Identification of members of Mycobacterium avium species by Accu-Probes, serotyping, and single IS900, IS901, IS1245 and IS901-flanking region PCR with internal standards

From Mycobacterium avium species Mycobacterium avium subsp. paratuberculosis (n=961), Mycobacterium a. avium (n=677), Mycobacterium a. silvaticum (n=5), and Mycobacterium a. hominissuis (n=1566) were examined, and from Mycobacterium tuberculosis complex M. tuberculosis (n=2), Mycobacterium bovis (n=13), M. bovis BCG (n=4), and Mycobacterium caprae (n=10) were examined. From other mycobacterial species Mycobacterium intracellulare (n=60) and atypical mycobacteria (n=256) including Mycobacterium fortuitum, Mycobacterium chelonae, Mycobacterium scrofulaceum, Mycobacterium gastri and other species of conditionally pathogenic mycobacteria were analysed. The internal standard molecules corresponding to insertion sequences IS900, IS901, IS1245, and flanking region (FR300) of IS901 were produced by PCR of alfalfa genome segment and inserted into plasmid vector. The resulting recombinant plasmid molecules were used as internal standards in coamplification with a total of 4729 mycobacterial collection strains and field isolates between 1996 and 2003. The size differences between amplicons obtained from IS900 (258 bp), IS901 (1108 bp), IS1245 (427 bp), and FR300 (300 bp) and from corresponding internal standard molecules ISIS900 (591 bp), ISIS901 (1 336 bp), ISIS1245 (583 bp), and IS901 flanking region of 300 bp ISFR300 (488 bp), respectively, allowed easy discrimination. The internal amplicons were visible by naked aye on agarose gel when 10(1), 10(3), 10(2), and 10(2) molecules for ISIS900, ISIS901, ISIS1245, and ISFR300 were used in the PCR, respectively, when no bacterial DNA was added to the reaction. The system was tested to define the amount of internal standards that could be used in the PCR without affecting the amplification of the specific segment. Non-specific amplifications were observed in M. fortuitum with IS1245 PCR and mixed infections with M. a. avium and M. a. hominissuis from pigs and cattle were found. PCR results of typing were compared with serotyping and Accu-Probes analyses in selected field isolates.     

Sequence and characteristics of IS900, an insertion element identified in a human Crohn''s disease isolate of Mycobacterium paratuberculosis.

The complete sequence of an insertion element IS900 in Mycobacterium paratuberculosis is reported. This is the first characterised example of a mycobacterial insertion element. IS900 consists of 1451bp of which 66% is G + C. It lacks terminal inverted and direct repeats, characteristic of Escherichia coli insertion elements but shows a degree of target sequence specificity. A single open reading frame (ORF 1197) coding for 399 amino acids is predicted. This amino acid sequence, and to a lesser extent the nucleotide sequence, show significant homologies to IS110, an insertion element of Streptomyces coelicolor A3(2). It is proposed that IS900, IS110, and similar insertion elements recently identified in disease isolates of Mycobacterium avium are members of a phylogenetically related family. IS900 will provide highly specific markers for the precise identification of Mycobacterium paratuberculosis, useful in defining its relationship to animal and human diseases.     

Infections caused by Mycobacterium avium subspecies avium, hominissuis, and paratuberculosis in free-ranging red deer (Cervus elaphus hippelaphus) in Austria, 2001-2004

Between 2001 and 2004, 14 Austrian free-ranging red deer (Cervus elaphus hippelaphus) infected by Mycobacterium avium species were observed. Eight of the cases were from different geographical regions, and six originated from the same hunting area. The affected animals had signs of diarrhea, severe weight loss, and emaciation. On post-mortem examination, lymphadenitis associated with grossly enlarged mesenteric lymph nodes as well as multiple caseous or purulent nodular lesions in the thickened wall of the intestines were present in all animals. In 10 cases M. avium subsp. avium and in four cases M. a. hominissuis were isolated. In three red deer, a mixed infection with M. a. hominissuis and M. a. paratuberculosis was evident. Typing of M. a. avium and M. a. hominissuis isolates was performed by polymerase chain reaction (PCR) detection of insertion sequence IS901 and the virulence-associated macrophage-induced gene (mig), inverted repeat (IR) typing (IS1245/IS1311), and random amplified polymorph DNA (RAPD) analysis. While all M. a. avium and M. a. hominissuis contained the mig gene, IS901 was detected only in M. a. avium. The prevalence of IS901-positive isolates correlated well with the geographic location of affected animals. The IS901-containing isolates were shown to be genotypically closely related, as they exhibit similar patterns in IR-typing and in RAPD analysis. In contrast, IS901-negative isolates (M. a. hominissuis) displayed distinct profiles in both molecular systems.     

