Ribotyping is the most widely used method for differentiating strains of Clostridium difficile fo... more Ribotyping is the most widely used method for differentiating strains of Clostridium difficile for epidemiological studies and infection control. Recently there have been calls for standardisation of the technique to which sophisticated technical solutions have been offered. This note offers a solution for standardisation based on conserved rrn operon Type-specific flanking genes. Furthermore, this technique can be used to detect Type-specific rrn operon deletions in passages from a single strain of C. difficile
The marked variability of irinotecan (Ir) clearance warrants individualized dosing based on hepat... more The marked variability of irinotecan (Ir) clearance warrants individualized dosing based on hepatic drug handling. The aims of this trial were to identify parameters from functional hepatic nuclear imaging (HNI) that correlate with (1) Ir pharmacology, and (2) single-nucleotide polymorphisms (SNPs) for the ABCB1 (P-glycoprotein) and UGT-1A1 genes, known to influence Ir handling.
International Journal of Systematic and Evolutionary Microbiology, 2001
The current literature on bacterial taxonomy, typing and evolution will be critically examined fr... more The current literature on bacterial taxonomy, typing and evolution will be critically examined from the perspective of whole-genome structure, function and organization. The following three categories of DNA band pattern studies will be reviewed : (i) random whole-genome analysis ; (ii) specific gene variation and (iii) mobile genetic elements. (i) The use of RAPD, PFGE and AFLP to analyse the whole genome will provide a skeleton of polymorphic sites with exact genomic positions as whole-genome sequence data become available. (ii) Different genes provide different levels of evolutionary information for determining isolate relatedness depending on whether they are highly variable (prone to recombination events and horizontal transfer), housekeeping genes with only a small number of single nucleotide differences between isolates or part of the rrn multigene family that is prone to intragenomic recombination and concerted evolution. Comparative analyses of these different gene classes can provide enhanced information about isolate relatedness. (iii) Mobile genetic elements such as insertion sequences, transposons, plasmids and bacteriophages integrate into the bacterial genome at specific (e.g. tRNA genes) or non-specific sites to alter band patterns produced by PFGE, RAPD or AFLP. From the literature it is not clear what level of genetic element duplication constitutes non-relatedness of isolates. A model is presented that incorporates all of the above genomic characteristics for the determination of isolate relatedness in taxonomic, typing and evolutionary studies.
Campylobacter concisus is an emerging pathogen of the human intestinal tract. This heterogeneous ... more Campylobacter concisus is an emerging pathogen of the human intestinal tract. This heterogeneous species of phenotypically indistinguishable strains has been divided into genomospecies A and genomospecies B by amplification of the 23S rRNA gene. The diversity of the ribosomal RNA (rrn) operon can be a useful tool for differentiation of C. concisus. In this study we investigated the rrn operon (5S rRNA, 16S rRNA, 23S rRNA genes, and the ITS regions) to differentiate and establish a systematic relationship among C. concisus isolates. The rrn operons were identified from two oral and two intestinal C. concisus whole genomes sequenced at RMIT University, Melbourne. The rrn operon sequences of eight C. concisus strains downloaded from public databases were also included in the analysis. We examined the potential of the rrn operon to be used in strain typing and delineation of phylogenetic relationships within C. concisus rrn operons. We have identified 38 indels in the rrn operon of the C. concisus genome. Five indels in the 23S rRNA gene were significantly associated with the genomospecies (p < 0.05). The phylogenetic tree generated from the rrn operons demonstrated sequence differences between strains within the 5S rRNA, the 16S rRNA, the 23S rRNA and other intergenic regions. Hence, C. concisus can be classified into two genomospecies (A & B) based on the presence of the indels in the rrn operon. The 23S rRNA gene was found to be more reliable for C. concisus typing than the 16S rRNA gene.
To develop a rapid and accurate method of typing large numbers of clinical isolates of CZostridiu... more To develop a rapid and accurate method of typing large numbers of clinical isolates of CZostridium dz$ciZe, four regions of the rRNA operon [A, 15-1407 and B, 907-1407 (16s-16s); C, 1392-507 and D, 907-507 (16S-23S)I were enzymically amplified from 24 strains. When region A was hybridized to HindIII-digested genomic DNA isolated from C. dz$ciZe strains, all of the variable length restriction fragments hybridized. When region B was hybridized to HindIII-digested genomic DNA isolated from C. dificife strains, a set of variable length restriction fragments (Group 11) hybridized predominantly. When region C was separated by agarose gel electrophoresis, a series of products ranging in size from approximately 800-1300 bp was obtained. When regions C and D were digested with HindIII, a constant region of 430 bp was found in both products and in all strains. From the above experiments it was concluded that the variable length Group I1 restriction fragments and the variable length region C amplification products were due to variable length 16S-23S spacer regions between alleles of the one strain. When region C amplification products were separated by denaturing PAGE, 16 variable length rRNA alleles (rrnA-P) were demonstrated from 24 C. dz$ciZe strains ranging in size from 852-1210 bp. After analysis with maximum parsimony, the 24 strains were divided into 14 ribotypes. The product C ribotypes and band sizes were stable after 14 single colony passages on horse blood agar plates and stable in vivo, since ribotype G was isolated twice from one patient and ribotype E was isolated three times from another patient (all on separate occasions). The ribotyping method described here has clear advantages over existing C. dificile typing methods; it has universal applicability, it is objective and is moderately rapid.
