Tn3-like Transposon Research Articles

The Macrolide Resistance Genes erm (B) and mef (E) Are Carried by Tn 2010 in Dual-Gene Streptococcus pneumoniae Isolates Belonging to Clonal Complex CC271

The genetic elements carrying macrolide resistance genes in Streptococcus pneumoniae isolates belonging to CC271 were investigated. The international clone Taiwan(19F)-14 was found to carry Tn2009, a Tn916-like transposon containing tet(M) and mef(E). The dual erm(B) mef(E) isolates carried Tn2010, which is similar to Tn2009 with the addition of a putative new transposon, the erm(B) genetic element.

A preliminary study investigating the survival of tetracycline resistant Enterococcus faecalis after root canal irrigation with high concentrations of tetracycline

To compare the ability of two Enterococcus faecalis strains to survive exposure to an irrigation solution containing a high concentration of tetracycline in the root canals of bovine teeth. The root canals of twelve bovine incisor root sections were chemo-mechanically prepared using commercially available drills, sodium hypochlorite and ethylenediamine tetra-acetic acid. The root sections were divided into two groups and inoculated with either a tetracycline sensitive or resistant strain of E. faecalis. The strains are isogenic, however one contains a conjugative transposon related to Tn916 which confers resistance to tetracycline, and the other strain is sensitive to the antibiotic. After 26 days of incubation the root canals were irrigated using one of three solutions (sterile distilled water, 50% ethanol or tetracycline at a concentration of 30 mg mL(-1)). The roots were sampled by grinding dentine and canal contents and the debris collected were incubated in broth to assess growth. Irrigation with sterile distilled water or 50% ethanol did not remove all of the cells present. The tetracycline containing solution was efficient in preventing any growth of sensitive E. faecalis, however the resistant strain was able to survive a 5 min exposure at 30 mg mL(-1). The presence of the Tn916-like conjugative transposon containing the tetracycline resistance gene tet(M) allowed an E. faecalis strain to survive irrigation using a solution containing an extremely high concentration of tetracycline in a root canal model.

Novel Macrolide Resistance Module Carried by the IncP-1β Resistance Plasmid pRSB111, Isolated from a Wastewater Treatment Plant

The macrolide resistance plasmid pRSB111 was isolated from bacteria residing in the final effluents of a wastewater treatment plant. The 47-kb plasmid confers resistance to azithromycin, clarithromycin, erythromycin, roxithromycin, and tylosin when it is carried by Pseudomonas sp. strain B13 and is very similar to prototype IncP-1beta plasmid pB3, which was previously isolated from an activated-sludge bacterial community of a wastewater treatment plant. The two plasmids differ in their accessory regions, located downstream of the conjugative transfer module gene traC. Nucleotide sequence analysis of the pRSB111 accessory region revealed that it contains a new macrolide resistance module composed of the genes mphR(E), mph(E), and mrx(E), which putatively encode a transcriptional regulator, a macrolide phosphotransferase, and a transmembrane transport protein, respectively. Analysis of the contributions of the individual genes of the macrolide resistance module revealed that mph(E) and mrx(E) are required for high-level macrolide resistance. The resistance genes are flanked by two insertion sequences, namely, ISPa15 and ISRSB111. Two truncated transposable elements, IS6100 and remnants of a Tn3-like transposon, were identified in the vicinity of ISRSB111. The accessory element of pRSB111 apparently replaced the Tn402-like element present on the sister plasmid, pB3, as suggested by the conservation of Tn402-specific terminal inverted repeats on pRSB111.

Class 1 and class 2 integrons in non-prevalent serovars of Salmonella enterica: structure and association with transposons and plasmids

To characterize class 1 and class 2 integrons which were simultaneously detected in non-typhoid Salmonella enterica strains of non-prevalent serovars, and to investigate their possible association with transposons and/or plasmids. Eight multidrug-resistant S. enterica strains belonging to serovars Virchow (4), Panama (2), Grumpensis (1) and Worthington (1), each containing a class 1 and a class 2 integron, were analysed. Nested PCR amplification was used to determine the gene-cassette configuration of the integrons. Overlapping PCR amplifications were applied in integron-transposon linkage experiments. Conjugation and hybridization experiments were used to localize integrons and transposons in the bacterial genome (plasmid and chromosome associated). One of two different class 1 integrons (with variable regions of 1000 bp/aadA1 and 2300 bp/sat-smr-aadA1) inserted into Tn21-like transposons, were found to coexist with the class 2 integron (2300 bp/dfrA1-sat1-aadA1) of Tn7 in the analysed strains. Class 1 integrons were always found in large conjugative plasmids whereas apparently intact or defective copies of the Tn7 integron could be located on the same plasmid and/or the bacterial chromosome. This report describes different associations between mobile genetic elements that play a crucial role in the capture and spread of antimicrobial drug resistance. As far as we are aware, this is the first description of class 2 integrons in serovars Panama, Grumpensis and Worthington.