Characterisation of IS901 integration sites in the Mycobacterium avium genome

Data are presented on the identification and characterisation of 17 chromosomal integration loci of the insertion element IS901 in the Mycobacterium avium (cervine strain JD88/118) genome. Thirteen of these integration loci have been mapped to their corresponding positions on the M. avium strain 104 (an IS901(-) strain) genome (The Institute for Genome Research (TIGR) unfinished genome-sequencing project). Sequence data for both upstream and downstream sequence flanking regions were obtained for 12 insertion loci, while upstream sequence was obtained for five others. A consensus IS901 insertion target sequence compiled from all 17 integration sites was in broad agreement with earlier reports that were based on only two such loci. Analysis of IS901 integration site flanking sequences revealed that, like IS900 in M. avium subspecies paratuberculosis, IS901 inserts preferentially between a putative ribosome-binding sequence (RBS) and the translational start codon of an open reading frame (ORF). In BLAST X and BLAST P searches of the GenBank database, these ORFs were shown to share significant homologies with a number of other prokaryotic genes.     

Isolation and diagnostic potential of ISMav2, a novel insertion sequence-like element from Mycobacterium avium subspecies paratuberculosis

A novel Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis) specific insertion sequence has been identified by representational difference analysis and designated as ISMav2. ISMav2 has no similarity to known mycobacterial IS elements but shows more than 50% identity to a non-composite transposon of Streptomyces coelicolor at the DNA and protein level. ISMav2 is present in at least three copies on the genome as assessed by Southern blot analysis and its potential value as a diagnostic tool was confirmed by PCR analyses on 79 M. paratuberculosis field isolates, nine M. avium ssp. avium isolates, and the reference strains of nine other mycobacterial species.     

Characterization of IS666, a newly described insertion element of Mycobacterium avium

The insertion sequence IS666 was isolated from Mycobacterium avium strain 101. IS666 is a 1474 bp insertion sequence belonging to the IS256 family, that includes IS6120 from Mycobacterium smegmatis, IS1166 and IS1295 from Rhodococcus sp. IGTS8, IST2 from Thiobacillus ferrooxidans, IS256 from Staphylococcus aureus, and ISRm3 from Rhizobium meliloti. IS666 has 24 bp imperfect inverted repeats that fit the consensus described for the family, and generates 9 bp duplications upon insertion into the host DNA with no apparent specificity in the target sequence. In contrast with its two closest homologues, IS1166 and IS6120, IS666 contains a single ORF that would codify a transposase of 434 aa. IS666 is restricted to M. avium, where it is present in 21% of the isolates in a number ranging between 1 to 7 copies.     

A standardised restriction fragment length polymorphism (RFLP) method for typing Mycobacterium avium isolates links IS901 with virulence for birds

A standardised method for PvuII-PstI-IS901 restriction fragment length polymorphism (RFLP) typing was developed and evaluated against 173 isolates of Mycobacterium avium subsp. avium and M. avium subsp. silvaticum originating from birds (N=46) and their aviaries (N=5), pigs (N=85), cattle (N=18), reference serotype strains (N=9), humans (N=7), a horse (N=1), a nutria (N=1), and strain M. avium subsp. avium ST 18 (formerly M. avium subsp. paratuberculosis ST 18). PvuII-IS1245 RFLP typing was also performed on all isolates. DNA was digested in parallel by restriction endonucleases PvuII or PstI and hybridised to standard probes prepared by PCR. DNA fingerprints were scanned by CCD camera and analysed by the Gel Compar (Applied Maths, Version 4.1, Kortrijk, Belgium) software using a standard isolate control profile. A total of 52 PvuII-PstI RFLP profiles was described including 25 PvuII RFLP profiles designated A to Y and 25 PstI RFLP profiles designated A1-L3. Profiles were found to be stable in vivo and in vitro after multiple subcultures. High IS901 copy number was associated with a "bird" PvuII-IS1245 RFLP profile and low IS901 copy number with M. avium subsp. avium isolates from humans and the nutria. A virulence assay of 100 IS901-positive isolates using intramuscular infections of pullets showed 83 isolates differentiated into 32 RFLP types to be virulent and 17 isolates differentiated into 12 RFLP types as nonvirulent. Attenuation of virulence for pullets could be attributed to either multiple in vitro subculture, polyclonal infection or human passage and was not related to IS901 or IS1245 profiles.     

