The impact of horizontal gene transfer on targeting the internal transcribed spacer region (ITS) to identify Acinetobacter junii strains.

AIMS: 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 Ac. junii strains. METHODS AND RESULTS: ITS sequences from a number of strains of Ac. junii were either downloaded from public databases or generated here by cloning and sequencing ITS PCR amplicons. ITS copies of Ac. junii strain 97338 were all 666 bp long, with identical sequences. In Ac. junii ATCC 17908(T) /BCRC 14854(T) ), ITS copies were also all identical in their lengths but now were 706/7 bp long. Two sequence variants of these 707 bp ITS were detected. One was identical in its sequence to the nine ITS copies downloaded from the whole genome sequence of Ac. junii CIP 64·5, and those in several other Ac. junii strains. The other 707 bp ITS variant occurred elsewhere only in Ac. junii strain DSM 14968 of those examined. The six ITS copies from the genome sequence of Ac. junii NIPH 182 were all 685 bp, and with identical sequences. Ac. junii strain 178 also possessed this same 685 bp ITS variant, one of six variants detected there. At least five ITS sequence variants were seen in Ac. junii strain 97380, four in strain DSM 14968 and two in the whole genome of strain 107470. CONCLUSIONS: As with those of other Acinetobacter species, such ITS variants arise not from intragenomic recombination events but from the presence of different length indels. These arise from horizontal gene transfers involving ITS fragments of other Acinetobacter species. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of these indels compromises the reliability of the ITS targeted methods available for typing Acinetobacter junii. It also precludes the value of using ITS sequences as phylogenetic markers in members of the genus Acinetobacter, since the outcomes in both cases depends on which copy variant is chosen.

Value of high resolution compression elastography and color doppler sonography in characterisation of breast lesions: comparison of different high-frequency transducers.

PURPOSE: To evaluate the additional effect of higher frequent linear probes than 12.5 MHz in color Doppler sonography and free hand sonoelastography of benign and malignant breast masses and to compare different color encodings in sonoelastography. MATERIALS AND METHODS: From December 2012 to March 2013, 37 patients with benign or malignant breast masses were prospectively included in this study. All solid masses have been histologically proven. Two readers assessed sonoelastographic findings at 12.5 MHz vs. 17 MHz according to the tsukuba elasticity score and additionally different color encodings were compared. Results of Doppler sonography using a score of 0, 1 or 2, depending on the degree of perfusion, also were assessed at 12.5 MHz vs. 17 MHz. RESULTS: Among the 37 examined breast masses there were 10 cysts, 16 fibroadenomas and 11 carcinomas. Median participant age was 49.0 years. Use of color Doppler sonography enabled to distinguish cysts from solid breast masses (p < 0.001), without an improvement at 17 MHz. Additional sonoelastography significantly improved the specificity in solid breast masses (p < 0.001). No changes could be seen using different colors in sonoelastography. CONCLUSION: Combination of color Doppler sonography and sonoelastography can increase the accuracy in distinguishing benign from malignant breast masses. The use of linear probes with a higher frequency than 12.5 MHz does not show any benefit, neither in color Doppler sonography nor in sonoelastography.

Comparison of contrast-enhanced ultrasound and compression elastography in the follow-up after endovascular aortic aneurysm repair.

PURPOSE: To compare compression elastography and contrast enhanced ultrasound in the follow-up after endovascular aortic aneurysm repair. MATERIAL AND METHODS: In this retrospective study a cohort of 33 patients with both CEUS and elastography follow-up examinations after EVAR were included. The examinations were done with a Siemens S 2000 with curved array 4 MHz multi-frequency transducer. RESULTS: Regarding our inclusion and exclusion criteria we obtained 33 patients. CEUS was used as the preferred examination in determining the presence of an endoleak. The true positive rate for the detection of Endoleaks with compression elastography was 42.4% (14/33), the false positive rate was 12.1% (4/33), the true negative rate was 15.2% (5/33) and the false negative rate was 30.3% (10/33). The sensitivity of compression elastography was therefore 58.3% and the specifity was 55.6%. Kappa coefficient was 0.115. CONCLUSION: Compression elastography does not seem to have any additional advantages in the detection and classification of endoleaks in comparison to CEUS.

Classification of endoleaks in the follow-up after EVAR using the time-to-peak of the contrast agent in CEUS examinations.