Identification of the plasmid-borne quinolone resistance gene qnrS in Salmonella enterica serovar Infantis

A Salmonella enterica serovar Infantis isolate of avian origin was investigated for the presence of the gene qnrS, its transferability and its association with other resistance genes. The Salmonella Infantis isolate was investigated for its susceptibility to antimicrobial agents and its plasmid content. Hybridization experiments and PCR assays were performed to identify the resistance genes while transformation and conjugation studies were conducted to show their transferability. The quinolone resistance-determining regions of the genes gyrA, gyrB, parC and parE were sequenced. Moreover, extended sequence analysis was performed to gain insight into the structure and organization of the qnrS gene area. The Salmonella Infantis isolate exhibited the Asp87-->Tyr87 mutation in gyrA, but no resistance-mediating mutations in the other target genes. It carried a conjugative plasmid, pINF5, on which a qnrS gene was detected in close proximity to a Tn3-like, blaTEM-1-carrying transposon. Homology to the qnrS-carrying plasmid pAH0376 of Shigella flexneri was limited to the Tn3-qnrS region. Sequence analysis of an approximately 13.4 kb region of pINF5 identified truncated insertion sequences of types IS26 and IS2 as well as an internal segment of the CS12 fimbrial gene cluster of Escherichia coli up- and downstream of the qnrS gene. This is to the best of our knowledge the first report of a qnrS gene in a Salmonella isolate. The analysis of the regions flanking the qnrS gene suggested that this region developed in multiple steps that included not only the integration of insertion sequences and a Tn3-like transposon, but also interplasmid recombination events.

Identification of Tn5397-like and Tn916-like transposons and diversity of the tetracycline resistance gene tet(M) in enterococci from humans, pigs and poultry

To analyse the sequence diversity of the tetracycline resistance gene tet(M) and its location on mobile elements in Enterococcus faecium and Enterococcus faecalis from humans, pigs and poultry in Denmark. A total of 76 isolates were screened for Tn916/Tn1545-like and Tn5397-like transposons using PCR. tet(M) was sequenced in 15 of the isolates and compared with tet(M) sequences submitted to GenBank (phylogenetic analysis and signs of recombination). Plasmids were extracted, filter-mating experiments were performed and Tn5397-like transposons were further characterized in selected isolates. In 8 of 13 isolates of E. faecium from broilers, tet(M) was present on Tn5397-like transposons, whereas tet(M) was predominantly associated with Tn916/Tn1545-like transposons in E. faecium from pigs and humans, as well as in E. faecalis from humans, pigs and broilers (50 of 63 isolates). The tet(M) genes were divided into three major subgroups according to the phylogenetic analysis. Subgroup I consisted of tet(M) from Clostridium difficile and E. faecium associated with Tn5397-like elements, subgroup II consisted of tet(M) located on Tn916/Tn1545 family transposons and subgroup III consisted of tet(M) associated with composite elements containing several resistance genes. We found evidence of recombination both within and between these groups. Moreover, we identified an E. faecium isolate with both Tn916/Tn1545-like and Tn5397-like elements. This study showed that enterococci contain diverse tet(M) genes present on different mobile elements, which may suggest that enterococci play an important role in the evolution and horizontal spread of mobile elements carrying tet(M). This is the first report of Tn5397-like elements in enterococci.

Macrolide inactivation gene cluster mphA-mrx-mphR adjacent to a class 1 integron in Aeromonas hydrophila isolated from a diarrhoeic pig in Oklahoma