Despite electrophoretic patterns of ITS PCR amplicons often suggesting only a single ITS sequence... more Despite electrophoretic patterns of ITS PCR amplicons often suggesting only a single ITS sequence variant is present in strains of Acinetobacter junii, sequence data shows differences in ITS copies between and among them. This paper set out to explain why these ITS variants arise, and whether their presence compromises the reliability of the ITS targeted methods currently available for typing A. junii strains. ITS sequences from a number of strains of A. junii were either downloaded from public databases or generated here by cloning and sequencing ITS PCR amplicons. ITS copies of A. junii strain 97338 were all 666bp long, with identical sequences. In A. junii ATCC17908T /BCRC 14854T), ITS copies were also all identical in their lengths but now were 706/7bp long. Two sequence variants of these 707bp ITS were detected. One was identical in its sequence to the nine ITS copies downloaded from the whole genome sequence of A.junii CIP 64.5, and those in several other A.junii strains. The ...
To determine whether ITS sequences in the rrn operon are suitable for identifying individual Acin... more To determine whether ITS sequences in the rrn operon are suitable for identifying individual Acinetobacter Acb complex members, we analysed length and sequence differences between multiple ITS copies within the genomes of individual strains. Length differences in ITS reported previously between A. nosocomialis BCRC15417 T (615 bp) and other strains (607 bp) can be explained by presence of an insertion (indel 13i/1) in the longer ITS variant. The same Indel 13i/1 was also found in ITS sequences of ten strains of A. calcoaceticus, all 639 bp long, and the 628 bp ITS of Acinetobacter strain BENAB127. Four additional indels (13i/2-13i/5) were detected in Acinetobacter strain c/t13TU 10090 ITS length variants (608, 609, 620, 621 and 630 bp). These ITS variants appear to have resulted from horizontal gene transfer involving other Acinetobacter species or in some cases unrelated bacteria. Although some ITS copies in strain c/t13TU 10090 are of the same length (620 bp) as those in Acinetobacter strains b/n1&3, A. pittii (10 strains), A. calcoaceticus and A. oleivorans (not currently acknowledged as an Acb member), their individual ITS sequences differ. Thus ITS length by itself can not by itself be used to identify Acb complex strains. A shared indel in ITS copies in two separate Acinetobacter species compromises the specificity of ITS targeted probes, as shown with the Aun-3 probe designed to target the ITS in A. pitti. The presence of indel 13i/5 in the ITS of Acinetobacter strain c/t13TU means it too responded positively to this probe. Thus, neither ITS sequencing nor the currently available ITS targeted probes can distinguish reliably between Acb member species.
The genus Rhodococcus in the sub-order Corynebacteriniae has had a chequered taxonomic history, b... more The genus Rhodococcus in the sub-order Corynebacteriniae has had a chequered taxonomic history, but many of the early uncertainties and confusions have been resolved satisfactorily through the application of chemotaxonomic and phylogenetic characters. Such information has allowed the creation and formal recognition of the closely related genera Gordonia, Tsukamurella and Dietzia to include isolates once placed unconvincingly in the genus
ABSTRACT The rrn operon is the basic building block of all bacteria found in 1–15 copies per bact... more ABSTRACT The rrn operon is the basic building block of all bacteria found in 1–15 copies per bacterial genome in all bacteria. All bacterial genomes contain rrn operons that contain four basic components: (i) 16S rRNA gene; (ii) 16S–23S intergenic transcribed spacer (ITS); (iii) 23S rRNA gene; (iv) 5S rRNA gene. The chapter “Bacterial Typing and Identification By Genomic Analysis of 16S–23S rRNA Intergenic Transcribed Spacer (ITS) Sequences” by Volker Gürtler et al., describes the methodology used to construct a FileMaker database for analysing and presenting information about the rrn operon and how its different components relate to each other. The database, named “RiboTyping” uses a combination of graphical, text and numerical information to analyse differences between the components within and between genomes. RiboTyping uses the capabilities of FileMaker to present graphics files in multiple records as a “movie” and it summarises text information numerically by sorting specific subsets of data. This methodology enables the entire collection of thousands of bacterial genomes to be analysed at the level of multi-copy rrn operons.
The whole genomic typing of 21 isolates of Pseudomonas aeruginosa from 15 intensive care unit (IC... more The whole genomic typing of 21 isolates of Pseudomonas aeruginosa from 15 intensive care unit (ICU) patients was performed by pulsed-field gel electrophoresis (PFGE using SpeI) and Riboprinting (using EcoRI and PvuII), and then the results were compared with predictions made from the whole genome sequence of P. aeruginosa PAO1. The analysis of electronic images from PFGE and Riboprinting by GelComparII demonstrated similar discrimination between PFGE and Riboprinting with PvuII enzyme; however, Riboprinting by EcoRI had reduced banding patterns and was shown to be of lower discrimination than PvuII. When analyzing isolates from patients, both PFGE and Riboprinting using PvuII enzyme gave equivalent results, with the exception of two isolates that were closely related by PvuII Riboprinting and unrelated by PFGE. These discrepancies in typing results can be explained and adjusted for by comparisons with the rrn properties and the SpeI restriction fragments predicted from the whole genome of P. aeruginosa PAO1. Properties of the rrn operon that need to be taken into account include: (i) restriction enzyme sites that produce one or two fragments for each rrn operon; (ii) genomic variability in ISR sequence length; (iii) different enzymes need to be used to determine differences in rrn operon copy number from Riboprints; and (iv) choice of a restriction enzyme that produces riboprinter bands derived from rrn operon regions that are highly variable within the genome and between isolates. This knowledge has ramifications for PFGE and Riboprinter design and analysis so that for each new species to be typed comparisons can be made using the whole genome sequence.
