The emergence of SARS-CoV-2 variants of concern in Australia by haplotype coalescence reveals a continental link to COVID-19 seasonality.

SARS-CoV-2 continues to evolve, even after implementation of public-wide vaccination, as can be observed by an increasing number of mutations over time. Compared to responses by the United States and European countries, the disease mitigation strategies employed by the Australian government have been swift and effective. This provides a unique opportunity to study the emergence of variants of concern (VOCs) at many latitude levels in a country that has been able to control infection for the majority of the pandemic. In the present study, we explored the occurrence and accumulation of major mutations typical of VOCs in different regions of Australia and the effects that latitude has on the establishment of VOC-induced disease. We also studied the constellation of mutations characteristic of VOCs to determine if the mutation sets acted as haplotypes. Our goal was to explore processes behind the emergence of VOCs as the viral disease progresses towards becoming endemic. Most reported COVID-19 cases were in largest cities located within a -30°S to - 50°S latitude corridor previously identified to be associated with seasonal behavior. Accumulation plots of individual amino acid variants of major VOCs showed that the first major haplotypes reported worldwide were also present in Australia. A classification of accumulation plots revealed the existence of 18 additional haplotypes associated with VOCs alpha, delta and omicron. Core mutant constellations for these VOCs and curve overlaps for variants in each set of haplotypes demonstrated significant decoupling patterns, suggesting processes of emergence. Finally, construction of a "haplotype network" that describes the viral population landscape of Australia throughout the COVID-19 pandemic revealed significant and unanticipated seasonal patterns of emergence and diversification. These results provide a unique window into our evolutionary understanding of a human pathogen of great significance. They may guide future research into mitigation and prediction strategies for future VOCs.

Bioinformatics analysis of codon usage patterns and influencing factors in Penaeus monodon nudivirus.

Penaeus monodon nudivirus (PmNV) is one of the most important and most commonly reported shrimp viruses. In the present study, codon usage of PmNV was studied in detail. Based on effective number of codons (ENC) values, strong to low codon usage bias was observed in PmNV genes. Nucleotide composition-ENC correlation analysis and the GC3 versus ENC relationship indicated that compositional constraint has a major effect on codon usage of PmNV. At the whole-genome level, relative synonymous codon usage (RSCU) analysis showed almost complete antagonism between the codon usage pattern of PmNV and its host P. monodon. However, codon adaptive index (CAI) values indicated that forces of selective/translational constraints have been able to overcome this antagonism in some genes.

Unusual features of the sequences of copies of the 16S-23S rRNA internal transcribed spacer regions of Acinetobacter bereziniae, Acinetobacter guillouiae and Acinetobacter baylyi arise from horizontal gene transfer events.

The highly variable nature of the internal transcribed spacer region (ITS) has been claimed to represent an ideal target for designing species-specific probes/primers capable of differentiating between closely related Acinetobacter species. However, several Acinetobacter species contain multiple ITS copies of variable lengths, and these include Acinetobacter bereziniae, Acinetobacter guillouiae and Acinetobacter baylyi. This study shows these length variations result from inter-genomic insertion/deletion events (indels) involving horizontal transfer of ITS fragments of other Acinetobacter species and possibly unrelated bacteria, as shown previously by us. In some instances, indel incorporation results in the loss of probe target sites in the recipient cell ITS. In other cases, some indel sequences contain target sites for probes designed from a single ITS sequence to target other Acinetobacter species. Hence, these can generate false positives. The largest of the indels that remove probe sites is 683 bp (labelled bay/i1-0), and it derives from the horizontal transfer of a complete ITS between A. bereziniae BCRC15423(T) and A. baylyi strain ADP1. As a consequence, ITS sequencing or fingerprinting cannot be used to distinguish between the 683 bp ITS in these two strains.