PURPOSE: To evaluate the feasibility of the classification of endoleaks following endovascular aortic aneurysm repair using the time-to-peak of the contrast agent in CEUS examinations. MATERIAL AND METHODS: In this retrospective study, a cohort of 171 patients with a total of 489 CEUS follow-up examinations after EVAR were included. In 254 of the 489 examinations, an endoleak was seen and the time-to-peak was measured in seconds. Existence of an endoleak was confirmed by CT as the gold standard. RESULTS: We evaluated 254 CEUS video sequences showing an endoleak out of a total of 489 examinations. Kruskal-Wallis test revealed with p = 0.001 differences between the single endoleak types based on the time to peak. Correction after Bonferroni showed significant differences between type Ia compared to Ib and to IIa over inferior mesenteric artery (IMA) and IIa over lumbar artery (LA). There are also disparities between type Ib and type IIa IMA and type III, furthermore between type IIa IMA compared to IIa LA and type III as well as type IIa LA matched to type III. CONCLUSION: CEUS is an important method for the follow-up after EVAR. The time-to-peak does not seem to be a useful additional feature in classifying endoleaks, although there are differences between the time-to-peak of the single endoleak types and it is possible to make an order of the different endoleak types referring to the mean values.

Improving the follow up after EVAR by using ultrasound image fusion of CEUS and MS-CT.

PURPOSE: To evaluate whether the image fusion with contrast enhanced ultrasound (CEUS) and CT affects the diagnosis of endoleaks in unclear cases. METHODS AND MATERIALS: 35 patients with follow-up examinations after endovascular aneurysm repair (EVAR) were included in this retrospective study. Mean patient age was 73 years (range 54-83 y). B-scan, colour doppler and CEUS (1.2 ml SonoVue®, Bracco Imaging Germany) were performed in all patients by an experienced examiner using two different high-end ultrasound system (Siemens ACUSON S2000™, Siemens Healthcare, Erlangen, Germany or Logic E9, GE Healthcare, Milwaukee,WI, USA) with a multifrequency curved array transducer. The examiner was initially blinded to the CT results. Additional image fusion with CT-angiography (CTA) was then performed. The ultrasound examinations were later read by two blinded unbiased investigators with more than five years of clinical ultrasound in consensus. RESULTS: All patients were examined using all diagnostic ultrasound tools of the study. The results show that image fusion is easy and convenient to perform. Conventional ultrasound examination with B-scan and colour Doppler examination detected one Type I and one Type II endoleak, contrast enhanced ultrasound detected one Type I and three Type II endoleaks after EVAR whereas CTA depicted one Type I and two Type II endoleaks. Ultrasound image fusion with CT-angiography confirmed one Type I and three Type II endoleaks. CONCLUSION: In comparison to conventional ultrasound and CTA the use of CEUS improved the visualization and classification of endoleaks. CEUS shows even small blood flow which can be depicted due to the real time imaging of endoleaks. In unclear cases additional ultrasound image fusion with CEUS and CT angiography improves the visualisation of small endoleaks and this may cause a change in the follow-up interval. CEUS is a good alternative to CT in the detection and follow-up of endoleaks, especially in patients with contraindications to CT contrast agents due to allergies or renal failure, enabling reduced additional costs and exposure to radiation.

Genomic typing of Pseudomonas aeruginosa isolates by comparison of Riboprinting and PFGE: correlation of experimental results with those predicted from the complete genome sequence of isolate PAO1.

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.

Genomic fingerprinting of the 16S-23S gene spacer region suggests that novel Acinetobacter isolates are present in activated sludge.

The taxonomic status of the genus Acinetobacteris currently confused and the role of these organisms in activated sludge is poorly understood. Currently unidentified isolates of Acinetobacterfrom activated sludge were fingerprinted by making use of polymorphisms in their 16S-23S rDNA spacer region. The PCR amplified 16S-23S rDNA spacer region was digested with five different restriction enzymes to further differentiate between the isolates. The resulting band patterns were very diverse and the data suggests that the activated sludge isolates are different to the known genomic species of Acinetobacter which are predominantly clinical isolates. The results of this study imply the existence of yet unrecognised species of Acinetobacter in activated sludge.

Use of denaturing gradient gel electrophoresis to detect mutation in VS2 of the 16S-23S rDNA spacer amplified from Staphylococcus aureus isolates.