To obtain Mycobacterium species identification directly from clinical specimens and cultures, the... more To obtain Mycobacterium species identification directly from clinical specimens and cultures, the 16S-23S rDNA spacer (ISR) was amplified using previously published primers that detect all Mycobacterium species. The restriction enzyme that could potentially produce the most restriction fragment length polymorphisms (RFLPs) was determined from all available ISR DNA sequences in GenBank to produce a novel data set of RFLPs for 31 slowly growing Mycobacterium species. Subsequently a GelCompar II database was constructed from RFLPs for 10 enzymes that have been used in the literature to differentiate slowly growing Mycobacterium species. The combination of Sau96I and HaeIII were the best choice of enzymes for differentiating clinically relevant slowly growing Mycobacterium species. A total of 392 specimens were studied by PCR with 195 negative and 197 positive specimens. The ISR-PCR product was digested with HaeIII (previously reported) and Sau96I (new to this study) to obtain a Mycobacterium species identification based on the ISR-RFLPs. The species identification obtained by ISR-RFLP was confirmed by DNA sequencing (isolate numbers are shown in parentheses) for M. avium (3), M. intracellulare (4), M. avium complex (1), M. gordonae (2) and M. tuberculosis (1). The total number of specimens (99) identified were from culture (67), Bactectrade mark 12B culture bottles (11), EDTA blood (3), directly from smear positive specimens (13), tissue (4) and urine (1). Direct species identification was obtained from all 13/13 smear positive specimens. The total number of specimens (99) were identified as M. tuberculosis (41), M. avium (7), M. avium complex (11), M. intracellulare MIN-A (20), M. flavescens (2), M. fortuitum (10), M. gordonae (4), M. shimoidei (1), M. ulcerans (1) and M. chelonae (2). This method reduces the time taken for Mycobacterium species identification from 8-10 weeks for culture and biochemical identification; to 4-6 weeks for culture and ISR-RFLP; to 2 days for smear-positive specimens by ISR-RFLP. The precise 2 day identification obtained may provide significant advantages in clinical management.
Clostridium difficile causes outbreaks of infectious diarrhoea, most commonly occurring in health... more Clostridium difficile causes outbreaks of infectious diarrhoea, most commonly occurring in healthcare institutions. Recently, concern has been raised with reports of C. difficile disease in those traditionally thought to be at low risk i.e. community acquired rather than healthcare acquired. This has increased awareness for the need to track outbreaks and PCR-ribotyping has found widespread use to elucidate epidemiologically linked isolates. PCR-ribotyping uses conserved regions of the 16S rRNA gene and 23S rRNA gene as primer binding sites to produce varying PCR products due to the intergenic spacer (ITS1) regions of the multiple operons. With the explosion of whole genome sequence data it became possible to analyse the start of the 23S rRNA gene for a more accurate selection of regions closer to the end of the ITS1. However the following questions must still be asked: (i) Does the chromosomal organisation of the rrn operon vary between C. difficile strains? and (ii) just how conserved are the primer binding regions? Eight published C. difficile genomes have been aligned to produce a detailed database of indels of the ITS1&amp;amp;amp;amp;amp;amp;amp;amp;#39;s from the rrn operon sets. An iPad Filemaker Go App has been constructed and named RiboTyping (RT). It contains detail such as sequences, ribotypes, strain numbers, GenBank numbers and genome position numbers. Access to various levels of the database is provided so that details can be printed. There are three main regions of the rrn operon that have been analysed by the database and related to each other by strain, ribotype and operon: (1) 16S gene (2) ITS1 indels (3) 23S gene. This has enabled direct intra- and inter-genomic comparisons at the strain, ribotype and operon (allele) levels in each of the three genomic regions. This is the first time that such an analysis has been done. By using the RT App with search criteria it will be possible to select probe combinations for specific strains/ribotypes/rrn operons for experiments to do with diagnostics, typing and recombination of operons. Many more incomplete C. difficile whole genome sequencing projects are recorded in GenBank as underway and the rrn operon information from these can also be added to the RT App when available. The RT App will help simplify probe selection because of the complexity of the ITS1 in C. difficile even in a single genome and because other allele-specific regions (16S and 23S genes) of variability can be relationally compared to design extra probes to increase sensitivity.
Campylobacter insulaenigrae is a novel species that has been recently only isolated from marine m... more Campylobacter insulaenigrae is a novel species that has been recently only isolated from marine mammals. This is the first report of C. insulaenigrae causing enteritis and septicaemia in a patient with end-stage hepatic and renal disease.