An IS900-like sequence found in a Mycobacterium sp. other than Mycobacterium avium subsp. paratuberculosis

The insertion sequence IS900 has been considered specific for Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) and has, therefore, been used as the target gene for diagnostic PCR of M. paratuberculosis. From a healthy dairy cow we have isolated and characterised a mycobacterium harbouring one copy of a sequence with 94% identity to IS900 at the nucleic acid level. The isolate was shown to be related to Mycobacterium cookii, as assessed by 16S rRNA sequencing. Strong amplifications were obtained with several PCR primers described for detection of IS900. This finding shows the need of alternative PCR systems based on other genes than IS900 to confirm the presence of M. paratuberculosis.     

Characterization of a Mycobacterium bovis BCG insertion sequence related to the IS21 family

The structure and distribution of a Mycobacterium bovis BCG insertion element of the IS21 family were investigated. Several IS21-like elements found in mycobacterial genomes were separated in four types, following their nucleic acid similarities. The M. bovis BCG IS21 element is highly similar to IS1533 (class I), 70% similar to IS1534 (class II), 52% similar to IS1532 (class III) of Mycobacterium tuberculosis, and 54% similar to both an Mycobacterium avium serovar 2 and an M. avium silvaticum IS (class IV). The M. bovis BCG IS21 element of the class I appears to be present in a single copy in the genome of M. bovis BCG, M. bovis, M. tuberculosis and Mycobacterium africanum and to be absent from all other tested species of the Corynebacteria-Mycobacteria-Nocardia group.     

Characterization of IS 1110, a highly mobiie genetic element from Mycobacterium avium

A highly mobile Insertion sequence designated IS 1110 was detected in Mycobacterium avium strain LR541 following an observed increase in size of the plasmid pLR20. Genomic libraries of M. avium strains carrying either parental pLR20 or the modified plasmid (pLR20′) were constructed and the sequence of the relevant clones was determined to characterize the insertion sequence and the target region. IS 1110 is a 1457 bp element lacking terminal inverted repeats, and is related to IS900 (from Mycobacterium paratuberculosis), IS901 and IS902 (from M. avium) and to IS116 (from Streptomyces clavuligerus). LR541 carries several copies of IS 1110. Individual colonies from the same plate show differences in Southern blot patterns when tested with an IS1110-derived probe; the ability to detect transposition events in random colonies, without any selection pressure, indicates an exceptionally high degree of mobility, which will be invaluable for transposon mutagenesis. Analyses of M. avium isolates from human, veterinary, and environmental sources showed that IS1110-hybridizing sequences are present in some M. avium isolates but they were not detected in strains of other mycobacterial species. The polymorphism exhibited In M. avium isolates suggests that this element may be useful for molecular epidemiological studies of M. avium infections.     

Bicarbonate gradients modulate growth and colony morphology in Aspergillus nidulans

A new insertion sequence (IS), IS 1642 , was identified in a Mycobacterium avium strain isolated from a human patient. IS 1642 had a size of 1642 bp and contained a single ORF encoding a probable transposase of 503 amino acid residues homologous (79% identity) to that of IS 1549 found in Mycobacterium smegmatis . The IS 1642 included imperfect inverted repeats (5'-cctgacttttatca-3', 5'-tgataaaagtcggg-3') on its ends, and was flanked by direct repeats of variable length ranging from 5 to 161 bp. It was suggested that the IS 1642 was widely distributed in many M. avium strains of human patients, and the Southern blot profile of IS 1642 was very diverse among the strains examined. The transposition event of IS 1642 was observed by in vitro repeated passages, showing that the IS 1642 is actually a transposable element. In light of these characteristics, IS 1642 could be a new useful marker when genotyping with high discrimination is required.     

Characterization of IS1001, an insertion sequence element of Bordetella parapertussis.