To develop a double gradient denaturing gradient gel electrophoresis (DG-DGGE) based typing method that rapidly and accurately types clinical isolates of Staphylococcus aureus, the VS2 region of the 16S-23S rRNA spacer region (ISR) was chosen because of its potential high variation. The VS2 region was amplified with a 40-mer GC-clamp attached to the 5'-end of the reverse primer. The 145 bp PCR product was then separated by DG-DGGE using denaturant concentrations of 25-40% and polyacrylamide concentrations of 6-12%. Of the five mutations identified in 336 S. aureus isolates, one mutation was found to be highly specific for 161/171 (94%) of methicillin-resistant S. aureus (MRSA) isolates from different geographic locations and isolation times. This same mutation was found in 15/160 (9%) of penicillin- or methicillin-sensitive S. aureus isolates. In some isolates two mutations occured together in the one genome suggesting some S. aureus isolates have two copies of VS2. In these 336 isolates nine genotypes with different combinations of the five mutations were identified. In 18 coagulase-negative staphylococci (CNS), the MRSA-specific mutation was found along with two other mutations in all isolates demonstrating consistent differences in the presence of these mutations between CNS and S. aureus. The marked differences in VS2 sequences found between MRSA, methicillin- or penicillin-sensitive S. aureus (SSA), and CNS by DGGE in the present study may be useful in evolutionary studies and in the development of a specific assay for MRSA from clinical specimens.

Nocardia veterana sp. nov., isolated from human bronchial lavage.

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(H4cycl.); 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 C56, C58 and C60 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 M157222T (DSM 44445T = NRRL B-24136T).

Genomic approaches to typing, taxonomy and evolution of bacterial isolates.

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.

Pyrolysis mass spectrometry (PyMS) and 16S-23S rDNA spacer region fingerprinting suggests the presence of novel acinetobacters in activated sludge.

Screening of large numbers of Acinetobacter spp. from activated sludge systems with Pyrolysis Mass Spectrometry (PyMS) showed that many did not cluster tightly with the currently described genomic species which have been obtained mainly from clinical sources. Selected isolates were then genotypically fingerprinted using their 16S-23S rDNA spacer region, and again the data revealed considerable differences in the genomic fingerprints of many of these activated sludge isolates to the predominantly clinical genomic species. In fact, few could be identified from them. The possibility that the current speciation within this genus is not adequate to encompass all these environmental isolates is addressed in relation to the methods used to study the population dynamics of Acinetobacter in activated sludge.

The role of recombination and mutation in 16S-23S rDNA spacer rearrangements.

The intragenomic heterogeneity of the bacterial intergenic (16S-23S rDNA) spacer region (ISR) was analysed from the following species in which sequences for the complete rRNA operon (rrn) set have been determined (rrn number): Enterococcus faecalis (6) and E. faecium (6), Bacillus subtilis (10), Staphylococcus aureus (9), Vibrio cholerae (4), Haemophilus influenzae (6) and Escherichia coli (7). It was found that some spacer sequence blocks were highly conserved between operons of a genome, whereas the presence of others was variable. When these variations were analysed using the program PLATO and partial likelihood phylogenies determined by DNAml for each operon set, three regions showed significant (Z>3.3) spatial variation [Region I was 78-184 nt long (2.14.4) possibly due to recombination or selection. Within Region I, there was sequence block variation in all operon sets [some operons contained tRNA genes (tRNAala, tRNAile or tRNAglu), whereas others had sequence blocks such as VS2 (S. aureus) or rsl (E. coli)]. Q Analysis of the ISR sequence from E. faecalis and E. faecium showed that there was more interspecies than intraspecies variation (both in DNA sequence and in the presence or absence of blocks). Dot matrix analysis of the sequence blocks in the nine rrn ISRs from S. aureus showed that there was significant homology between VS2 and VS5/VS6. Furthermore, repeat motifs with only A or T were present in higher copy numbers in VS5/VS6 than in VS2. Since these sequence blocks (VS2 and VS5-VS6) are related, intragenic evolution resulting in AT expansion may have occurred between these two regions. A model is proposed that postulates a role for recombination and AT-expansion in intra-genomic ISR variations. This process may represent a general mechanism of concerted evolution for bacterial ISR rearrangements.

Use of double gradient denaturing gradient gel electrophoresis to detect (AT)n polymorphisms in the UDP-glucuronosyltransferase 1 gene promoter associated with Gilbert's syndrome.

Gilbert'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'-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.

Identification of Mycobacterium shimoidei by molecular techniques: case report and summary of the literature.