International Journal of Systematic and Evolutionary Microbiology, 2001
A nocardioform bacterium was isolated from the bronchoscopic lavage of a 78-year-old man with a p... more A nocardioform bacterium was isolated from the bronchoscopic lavage of a 78-year-old man with a past history of tuberculous pleurisy, who presented with bilateral upper lobe lesions at Austin and Repatriation Medical Centre, Heidelberg, Australia. The strain was aerobic, Gram-positive, produced beige substrate mycelium and scant white aerial mycelium. It showed chemotaxonomic markers which were consistent with the classification of Nocardia : i.e. meso-diaminopimelic acid, N-glycolylmuramic acid, arabinose and galactose as diagnostic sugars ; phospholipids phosphatidylinositol mannosides, phosphatidylinositol, phosphatidylethanolamine and diphosphatidylglycerol ; a menaquinone with a cyclic isoprene side chain, MK-8(H 4cycl.) ; a fatty acid pattern composed of unbranched saturated and monounsaturated fatty acids with a considerable amount of tuberculostearic acid ; and mycolic acids composed of 54-62 carbon atoms with three principal mycolic acids which were mono-and polyunsaturated, showing a chain length C 56 , C 58 and C 60 and accounting for over 70 % of the entire pattern. The 16S rDNA sequence showed the highest similarity to the type strain of Nocardia vaccinii ; the DNA-DNA similarity of the two strains was 31 %. These data, together with distinct physiological traits and molecular biological analyses, as well as chemotaxonomic results, led to the conclusion that the novel isolate represents a new species within the genus Nocardia for which the name Nocardia veterana sp. nov. is proposed. The type strain is M157222 T (DSM 44445 T l NRRL B-24136 T).
Gilbert&#39;s syndrome, due to reduced hepatic bilirubin glucuronidation is associated with t... more Gilbert&#39;s syndrome, due to reduced hepatic bilirubin glucuronidation is associated with the presence of two extra nucleotides (TA) in the promoter region of the UDP-glucuronosyltransferase 1 (UGT1A1) gene. A rapid method was developed to detect this genetic polymorphism, using double gradient denaturing gradient gel electrophoresis (DG-DGGE). The promoter region of the UGT1A1 gene was amplified with a 40-mer GC-clamp attached to the 5&#39;-end of the reverse primer. The polymerase chain reaction (PCR) product was then separated by DG-DGGE using denaturant concentrations of 15-25% and polyacrylamide concentrations of 6-12%. The (TA)6/(TA)6 homozygotes were clearly distinguished from both (TA)7/(TA)7 homozygotes and (TA)6/(TA)7 heterozygotes. The (TA)7 allele frequency was consistent with that previously reported and elevated bilirubin levels correlated with the presence of the (TA)7 allele. The DG-DGGE method described will make detection for this polymorphism fast, simple, nonradioactive and suitable for a clinical routine diagnostic laboratory, helping to establish the role of this polymorphism in individuals with jaundice due to multiple causes.
Clostridium difficile is a major spore-forming environmental pathogen that causes serious health ... more Clostridium difficile is a major spore-forming environmental pathogen that causes serious health problems in patients undergoing antibiotic therapy. Consequently, reliable and sensitive methods for typing individual strains are required for epidemiological and environmental studies. Ribotyping is generally considered the best method, but it fails to account for sequence diversity which might exist in intergenic 16S-23S rRNA spacer regions (ISRs) within and among strains of this organism. Therefore, this study was undertaken to compare the sequence of each individual ISR in five strains of C. difficile to explore the extent of this diversity and see whether such information might provide the basis for more sensitive and discriminatory strain typing methods. After targeted PCR amplification, cloning, and sequencing, the diversity of the ISRs was used as a measure of rRNA operon copy number. In C. difficile strains 630, ATCC 43593, A, and B, 11, 11, 7, and 8 ISR length variants, respec...
Restriction maps were constructed of enzymically amplified 16s rRNA genes (rDNA) isolated from ei... more Restriction maps were constructed of enzymically amplified 16s rRNA genes (rDNA) isolated from eight Clos?ridium species. Using maximum parsimony, a dendrogram was constructed from these and published 16s rRNA sequence data. Two distinct clusters were identified: cluster I contained C. dBcile, C. sordklli, and C. bifementans, and showed 30 of 35 restriction sites in common; cluster I1 contained C. tetani, C. perfingens C. sprogenes and C. botulinum C and G, and showed 20 of 35 restriction sites in common. Further analysis of cluster I organisms revealed that of five HpaII fragments, two were found in equal amounts in all organisms, one was found in varying amounts in all organisms, and two were found, in varying amounts, only in C. sordelli and C. bifevmentans. C. sordelli-specific and C. bifevmentans-specific HpaII fragments were demonstrated by Southern hybridization of rDNA. One HpaII site within the rDNA was present on most alleles in C. bifementans, present on a minority of alleles in C. sordelli and absent in C. di,,cile. This suggested that there were two 16s rRNA alleles with different sequences present within each of the genomes of C. bifermentans and C. sordklli.