To characterize a multidrug-resistant Aeromonas hydrophila isolate (CVM861) that possesses a high-level macrolide inactivation gene cluster (mphA-mrx-mphR), previously only reported in Escherichia coli. PCR fragment length mapping, gene sequencing and Southern blotting were used to map the mphA-mrx-mphR gene cluster and flanking elements in CVM861. Conjugation experiments were done to determine whether the multidrug resistance genetic element was mobile. The mphA-mrx-mphR gene cluster mapped downstream of a class 1 integron and upstream of an aph(3') gene, and was present on a Tn21-like element. The gene order determined by sequencing was intI1-dhfrXII-orfF-aadA2-qacDeltaE-sul1-orf5Delta178-tnpA-mphR-mrx-mphA. Horizontal transmission of high-level macrolide resistance from CVM861 to E. coli 47011 was inconsistent; however, a composite plasmid possessing the mphA gene cluster was transferred at a conjugation frequency of 2.02 x 10(-5) per recipient. An mphA-mrx-mphR gene cluster was present downstream of the In2 integron located on a Tn21-like transposon in an A. hydrophila isolate. Whether this recombination event resulted in the truncation of the orf5 sequence is unknown. The presence of other resistance genes downstream of the mphA-mrx-mphR gene cluster suggests that multiple recombination events have occurred on this genetic element. This is the first known report of the mphA-mrx-mphR gene cluster carried by A. hydrophila and the first known isolation of this cluster in the United States.

Molecular Characterization of Pneumococci with Efflux-Mediated Erythromycin Resistance and Identification of a Novel mef Gene Subclass, mef (I)

The molecular genetics of macrolide resistance were analyzed in 49 clinical pneumococci (including an "atypical" bile-insoluble strain currently assigned to the new species Streptococcus pseudopneumoniae) with efflux-mediated erythromycin resistance (M phenotype). All test strains had the mef gene, identified as mef(A) in 30 isolates and mef(E) in 19 isolates (including the S. pseudopneumoniae strain) on the basis of PCR-restriction fragment length polymorphism analysis. Twenty-eight of the 30 mef(A) isolates shared a pulsed-field gel electrophoresis (PFGE) type corresponding to the England14-9 clone. Of those isolates, 27 (20 belonging to serotype 14) yielded multilocus sequence type ST9, and one isolate yielded a new sequence type. The remaining two mef(A) isolates had different PFGE types and yielded an ST9 type and a new sequence type. Far greater heterogeneity was displayed by the 19 mef(E) isolates, which fell into 11 PFGE types, 12 serotypes (though not serotype 14), and 12 sequence types (including two new ones and an undetermined type for the S. pseudopneumoniae strain). In all mef(A) pneumococci, the mef element was a regular Tn1207.1 transposon, whereas of the mef(E) isolates, 17 carried the mega element and 2 exhibited a previously unreported organization, with no PCR evidence of the other open reading frames of mega. The mef gene of these two isolates, which did not match with the mef(E) gene of the mega element (93.6% homology) and which exhibited comparable homology (91.4%) to the mef(A) gene of the Tn1207.1 transposon, was identified as a novel mef gene variant and was designated mef(I). While penicillin-nonsusceptible isolates (three resistant isolates and one intermediate isolate) were all mef(E) strains, tetracycline resistance was also detected in three mef(A) isolates, due to the tet(M) gene carried by a Tn916-like transposon. A similar mechanism accounted for resistance in four of the five tetracycline-resistant isolates carrying mef(E), in three of which mega was inserted in the Tn916-like transposon, giving rise to the composite element Tn2009. In the fifth mef(E)-positive tetracycline-resistant isolate (the S. pseudopneumoniae strain), tetracycline resistance was due to the presence of the tet(O) gene, apparently unlinked to mef(E).

Novel Rearrangement of a Class 2 Integron in Two Non-Epidemiologically Related Isolates of Acinetobacter baumannii

Tn7::In2-8 contains sat2-aadB-catB2(deltaattC)-dfrA1-sat2-aadA1-orfX in the variable region of a class 2 integron embedded in the Tn7-like transposon. This novel transposon was inserted in its preferred site downstream of the glms gene in Acinetobacter baumannii. Acquisition of the pseudocassette catB2 could have arisen by a secondary-site integrase-mediated intermolecular recombination event.

An unusual Tn21-like transposon containing an ars operon is present in highly arsenic-resistant strains of the biomining bacterium Acidithiobacillus caldus