By analysis of repetitive DNA in Bordetella parapertussis, an insertion sequence element, designated IS1001, was identified. Sequence analysis revealed that IS1001 comprised 1,306 bp and contained inverted repeats at its termini. Furthermore, several open reading frames that may code for transposition functions were identified. The largest open reading frame coded for a protein comprising 406 amino acid residues and showed homology to TnpA, which is encoded by an insertion sequence element (IS1096) found in Mycobacterium smegmatis. Examination of flanking sequences revealed that insertion of IS1001 occurs preferentially in stretches of T's or A's and results in a duplication of target sequences of 6 to 8 bases. IS1001 was found in about 20 copies in 10 B. parapertussis strains analyzed. No restriction fragment length polymorphism was observed in B. parapertussis when IS1001 was used as a probe. An insertion sequence element similar or identical to IS1001 was found in B. bronchiseptica strains isolated from pigs and a rabbit. In these strains, about five copies of the IS1001-like element were present at different positions in the bacterial chromosome. Neither B. pertussis nor B. bronchiseptica strains isolated from humans and dogs contained an IS1001-like element. Therefore, IS1001 may be used as a specific probe for the detection of B. parapertussis in human clinical samples.     

Isolation and analysis of IS6120, a new insertion sequence from Mycobacterium smegmatis

Insertion sequence IS6120 from Mycobacterium smegmatis was identified by its ability to transpose into different sites in the lambda repressor gene, cl857, carried on an Escherichia coli/mycobacteria shuttle plasmid. IS6120 is a novel 1.5 kb insertion sequence, which has 24-bp imperfect terminal inverted repeats and generates 9-bp duplications of the target DNA following insertion. IS6120 is present in at least three copies in M. smegmatis but was not found in other species, including Mycobacterium tuberculosis. Nucleotide sequence analysis revealed that IS6120 contains two open reading frames, one of which encodes a putative transposase with similarities to those found in IS256 from Staphylococcus aureus, IST2 from Thiobacillus ferrooxidans, and ISRm3 from Rhizobium meliloti. The fact that IS6120 does not recognize a consensus target sequence for insertion and has no homologous sequences in the other strains studied makes IS6120 useful for transposon mutagenesis in mycobacteria.     

IS150: distribution, nucleotide sequence and phylogenetic relationships of a new E. coli insertion element.

Recently we identified the new insertion (IS) sequence IS150 in various strains of Escherichia coli K-12. We have screened other strains of E. coli and Salmonella typhimurium for the presence of homologous sequences. The strains of E. coli K-12 and W tested contain one or more copies of homology to IS150. We have also determined the complete nucleotide sequence of a copy of IS150 inserted into IS1. Comparison of nucleotide and deduced amino acid sequences of IS150, IS2, IS3, IS51, IS600 and IS629 reveals significant homologies suggesting that these elements are members of a family of phylogenetically related insertion sequences.     

IS902, an insertion element of the chronic-enteritis-causing Mycobacterium avium subsp. silvaticum.

An insertion sequence element of Mycobacterium avium subsp. silvaticum was isolated and its complete nucleotide sequence determined. IS902 is 1470 bp in size and is repeated 10-12 times per genome. An open reading frame of 1200 bp was identified, encoding a protein product of Mr 43932. This protein is highly similar to the predicted proteins of IS900 of Mycobacterium paratuberculosis, IS116 of Streptomyces clavuligerus and IS110 of Streptomyces coelicolor. IS902 lacks terminal inverted repeats and flanking direct repeats but displays insertion site specificity.     

A genetic mechanism for deletion of the ser2 gene cluster and formation of rough morphological variants of Mycobacterium avium

A major phenotypic trait of the Mycobacterium avium complex is the ability to produce rough and smooth colony variants. The chemical basis of this morphological variation is the loss of an antigenic surface structure, termed glycopeptidolipid (GPL), by rough variants. Using M. avium serovar 2 strain 2151 as a model system, this laboratory previously reported that rough variants arise via the deletion of large genomic regions encoding GPL biosynthesis. One such deletion encompasses the gene cluster (ser2) responsible for production of the serovar 2 GPL haptenic oligosaccharide. In this study, nucleotide sequencing revealed that both ends of the ser2 gene cluster are flanked by a novel insertion sequence (IS1601) oriented as direct repeats. Detailed analyses of the site of deletion in the genome of M. avium 2151 Rg-1 demonstrated that a single copy of IS1601 remained and that the ser2 gene cluster was deleted by homologous recombination. This same deletion pattern was observed for 10 out of 15 rough colony variants tested. Additionally, these studies revealed that IS1601 contains portions of three independent insertion sequences. This report is the first to define the precise genetic basis of colony variation in Mycobacterium spp. and provides further evidence that homologous recombination between insertion sequence elements can be a primary determinant of genome plasticity in these bacteria.     