Insertions or deletions (Indels) in the rrn 16S-23S rRNA gene internal transcribed spacer region (ITS) compromise the typing and identification of strains within the Acinetobacter calcoaceticus-baumannii (Acb) complex and closely related members.

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 BCRC15417T (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.

Draft Genome Sequence of trh+ Vibrio parahaemolyticus VP-49, Isolated from Seafood Harvested along the Mangalore Coast, India.

Vibrio parahaemolyticus is a seafood-borne pathogen autochthonous to the marine and estuarine ecosystem, which is responsible for gastroenteritis due to the consumption of contaminated raw seafood. Here, we report the draft genome sequence of V. parahaemolyticus VP-49, isolated from seafood, to identify the different virulence attributes and to study the mechanisms that enhance its environmental fitness.

The increased sensitivity of human cytomegalovirus (HCMV) PCR quantitation in whole blood affects reproductive rate (Ro) measurement.

In order to determine the effect of the increase in sensitivity of HCMV detection in whole blood compared to plasma on reproductive rate (Ro) measurement, an optimized human cytomegalovirus (HCMV) quantitative PCR assay was developed. The results presented in this study are summarized by the following three methodological improvements: (i) at values below the limit of quantitation (LOQ) of 60copies/ml, determination of HCMV load was more sensitive with whole blood than plasma, (ii) for the determination of viral load, whole blood was more sensitive than plasma below 1000copies/ml but little difference was observed above 1000copies/ml and (iii) the measurement of "Reproductive Rate" can be affected by imprecise measurement of HCMV viral load in either plasma or whole blood compartments depending on whether samples were taken from patients on antiviral treatment or from patients where HCMV load was rising. Taken together this study provides methodological improvements suggesting that below HCMV viral load levels of 1000copies/ml (1640IU/ml) both plasma and whole blood should be tested.

Reprint of New opportunities for improved ribotyping of C. difficile clinical isolates by exploring their genomes.

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'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.

New opportunities for improved ribotyping of C. difficile clinical isolates by exploring their genomes.

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'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.

A novel method for simultaneous Enterococcus species identification/typing and van genotyping by high resolution melt analysis.

In order to develop a typing and identification method for van gene containing Enterococcus faecium, two multiplex PCR reactions were developed for use in HRM-PCR (High Resolution Melt-PCR): (i) vanA, vanB, vanC, vanC23 to detect van genes from different Enterococcus species; (ii) ISR (intergenic spacer region between the 16S and 23S rRNA genes) to detect all Enterococcus species and obtain species and isolate specific HRM curves. To test and validate the method three groups of isolates were tested: (i) 1672 Enterococcus species isolates from January 2009 to December 2009; (ii) 71 isolates previously identified and typed by PFGE (pulsed-field gel electrophoresis) and MLST (multi-locus sequence typing); and (iii) 18 of the isolates from (i) for which ISR sequencing was done. As well as successfully identifying 2 common genotypes by HRM from the Austin Hospital clinical isolates, this study analysed the sequences of all the vanB genes deposited in GenBank and developed a numerical classification scheme for the standardised naming of these vanB genotypes. The identification of Enterococcus faecalis from E. faecium was reliable and stable using ISR PCR. The typing of E. faecium by ISR PCR: (i) detected two variable peaks corresponding to different copy numbers of insertion sequences I and II corresponding to peak I and II respectively; (ii) produced 7 melt profiles for E. faecium with variable copy numbers of sequences I and II; (iii) demonstrated stability and instability of peak heights with equal frequency within the patient sample (36.4±4.5 days and 38.6±5.8 days respectively for 192 patients); (iv) detected ISR-HRM types with as much discrimination as PFGE and more than MLST; and (v) detected ISR-HRM types that differentiated some isolates that were identical by PFGE and MLST. In conjunction with the rapid and accurate van genotyping method described here, this ISR-HRM typing and identification method can be used as a stable identification and typing method with predictable instability based on recombination and concerted evolution of the rrn operon that will complement existing typing methods.