A 53-year-old woman from Melbourne, Australia, with squamous cell carcinoma of the oesophagus was shown by computed tomography (CT) scan to have a left apical cavity and inflammatory changes in the right lung consistent with aspiration. Acid-fast bacilli isolated from bronchial washings were identified biochemically first as Mycobacterium terrae, but later as M. shimoidei on the basis of 1) restriction fragment analysis and 2) sequencing of polymerase chain reaction (PCR) amplified 16S rDNA. Nine other descriptions of patients with M. shimoidei isolates were collated. The salient feature of isolates considered to be pathogenic was pulmonary cavitation. Most patients had underlying lung disease, including past tuberculosis or malignancy. Six of eight patients died of progressive respiratory illness, although the contribution of M. shimoidei was not always clear, and two patients improved. One patient with the acquired immune-deficiency syndrome (AIDS) died with Salmonella enteritidis and M. shimoidei isolated from blood cultures. One isolate was regarded as a coloniser. There are insufficient clinical or sensitivity data on which to base recommendations for therapy, but a combination of ethambutol, rifabutin and pyrazinamide could be considered.

Aetiology of severe iron overload in a family with hereditary haemolytic anaemia.

Severe iron overload is a reported complication of certain erythroid disorders which are characterized by increased erythropoietic activity. Proposed mechanisms include enhancement of iron absorption secondary to increased erythroid activity and coexistent heterozygosity or homozygosity for haemochromatosis. We performed PCR-based analysis for the haemochromatosis-related HFE C282Y mutation in an extended family with inherited haemolytic anaemia in which several members exhibited iron overload. The results demonstrated iron overload was associated with homozygosity but not heterozygosity for this mutation. Such an association may also exist in other erythroid disorders in which iron overload has been reported.

Typing of Clostridium difficile strains by PCR-amplification of variable length 16S-23S rDNA spacer regions.

To develop a rapid and accurate method of typing large numbers of clinical isolates of Clostridium difficile, four regions of the rRNA operon [A, 15-1407 and B, 907-1407 (16S-16S); C, 1392-507 and D, 907-507 (16S-23S)] were enzymically amplified from 24 strains. When region A was hybridized to HindIII-digested genomic DNA isolated from C. difficile strains, all of the variable length restriction fragments hybridized. When region B was hybridized to HindIII-digested genomic DNA isolated from C. difficile strains, a set of variable length restriction fragments (Group II) 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 II 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. difficile strains ranging in size from 852-1210 bp. After analysis with maximum parsimony, the 24 strains were divided into 14 ribotypes.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of Clostridium difficile enterotoxin gene in clinical specimens by the polymerase chain reaction.

A rapid assay was developed for detection of the Clostridium difficile enterotoxin gene in stool specimens by means of the polymerase chain reaction (PCR). The PCR primers amplified a 63-bp repetitive sequence of the enterotoxin gene, thereby generating a distinctive ladder pattern of DNA bands following electrophoresis. Crude DNA extracts from stools containing C. difficile produced one (63-bp) or more bands of the characteristic ladder. Of 172 stool specimens from 58 patients, 37 gave positive results by culture (15 specimens) or cytotoxin assay (36 specimens). When 36 available "positive" specimens were tested by the PCR assay, 34 (94%) gave positive results--24 by direct testing, and 10 after extraction of DNA by the Qiagen procedure. Insufficient material of the remaining two specimens was available for DNA extraction. Of 21 stools "negative" for C. difficile by culture or cytotoxin assay, one gave a positive result by PCR and seven produced atypical bands. The rapid PCR detection technique for C. difficile was more sensitive than standard culture, and of a sensitivity similar to cytotoxin testing. The method has the potential for adoption in routine laboratory practice.

Classification of medically important clostridia using restriction endonuclease site differences of PCR-amplified 16S rDNA.

Restriction maps were constructed of enzymically amplified 16S rRNA genes (rDNA) isolated from eight Clostridium 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. difficile, C. sordelli, and C. bifermentans, and showed 30 of 35 restriction sites in common; cluster II contained C. tetani, C. perfringens C. sporogenes 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. bifermentans. C. sordelli-specific and C. bifermentans-specific HpaII fragments were demonstrated by Southern hybridization of rDNA. One HpaII site within the rDNA was present on most alleles in C. bifermentans, present on a minority of alleles in C. sordelli and absent in C. difficile. This suggested that there were two 16S rRNA alleles with different sequences present within each of the genomes of C. bifermentans and C. sordelli.