar conversion in Leptospira biflexa strains.

A bacterial population change involving chromosomal rearrangement and phenotypic changes in antigens, proteins and lipopolysaccharides is described for strains of Leptospira biflexa that were previously grown in media containing homologous oligoclonal antibodies. The chromosomal rearrangement phenomenon showed that the variants differed from the parent strains, yet they were similar to phenotypically related serovars already occurring in nature. Accordingly, in vitro serovar conversion mediated by chromosomal rearrangement, due to as yet unknown genetic mechanisms, had occurred.

Detection of Leptospiraceae by amplification of 16S ribosomal DNA.

The polymerase chain reaction (PCR) was developed to detect Leptospiraceae. Primers were used to amplify 1 631 base-pair (bp) 5'-region of 16S rDNA. Representative strains from the species, Leptospira interrogans sensu stricto, L. borgpetersenii, L. noguchii, L. santarosai, L. weilii, L. inadai, L. meyeri and the single member strain of Leptonema were amplified. In contrast, strains representing the saprophytic species. L. biflexa, L. wolbachii and L. parva were not amplified. There was no PCR product from 23 phylogenetically unrelated species of bacteria. As little as 10-1 pg of purified DNA and as few as 10-1 leptospires could be detected using the PCR analysis. Isolates of leptospires from clinical sources gave a positive PCR band, but those from surface waters did not.

A comparative investigation and identification of Leptospira interrogans serogroup icterohaemorrhagiae strains by monoclonal antibody and DNA fingerprint analyses.

The identification of Leptospira interrogans icterohaemorrhagiae strains from a number of Reference Laboratories were confirmed using monoclonal antibody (MoAb) and DNA restriction endonuclease (EcoR1) analyses. With a few exceptions, strain fidelity was demonstrated. Three clinical isolates and one isolate from a rat (Rattus norvegicus) were identified on DNA fragment patterns and found to be similar to the reference strains, icterohaemorrhagiae copenhageni, I. "icterohaemorrhagiae" Ictero I and I. icterohaemorrhagiae RGA.

The detection of genetic variation in Leptospira interrogans serogroup ICTEROHAEMORRHAGIAE by ribosomal RNA gene restriction fragment patterns.

Deoxyribonucleic acid from Leptospira interrogans serogroup ICTEROHAEMORRHAGIAE reference strains together with clinical and animal isolates were cleaved with EcoRI, electrophoresed and transferred to nylon membranes. An Escherichia coli MRE 600, 16S + 23S ribosomal RNA template was used in the synthesis of a single strand biotin-labelled cDNA probe using a reverse transcriptase. Ribosomal RNA cistrons present in the DNA fragments of strains were located using the cDNA probe. Between 4 and 7 well defined ribosomal RNA restriction bands were detected for the leptospires studied that varied in size from 12.1 to 0.57 kb. The hybridisation patterns were distinctive and unique for the reference strains that allowed for their characterisation. Similar patterns were observed for I. budapest M 20, I. "icterohaemorrhagiae" Ictero I and the type strain, I. icterohaemorrhagiae RGA. Clinical isolates and a isolate from a rat were compared with reference strain DNA patterns and identified as being similar to I. copenhageni M 20, I. "icterohaemorrhagiae" Ictero I and I. icterohaemorrhagiae RGA. These results indicate that ribosomal DNA fingerprinting may be useful in the classification and molecular epidemiology of pathogenic leptospires.

Numerical classification of campylobacters by DNA-restriction endonuclease analysis.

Reference strains from five different species of Campylobacter were examined by DNA-restriction endonuclease analysis. The DNA profiles were resolved as absorbence peaks and converted to numerical values which were analysed by a Sirius microprocessor. The percentage similarity between each profile was calculated and the strains clustered together by the computer on shared similarities and the results displayed as an abbreviated dendrogram. Each of the species of Campylobacter were clearly separated from each other and the different biotypes within a species could also be differentiated.