HRM confirmation of Neisseria gonorrhoeae in clinical specimens by G→A (position 857) mutation detection in the 16S rRNA gene before sequencing and after porA confirmation.

A total of 2273 specimens submitted to the Austin Hospital Pathology Service for Neisseria gonorrhoeae screening between September 1, 2009 and May 11, 2011 were used in this study. Specimens were simultaneously screened and confirmed with a previously published real time PCR assay for the opa gene (extra primers were included to increase sensitivity) and the porA gene respectively. The opa gene screen and initial porA gene confirmation yielded an N. gonorrhoeae positivity rate of 0.88% (20/2273) and 0.49% (11/2191) for specimens and patients respectively. A 16S rDNA High Resolution Melt confirmatory PCR was developed subsequently; this reduced the N. gonorrhoeae positivity rate to 0.35% (8/2273) and 0.27% (6/2191) for specimens and patients respectively (not altered by 16S sequencing). The higher rate of secondary confirmation (16S HRM) in patients compared with samples was due to the detection of species other than N. gonorrhoeae detected by the initial screening and confirmation test. This underlines the importance of performing the secondary confirmatory test that has been developed in this study.

High resolution melt analysis to track infections due to ribotype 027 Clostridium difficile.

The increased prevalence of hypervirulent ribotype 027 Clostridium difficile requires rapid identification of isolates in order to implement timely infection control strategies. High resolution melt (HRM) analysis of PCR products can identify strain variation amongst genera of bacteria. The intergenic (16S-23S rDNA) spacer region contains sequence regions conserved within genera and other sequence region variables between species within genera. We wished to investigate whether HRM analysis of PCR ribotyping products could identify ribotype 027 C. difficile. Ribotyping was performed on 93 clinical isolates and five control strains and band patterns were analysed using GelCompar II (Applied Maths, USA). Real-time PCR using ribotyping primers was performed and normalised melt curves were generated. The HRM data was then imported into ScreenClust software (QIAGEN) to generate principal component analysis graphs depicting clustered relationships of strains. Ribotyping produced clear PCR bands for 88/98 isolates tested. Dendrograms generated by GelCompar showed a diversity of ribotype patterns amongst these 88 isolates with 18 groups identified with 70% homology. One clinical isolate showed 100% homology with the control 027 strains. ScreenClust analysis of the same 88 HRM results showed clustering of isolates, with 027 strains identifiable as a unique cluster. HRM analysis correctly identified the control 027 stains and the clinical isolate shown to be 027. HRM combined with ScreenClust analysis of real-time PCR products of the 16S-23S rDNA spacer region successfully identified ribotype 027 strains. For infection control purposes this was achieved within 2-3 h of colony isolation.

Developments in immunological technologies leading to improvements in point-of-care diagnostic testing.

Rapid and accurate point of care testing is of great concern, especially in terms of detecting infectious disease outbreaks in developing countries having high population burdens. While numerous detection systems are currently available, care must be taken in choosing those that are reliable and have proven acceptable levels of sensitivity and specificity, based on adequate laboratory and pre-clinical trial testing. Two papers in the current issue show significant advances in technology that could bring Point-of-Care Diagnostic Testing closer to reality for cholera and environmental mycobacteria.

Unique organization of the 16S-23S intergenic spacer regions of strains of Acinetobacter baylyi provides a means for its identification from other Acinetobacter species.

This paper extends an earlier report on rrn operon characteristics in members of the genus Acinetobacter. It describes a systematic approach towards developing and validating a protocol for elucidating how the intergenic spacer regions (ISR) in Acinetobacter baylyi strains are organized and allows the numbers of long and short ISRs to be determined. Experimental data confirmed the in silico predictions based on available A. baylyi rrn sequence data. All were shown to possess three long ISRs and 4 short ISRs, differing in most cases in length by about 90nt. However, the ISR arrangement in A. baylyi strain 93A2 was different. Although it also possessed 4 SISRs and three LISRs, their length difference was less (39nt) which was confirmed from its ISR sequence data. Primer sets for PCR identification of A. baylyi could then be determined. Applying the same approach to other species of Acinetobacter showed none shared the same ISR organization as A. baylyi. Its value in typing members of this genus is discussed.