Identification of an insertion sequence, IS1081, in Mycobacterium bovis

Abstract: An insertion sequence, IS1081, in the genome of Mycobacterium bovis has been identified and sequenced. It is 1324 bp long with 15 bp inverted repeat ends and contains a large ORF. There are six copies of IS1081 in the genome of M. bovis and the element is also present in Mycobacterium tuberculosis . IS1081 is not closely related to other DNA elements described in actinomycetes but its putative transposase bears some resemblance to that of IS256 from Staphylococcus aureus . IS1081 may be useful for genetic manipulations and for developing a diagnostic test for bovine tuberculosis based on the polymerase chain reaction.     

Identification of an insertion sequence, IS1081, in Mycobacterium bovis.

An insertion sequence, IS1081, in the genome of Mycobacterium bovis has been identified and sequenced. It is 1324 bp long with 15 bp inverted repeat ends and contains a large ORF. There are six copies of IS1081 in the genome of M. bovis and the element is also present in Mycobacterium tuberculosis. IS1081 is not closely related to other DNA elements described in actinomycetes but its putative transposase bears some resemblance to that of IS256 from Staphylococcus aureus. IS1081 may be useful for genetic manipulations and for developing a diagnostic test for bovine tuberculosis based on the polymerase chain reaction.     

Mycobacterium avium subsp. paratuberculosis in the catchment area and water of the River Taff in South Wales, United Kingdom, and its potential relationship to clustering of Crohn's disease cases in the city of Cardiff

In South Wales, United Kingdom, a populated coastal region lies beneath hill pastures grazed by livestock in which Mycobacterium avium subsp. paratuberculosis is endemic. The Taff is a spate river running off the hills and through the principal city of Cardiff. We sampled Taff water above Cardiff twice weekly from November 2001 to November 2002. M. avium subsp. paratuberculosis was detected by IS900 PCR and culture. Thirty-one of 96 daily samples (32.3%) were IS900 PCR positive, and 12 grew M. avium subsp. paratuberculosis bovine strains. Amplicon sequences from colonies were identical to the sequence with GenBank accession no. X16293, whereas 16 of 19 sequences from river water DNA extracts had a single-nucleotide polymorphism at position 214. This is consistent with a different strain of M. avium subsp. paratuberculosis in the river, which is unculturable by the methods we used. Parallel studies showed that M. avium subsp. paratuberculosis remained culturable in lake water microcosms for 632 days and persisted to 841 days. Of four reservoirs controlling the catchment area of the Taff, M. avium subsp. paratuberculosis was present in surface sediments from three and in sediment cores from two, consistent with deposition over at least 50 years. Previous epidemiological research in Cardiff demonstrated a highly significant increase of Crohn's disease in 11 districts. These bordered the river except for a gap on the windward side. A topographical relief map shows that this gap is directly opposite a valley open to the prevailing southwesterly winds. This would influence the distribution of aerosols carrying M. avium subsp. paratuberculosis from the river.     

Identification, DNA sequence, and distribution of IS981, a new, high-copy-number insertion sequence in lactococci.

An insertion in the lactococcal plasmid pGBK17, which inactivated the gene(s) encoding resistance to the prolate-headed phage c2, was cloned, sequenced, and identified as a new lactococcal insertion sequence (IS). IS981 was 1,222 bp in size and contained two open reading frames, one large enough to encode a transposase. IS981 ended in imperfect inverted repeats of 26 of 40 bp and generated a 5-bp direct repeat of target DNA at the site of insertion. IS981 was present on the chromosome of Lactococcus lactis subsp. lactis LM0230 from where it transposed to pGBK17 during transformation. Twenty-three strains of lactococci examined for the presence of IS981 by Southern hybridization showed 4 to 26 copies per genome, with L. lactis subsp. cremoris strains containing the highest number of copies. Comparison of the DNA sequence and the amino acid sequence of the long open reading frame to other known sequences showed that IS981 is related to a family of IS elements that includes IS2, IS3, IS51, IS150, IS600, IS629, IS861, IS904, and ISL1.