Ribotyping is the most widely used method for differentiating strains of Clostridium difficile fo... more Ribotyping is the most widely used method for differentiating strains of Clostridium difficile for epidemiological studies and infection control. Recently there have been calls for standardisation of the technique to which sophisticated technical solutions have been offered. This note offers a solution for standardisation based on conserved rrn operon Type-specific flanking genes. Furthermore, this technique can be used to detect Type-specific rrn operon deletions in passages from a single strain of C. difficile
The marked variability of irinotecan (Ir) clearance warrants individualized dosing based on hepat... more The marked variability of irinotecan (Ir) clearance warrants individualized dosing based on hepatic drug handling. The aims of this trial were to identify parameters from functional hepatic nuclear imaging (HNI) that correlate with (1) Ir pharmacology, and (2) single-nucleotide polymorphisms (SNPs) for the ABCB1 (P-glycoprotein) and UGT-1A1 genes, known to influence Ir handling.
International Journal of Systematic and Evolutionary Microbiology, 2001
The current literature on bacterial taxonomy, typing and evolution will be critically examined fr... more The current literature on bacterial taxonomy, typing and evolution will be critically examined from the perspective of whole-genome structure, function and organization. The following three categories of DNA band pattern studies will be reviewed : (i) random whole-genome analysis ; (ii) specific gene variation and (iii) mobile genetic elements. (i) The use of RAPD, PFGE and AFLP to analyse the whole genome will provide a skeleton of polymorphic sites with exact genomic positions as whole-genome sequence data become available. (ii) Different genes provide different levels of evolutionary information for determining isolate relatedness depending on whether they are highly variable (prone to recombination events and horizontal transfer), housekeeping genes with only a small number of single nucleotide differences between isolates or part of the rrn multigene family that is prone to intragenomic recombination and concerted evolution. Comparative analyses of these different gene classes can provide enhanced information about isolate relatedness. (iii) Mobile genetic elements such as insertion sequences, transposons, plasmids and bacteriophages integrate into the bacterial genome at specific (e.g. tRNA genes) or non-specific sites to alter band patterns produced by PFGE, RAPD or AFLP. From the literature it is not clear what level of genetic element duplication constitutes non-relatedness of isolates. A model is presented that incorporates all of the above genomic characteristics for the determination of isolate relatedness in taxonomic, typing and evolutionary studies.
Campylobacter concisus is an emerging pathogen of the human intestinal tract. This heterogeneous ... more Campylobacter concisus is an emerging pathogen of the human intestinal tract. This heterogeneous species of phenotypically indistinguishable strains has been divided into genomospecies A and genomospecies B by amplification of the 23S rRNA gene. The diversity of the ribosomal RNA (rrn) operon can be a useful tool for differentiation of C. concisus. In this study we investigated the rrn operon (5S rRNA, 16S rRNA, 23S rRNA genes, and the ITS regions) to differentiate and establish a systematic relationship among C. concisus isolates. The rrn operons were identified from two oral and two intestinal C. concisus whole genomes sequenced at RMIT University, Melbourne. The rrn operon sequences of eight C. concisus strains downloaded from public databases were also included in the analysis. We examined the potential of the rrn operon to be used in strain typing and delineation of phylogenetic relationships within C. concisus rrn operons. We have identified 38 indels in the rrn operon of the C. concisus genome. Five indels in the 23S rRNA gene were significantly associated with the genomospecies (p < 0.05). The phylogenetic tree generated from the rrn operons demonstrated sequence differences between strains within the 5S rRNA, the 16S rRNA, the 23S rRNA and other intergenic regions. Hence, C. concisus can be classified into two genomospecies (A & B) based on the presence of the indels in the rrn operon. The 23S rRNA gene was found to be more reliable for C. concisus typing than the 16S rRNA gene.
To develop a rapid and accurate method of typing large numbers of clinical isolates of CZostridiu... more To develop a rapid and accurate method of typing large numbers of clinical isolates of CZostridium dz$ciZe, four regions of the rRNA operon [A, 15-1407 and B, 907-1407 (16s-16s); C, 1392-507 and D, 907-507 (16S-23S)I were enzymically amplified from 24 strains. When region A was hybridized to HindIII-digested genomic DNA isolated from C. dz$ciZe strains, all of the variable length restriction fragments hybridized. When region B was hybridized to HindIII-digested genomic DNA isolated from C. dificife strains, a set of variable length restriction fragments (Group 11) hybridized predominantly. When region C was separated by agarose gel electrophoresis, a series of products ranging in size from approximately 800-1300 bp was obtained. When regions C and D were digested with HindIII, a constant region of 430 bp was found in both products and in all strains. From the above experiments it was concluded that the variable length Group I1 restriction fragments and the variable length region C amplification products were due to variable length 16S-23S spacer regions between alleles of the one strain. When region C amplification products were separated by denaturing PAGE, 16 variable length rRNA alleles (rrnA-P) were demonstrated from 24 C. dz$ciZe strains ranging in size from 852-1210 bp. After analysis with maximum parsimony, the 24 strains were divided into 14 ribotypes. The product C ribotypes and band sizes were stable after 14 single colony passages on horse blood agar plates and stable in vivo, since ribotype G was isolated twice from one patient and ribotype E was isolated three times from another patient (all on separate occasions). The ribotyping method described here has clear advantages over existing C. dificile typing methods; it has universal applicability, it is objective and is moderately rapid.