Campylobacter insulaenigrae causing septicaemia and enteritis.

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.

The mosaic nature of intergenic 16S-23S rRNA spacer regions suggests rRNA operon copy number variation in Clostridium difficile strains.

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, respectively, were found (containing different combinations of sequence groups [i to xiii]), suggesting 11, 11, 7, and 8 rrn copies in the respective strains. Many ISRs of the same length differed markedly in their sequences, and some of these were restricted in occurrence to a single strain. Most of these ISRs did not contain any tRNA genes, and only single copies of the tRNA(Ala) gene were found in those that did. The presence of ISR sequence groups (i to xiii) varied between strains, with some found in one, two, three, four, or all five strains. We conclude that the intergenic 16S-23S rRNA spacer regions showed a high degree of diversity, not only among the rrn operons in different strains and different rrn copies in a single strain but also among ISRs of the same length. It appears that C. difficile ISRs vary more at the inter- and intragenic levels than those of other species as determined by empirical comparison of sequences. The precise characterization of these sequences has demonstrated a high level of mosaic sequence block rearrangements that are present or absent in multiple strain-variable rrn copies within and between five different strains of C. difficile.

Relationship of hepatic functional imaging to irinotecan pharmacokinetics and genetic parameters of drug elimination.

PURPOSE: 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. METHODS: Patients underwent genotyping for ABCB1 SNPs and UTUGT-1A1*28 carriage and HNI with 99mTc-DIDA (acetanilidoiminodiacetic acid)/99mTc-DISIDA (disofenin) and MIBI (99mTc-sestamibi) scans, probes for biliary transport proteins ABCC1 and -2, and ABCB1 function. HNI data were analyzed by noncompartmental and deconvolutional analysis to provide hepatic extraction and biliary excretion parameters. Patients received Ir, fluorouracil, and folinic acid using a weekly x2, every-3-weeks schedule. Plasma was taken for Ir and SN-38 analysis on day 1, cycle 1. RESULTS: Of the 21 patients accrued, Ir pharmacokinetics data were obtained from 16 patients. 99mTc-DIDA/DISIDA percent retention at 1 hour (1-hour RET) correlated to baseline serum bilirubin (P = .008). Both 99mTc-DIDA/DISIDA and MIBI 1-hour RET correlated with SN-38 area under the curve (AUC; P < .01). On multiple regression analysis, SN-38 AUC = -215 + 18.68 x bilirubin + 4.27 x MIBI 1-hour RET (P = .009, R2 = 44.2%). HNI parameters did not correlate with Ir toxicity or UGT1A1*28 carriage. MIBI excretion was prolonged in patients with the ABCB1 exon 26 TT variant allele relative to wild-type (P = .015). CONCLUSION: Functional imaging of hepatic uptake and excretory pathways may have potential to predict Ir pharmacokinetics. Evaluation of a larger cohort as well as polymorphisms in other biliary transporters and UGT1A1 alleles is warranted.

Estimation of ribosomal RNA operon (rrn) copy number in Acinetobacter isolates and potential of patterns of rrn operon-containing fragments for typing strains of members of this genus.

The copy number of the rrn operon in 70 strains of Acinetobacter including the type strains of almost all the genomic species with validated names was estimated after digestion of their genomic DNA by the restriction enzymes BglII and PstI, and Southern blotting. Copy number estimates varied between and among species, with between 3 and 7 rrn operon copies detected. Copy number estimates obtained from the same strain with the two enzymes sometimes varied. BglII generated RFLP patterns of the rrn containing fragments obtained from Southern blots after agarose gel electrophoresis were examined for their value in identifying Acinetobacter isolates. This method was very reproducible with the same fragment pattern always generated from the same isolate on repeated analysis. Often multiple strains of the same genomic species gave identical or very similar patterns (e.g. Acinetobacter baylyi), clustering closest together on the dendrogram generated after numerical analysis of these patterns. However, with some, like BG5 and BG8, the patterns derived from the different strains, some of which had been placed in this genomic species from DNA:DNA hybridization data, varied considerably to each other and to the type strain. Little similarity was seen when relationships between these strains based on these patterns were compared to those using DNA:DNA hybridization data. Often these patterns could be used to question earlier identification of strains using phenotypic characters. Thus, strain AB82 thought to belong to genomic species 5 gave an identical pattern to A. bouvetii(T) (DSM 14964). In some cases this pattern analysis suggested that novel species of Acinetobacter might exist among the strains examined.

Direct identification of slowly growing Mycobacterium species by analysis of the intergenic 16S-23S rDNA spacer region (ISR) using a GelCompar II database containing sequence based optimization for restriction fragment site polymorphisms (RFLPs) for 12 enzymes.

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.

Variation of 16S-23S rRNA intergenic spacer regions (ISRs) in Acinetobacter baylyi (strain B2) isolated from activated sludge.

To determine the variability of the 16S-23S rRNA intergenic spacer region (ISR) of the newly described Acinetobacter baylyi, 88 clones containing ISR amplicons were screened and 14 chosen for further analysis. Two different sized 16S-23S rRNA ISRs were distinguished comprising five variable and four conserved nucleotide blocks. The major regions of heterogeneity between the different sized ISRs were due to blocks of substitutions with unique secondary structures interspersed with nucleotide substitutions, rather than differences caused by presence or absence of tRNA genes, which is often the case. Recombination events causing shuffling of nucleotide blocks are considered the most likely explanation for the mosaic structure observed between the different copies of the ISR. Single base differences present in the long ISR (LISR) were then exploited in attempts to detect possible heterogeneity between rrn copies in Acinetobacter baylyi but variability was not detected by RFLP analysis of LISR-specific PCR products. These primers were shown to be highly specific for 3 Acinetobacter baylyi strains based on LISR sequence homogeneity.

Can whole genome analysis refine the taxonomy of the genus Rhodococcus?

The current systematics of the genus Rhodococcus is unclear, partly because many members were originally included before the application of a polyphasic taxonomic approach, central to which is the acquisition of 16S rRNA sequence data. This has resulted in the reclassification and description of many new species. Hence, the literature is replete with new species names that have not been brought together in an organized and easily interpreted form. This taxonomic confusion has been compounded by assigning many xenobiotic degrading isolates with phylogenetic positions but without formal taxonomic descriptions. In order to provide a framework for a taxonomic approach based on multiple genetic loci, a survey was undertaken of the known genome characteristics of members of the genus Rhodococcus including: (i) genetics of cell envelope biosynthesis; (ii) virulence genes; (iii) gene clusters involved in metabolic degradation and industrially relevant pathways; (iv) genetic analysis tools; (v) rapid identification of bacteria including rhodococci with specific gene RFLPs; (vi) genomic organization of rrn operons. Genes encoding virulence factors have been characterized for Rhodococcus equi and Rhodococcus fascians. Based on peptide signature comparisons deduced from gene sequences for cytochrome P-450, mono- and dioxygenases, alkane degradation, nitrile metabolism, proteasomes and desulfurization, phylogenetic relationships can be deduced for Rhodococcus erythropolis, Rhodococcus globerulus, Rhodococcus ruber and a number of undesignated Rhodococcus spp. that may distinguish the genus Rhodococcus into two further genera. The linear genome topologies that exist in some Rhodococcus species may alter a previously proposed model for the analysis of genomic fingerprinting techniques used in bacterial systematics.