Despite electrophoretic patterns of ITS PCR amplicons often suggesting only a single ITS sequence... more Despite electrophoretic patterns of ITS PCR amplicons often suggesting only a single ITS sequence variant is present in strains of Acinetobacter junii, sequence data shows differences in ITS copies between and among them. This paper set out to explain why these ITS variants arise, and whether their presence compromises the reliability of the ITS targeted methods currently available for typing A. junii strains. ITS sequences from a number of strains of A. junii were either downloaded from public databases or generated here by cloning and sequencing ITS PCR amplicons. ITS copies of A. junii strain 97338 were all 666bp long, with identical sequences. In A. junii ATCC17908T /BCRC 14854T), ITS copies were also all identical in their lengths but now were 706/7bp long. Two sequence variants of these 707bp ITS were detected. One was identical in its sequence to the nine ITS copies downloaded from the whole genome sequence of A.junii CIP 64.5, and those in several other A.junii strains. The ...
To determine whether ITS sequences in the rrn operon are suitable for identifying individual Acin... more To determine whether ITS sequences in the rrn operon are suitable for identifying individual Acinetobacter Acb complex members, we analysed length and sequence differences between multiple ITS copies within the genomes of individual strains. Length differences in ITS reported previously between A. nosocomialis BCRC15417 T (615 bp) and other strains (607 bp) can be explained by presence of an insertion (indel 13i/1) in the longer ITS variant. The same Indel 13i/1 was also found in ITS sequences of ten strains of A. calcoaceticus, all 639 bp long, and the 628 bp ITS of Acinetobacter strain BENAB127. Four additional indels (13i/2-13i/5) were detected in Acinetobacter strain c/t13TU 10090 ITS length variants (608, 609, 620, 621 and 630 bp). These ITS variants appear to have resulted from horizontal gene transfer involving other Acinetobacter species or in some cases unrelated bacteria. Although some ITS copies in strain c/t13TU 10090 are of the same length (620 bp) as those in Acinetobacter strains b/n1&3, A. pittii (10 strains), A. calcoaceticus and A. oleivorans (not currently acknowledged as an Acb member), their individual ITS sequences differ. Thus ITS length by itself can not by itself be used to identify Acb complex strains. A shared indel in ITS copies in two separate Acinetobacter species compromises the specificity of ITS targeted probes, as shown with the Aun-3 probe designed to target the ITS in A. pitti. The presence of indel 13i/5 in the ITS of Acinetobacter strain c/t13TU means it too responded positively to this probe. Thus, neither ITS sequencing nor the currently available ITS targeted probes can distinguish reliably between Acb member species.
The genus Rhodococcus in the sub-order Corynebacteriniae has had a chequered taxonomic history, b... more The genus Rhodococcus in the sub-order Corynebacteriniae has had a chequered taxonomic history, but many of the early uncertainties and confusions have been resolved satisfactorily through the application of chemotaxonomic and phylogenetic characters. Such information has allowed the creation and formal recognition of the closely related genera Gordonia, Tsukamurella and Dietzia to include isolates once placed unconvincingly in the genus
ABSTRACT The rrn operon is the basic building block of all bacteria found in 1–15 copies per bact... more ABSTRACT The rrn operon is the basic building block of all bacteria found in 1–15 copies per bacterial genome in all bacteria. All bacterial genomes contain rrn operons that contain four basic components: (i) 16S rRNA gene; (ii) 16S–23S intergenic transcribed spacer (ITS); (iii) 23S rRNA gene; (iv) 5S rRNA gene. The chapter “Bacterial Typing and Identification By Genomic Analysis of 16S–23S rRNA Intergenic Transcribed Spacer (ITS) Sequences” by Volker Gürtler et al., describes the methodology used to construct a FileMaker database for analysing and presenting information about the rrn operon and how its different components relate to each other. The database, named “RiboTyping” uses a combination of graphical, text and numerical information to analyse differences between the components within and between genomes. RiboTyping uses the capabilities of FileMaker to present graphics files in multiple records as a “movie” and it summarises text information numerically by sorting specific subsets of data. This methodology enables the entire collection of thousands of bacterial genomes to be analysed at the level of multi-copy rrn operons.
The whole genomic typing of 21 isolates of Pseudomonas aeruginosa from 15 intensive care unit (IC... more The whole genomic typing of 21 isolates of Pseudomonas aeruginosa from 15 intensive care unit (ICU) patients was performed by pulsed-field gel electrophoresis (PFGE using SpeI) and Riboprinting (using EcoRI and PvuII), and then the results were compared with predictions made from the whole genome sequence of P. aeruginosa PAO1. The analysis of electronic images from PFGE and Riboprinting by GelComparII demonstrated similar discrimination between PFGE and Riboprinting with PvuII enzyme; however, Riboprinting by EcoRI had reduced banding patterns and was shown to be of lower discrimination than PvuII. When analyzing isolates from patients, both PFGE and Riboprinting using PvuII enzyme gave equivalent results, with the exception of two isolates that were closely related by PvuII Riboprinting and unrelated by PFGE. These discrepancies in typing results can be explained and adjusted for by comparisons with the rrn properties and the SpeI restriction fragments predicted from the whole genome of P. aeruginosa PAO1. Properties of the rrn operon that need to be taken into account include: (i) restriction enzyme sites that produce one or two fragments for each rrn operon; (ii) genomic variability in ISR sequence length; (iii) different enzymes need to be used to determine differences in rrn operon copy number from Riboprints; and (iv) choice of a restriction enzyme that produces riboprinter bands derived from rrn operon regions that are highly variable within the genome and between isolates. This knowledge has ramifications for PFGE and Riboprinter design and analysis so that for each new species to be typed comparisons can be made using the whole genome sequence.
To obtain Mycobacterium species identification directly from clinical specimens and cultures, the... more To obtain Mycobacterium species identification directly from clinical specimens and cultures, the 16S-23S rDNA spacer (ISR) was amplified using previously published primers that detect all Mycobacterium species. The restriction enzyme that could potentially produce the most restriction fragment length polymorphisms (RFLPs) was determined from all available ISR DNA sequences in GenBank to produce a novel data set of RFLPs for 31 slowly growing Mycobacterium species. Subsequently a GelCompar II database was constructed from RFLPs for 10 enzymes that have been used in the literature to differentiate slowly growing Mycobacterium species. The combination of Sau96I and HaeIII were the best choice of enzymes for differentiating clinically relevant slowly growing Mycobacterium species. A total of 392 specimens were studied by PCR with 195 negative and 197 positive specimens. The ISR-PCR product was digested with HaeIII (previously reported) and Sau96I (new to this study) to obtain a Mycobacterium species identification based on the ISR-RFLPs. The species identification obtained by ISR-RFLP was confirmed by DNA sequencing (isolate numbers are shown in parentheses) for M. avium (3), M. intracellulare (4), M. avium complex (1), M. gordonae (2) and M. tuberculosis (1). The total number of specimens (99) identified were from culture (67), Bactectrade mark 12B culture bottles (11), EDTA blood (3), directly from smear positive specimens (13), tissue (4) and urine (1). Direct species identification was obtained from all 13/13 smear positive specimens. The total number of specimens (99) were identified as M. tuberculosis (41), M. avium (7), M. avium complex (11), M. intracellulare MIN-A (20), M. flavescens (2), M. fortuitum (10), M. gordonae (4), M. shimoidei (1), M. ulcerans (1) and M. chelonae (2). This method reduces the time taken for Mycobacterium species identification from 8-10 weeks for culture and biochemical identification; to 4-6 weeks for culture and ISR-RFLP; to 2 days for smear-positive specimens by ISR-RFLP. The precise 2 day identification obtained may provide significant advantages in clinical management.
Clostridium difficile causes outbreaks of infectious diarrhoea, most commonly occurring in health... more Clostridium difficile causes outbreaks of infectious diarrhoea, most commonly occurring in healthcare institutions. Recently, concern has been raised with reports of C. difficile disease in those traditionally thought to be at low risk i.e. community acquired rather than healthcare acquired. This has increased awareness for the need to track outbreaks and PCR-ribotyping has found widespread use to elucidate epidemiologically linked isolates. PCR-ribotyping uses conserved regions of the 16S rRNA gene and 23S rRNA gene as primer binding sites to produce varying PCR products due to the intergenic spacer (ITS1) regions of the multiple operons. With the explosion of whole genome sequence data it became possible to analyse the start of the 23S rRNA gene for a more accurate selection of regions closer to the end of the ITS1. However the following questions must still be asked: (i) Does the chromosomal organisation of the rrn operon vary between C. difficile strains? and (ii) just how conserved are the primer binding regions? Eight published C. difficile genomes have been aligned to produce a detailed database of indels of the ITS1&amp;amp;amp;amp;amp;amp;amp;amp;#39;s from the rrn operon sets. An iPad Filemaker Go App has been constructed and named RiboTyping (RT). It contains detail such as sequences, ribotypes, strain numbers, GenBank numbers and genome position numbers. Access to various levels of the database is provided so that details can be printed. There are three main regions of the rrn operon that have been analysed by the database and related to each other by strain, ribotype and operon: (1) 16S gene (2) ITS1 indels (3) 23S gene. This has enabled direct intra- and inter-genomic comparisons at the strain, ribotype and operon (allele) levels in each of the three genomic regions. This is the first time that such an analysis has been done. By using the RT App with search criteria it will be possible to select probe combinations for specific strains/ribotypes/rrn operons for experiments to do with diagnostics, typing and recombination of operons. Many more incomplete C. difficile whole genome sequencing projects are recorded in GenBank as underway and the rrn operon information from these can also be added to the RT App when available. The RT App will help simplify probe selection because of the complexity of the ITS1 in C. difficile even in a single genome and because other allele-specific regions (16S and 23S genes) of variability can be relationally compared to design extra probes to increase sensitivity.
Campylobacter insulaenigrae is a novel species that has been recently only isolated from marine m... more Campylobacter insulaenigrae is a novel species that has been recently only isolated from marine mammals. This is the first report of C. insulaenigrae causing enteritis and septicaemia in a patient with end-stage hepatic and renal disease.
International Journal of Systematic and Evolutionary Microbiology, 2001
A nocardioform bacterium was isolated from the bronchoscopic lavage of a 78-year-old man with a p... more A nocardioform bacterium was isolated from the bronchoscopic lavage of a 78-year-old man with a past history of tuberculous pleurisy, who presented with bilateral upper lobe lesions at Austin and Repatriation Medical Centre, Heidelberg, Australia. The strain was aerobic, Gram-positive, produced beige substrate mycelium and scant white aerial mycelium. It showed chemotaxonomic markers which were consistent with the classification of Nocardia : i.e. meso-diaminopimelic acid, N-glycolylmuramic acid, arabinose and galactose as diagnostic sugars ; phospholipids phosphatidylinositol mannosides, phosphatidylinositol, phosphatidylethanolamine and diphosphatidylglycerol ; a menaquinone with a cyclic isoprene side chain, MK-8(H 4cycl.) ; a fatty acid pattern composed of unbranched saturated and monounsaturated fatty acids with a considerable amount of tuberculostearic acid ; and mycolic acids composed of 54-62 carbon atoms with three principal mycolic acids which were mono-and polyunsaturated, showing a chain length C 56 , C 58 and C 60 and accounting for over 70 % of the entire pattern. The 16S rDNA sequence showed the highest similarity to the type strain of Nocardia vaccinii ; the DNA-DNA similarity of the two strains was 31 %. These data, together with distinct physiological traits and molecular biological analyses, as well as chemotaxonomic results, led to the conclusion that the novel isolate represents a new species within the genus Nocardia for which the name Nocardia veterana sp. nov. is proposed. The type strain is M157222 T (DSM 44445 T l NRRL B-24136 T).
Gilbert&#39;s syndrome, due to reduced hepatic bilirubin glucuronidation is associated with t... more Gilbert&#39;s syndrome, due to reduced hepatic bilirubin glucuronidation is associated with the presence of two extra nucleotides (TA) in the promoter region of the UDP-glucuronosyltransferase 1 (UGT1A1) gene. A rapid method was developed to detect this genetic polymorphism, using double gradient denaturing gradient gel electrophoresis (DG-DGGE). The promoter region of the UGT1A1 gene was amplified with a 40-mer GC-clamp attached to the 5&#39;-end of the reverse primer. The polymerase chain reaction (PCR) product was then separated by DG-DGGE using denaturant concentrations of 15-25% and polyacrylamide concentrations of 6-12%. The (TA)6/(TA)6 homozygotes were clearly distinguished from both (TA)7/(TA)7 homozygotes and (TA)6/(TA)7 heterozygotes. The (TA)7 allele frequency was consistent with that previously reported and elevated bilirubin levels correlated with the presence of the (TA)7 allele. The DG-DGGE method described will make detection for this polymorphism fast, simple, nonradioactive and suitable for a clinical routine diagnostic laboratory, helping to establish the role of this polymorphism in individuals with jaundice due to multiple causes.
Clostridium difficile is a major spore-forming environmental pathogen that causes serious health ... more Clostridium difficile is a major spore-forming environmental pathogen that causes serious health problems in patients undergoing antibiotic therapy. Consequently, reliable and sensitive methods for typing individual strains are required for epidemiological and environmental studies. Ribotyping is generally considered the best method, but it fails to account for sequence diversity which might exist in intergenic 16S-23S rRNA spacer regions (ISRs) within and among strains of this organism. Therefore, this study was undertaken to compare the sequence of each individual ISR in five strains of C. difficile to explore the extent of this diversity and see whether such information might provide the basis for more sensitive and discriminatory strain typing methods. After targeted PCR amplification, cloning, and sequencing, the diversity of the ISRs was used as a measure of rRNA operon copy number. In C. difficile strains 630, ATCC 43593, A, and B, 11, 11, 7, and 8 ISR length variants, respec...
Restriction maps were constructed of enzymically amplified 16s rRNA genes (rDNA) isolated from ei... more Restriction maps were constructed of enzymically amplified 16s rRNA genes (rDNA) isolated from eight Clos?ridium species. Using maximum parsimony, a dendrogram was constructed from these and published 16s rRNA sequence data. Two distinct clusters were identified: cluster I contained C. dBcile, C. sordklli, and C. bifementans, and showed 30 of 35 restriction sites in common; cluster I1 contained C. tetani, C. perfingens C. sprogenes and C. botulinum C and G, and showed 20 of 35 restriction sites in common. Further analysis of cluster I organisms revealed that of five HpaII fragments, two were found in equal amounts in all organisms, one was found in varying amounts in all organisms, and two were found, in varying amounts, only in C. sordelli and C. bifevmentans. C. sordelli-specific and C. bifevmentans-specific HpaII fragments were demonstrated by Southern hybridization of rDNA. One HpaII site within the rDNA was present on most alleles in C. bifementans, present on a minority of alleles in C. sordelli and absent in C. di,,cile. This suggested that there were two 16s rRNA alleles with different sequences present within each of the genomes of C. bifermentans and C. sordklli.
Uploads
Papers by Volker GURTLER