A transposon, TnAtcArs, that carries a set of arsenic-resistance genes was isolated from a strain of the moderately thermophilic, sulfur-oxidizing, biomining bacterium Acidithiobacillus caldus. This strain originated from a commercial plant used for the bio-oxidation of gold-bearing arsenopyrite concentrates. Continuous selection for arsenic resistance over many years had made the bacterium resistant to high concentrations of arsenic. Sequence analysis indicated that TnAtcArs is 12 444 bp in length and has 40 bp terminal inverted repeat sequences and divergently transcribed resolvase and transposase genes that are related to the Tn21-transposon subfamily. A series of genes consisting of arsR, two tandem copies of arsA and arsD, two ORFs (7 and 8) and arsB is situated between the resolvase and transposase genes. Although some commercial strains of At. caldus contained the arsDA duplication, when transformed into Escherichia coli, the arsDA duplication was unstable and was frequently lost during cultivation or if a plasmid containing TnAtcArs was conjugated into a recipient strain. TnAtcArs conferred resistance to arsenite and arsenate upon E. coli cells. Deletion of one copy of arsDA had no noticeable effect on resistance to arsenite or arsenate in E. coli. ORFs 7 and 8 had clear sequence similarity to an NADH oxidase and a CBS-domain-containing protein, respectively, but their deletion did not affect resistance to arsenite or arsenate in E. coli. TnAtcArs was actively transposed in E. coli, but no increase in transposition frequency in the presence of arsenic was detected. Northern hybridization and reporter gene studies indicated that although ArsR regulated the 10 kb operon containing the arsenic-resistance genes in response to arsenic, ArsR had no effect on the regulation of genes associated with transposition activity.

The Gene Encoding the Glutamate Dehydrogenase in Lactococcus lactis Is Part of a Remnant Tn 3 Transposon Carried by a Large Plasmid

The gene responsible for the uncommon glutamate dehydrogenase (GDH) activity of Lactococcus lactis was identified and characterized. It encodes a GDH of family I that is mainly active in glutamate biosynthesis, is carried by a large plasmid, and is included, with functional cadmium resistance genes, in a remnant Tn3-like transposon.

Physical linkage of Tn3 and part of Tn1721 in a tetracycline and ampicillin resistance plasmid from Salmonella Typhimurium

The complete nucleotide sequence of the 12 656 bp plasmid pFPTB1 from Salmonella enterica subsp. enterica serovar Typhimurium, which mediates resistance to tetracyclines and ampicillin, was determined. The plasmid was analysed for potential reading frames and structural features indicative of transposons and transposon relics. Plasmid pFPTB1 was transformed into Escherichia coli JM109, overlapping restriction fragments were cloned into E. coli plasmid vectors and sequenced. In vitro susceptibility testing was carried out to confirm the resistance phenotype mediated by this plasmid. Plasmid pFPTB1 contains a complete Tn3-like transposon of 4950 bp consisting of the left terminal repeat, Tn3-related tnpR and tnpA genes for transposition functions, a novel gene for ampicillin resistance bla(TEM-135), and the right terminal repeat. Immediately downstream, the terminal 5215 bp at the right end of a Tn1721-like transposon, including the right terminal repeat, a truncated transposase gene, as well as the genes tet(A) and tetR for tetracycline resistance, were detected. A 5 bp direct repeat, TAAAA, was seen immediately upstream of the Tn3 part and immediately downstream of the Tn1721 part. Plasmid pFPTB1 also carries a replication region similar to that of the Klebsiella pneumoniae plasmid pJHCMW1. Plasmid pFPTB1 is one of the few completely sequenced plasmids from S. Typhimurium and harbours a novel transposon-like structure consisting of a Tn3-related part containing the bla(TEM-135) gene for ampicillin resistance and a Tn1721-related part containing the tetR-tet(A) genes for tetracycline resistance.

Multiple sets of arsenic resistance genes are present within highly arsenic-resistant industrial strains of the biomining bacterium, Acidithiobacillus caldus

The moderately thermophilic, sulfur-oxidizing bacterium Acidithiobacillus caldusis an important role player in continuous-flow biooxidation processes associated with the biooxidation of arsenopyrite concentrates. Certain isolates of these bacteria have been exposed to high concentrations of arsenic for many years and have become highly arsenic-resistant. We investigated the arsenic resistance genes of the highly resistant strains of At. caldus and compared these with those of less arsenic-resistant isolates. Up to three sets of arsenic resistance genes were found in the most resistant strains. One set of genes was located on a 12-kb Tn 21-like transposon flanked by Tn 21-like 40-bp inverted repeats (IR) and the transposon was active in the bacterium, Escherichia coli. This transposon was present only in highly arsenic-resistant strains and contained an unusual ars operon with arsR, arsC, two arsD-like, two arsA-like and arsB genes. Situated between the arsA and arsB genes are two open reading frames encoding for NADH oxidase-like enzyme and a protein with a CBS domain that is also present in IMP dehydrogenases. Deletion of one copy of the arsDA genes occurred readily but there were no marked differences in arsenic resistance in the deleted clones. The second set of arsenic resistance genes appeared to be related to the first set although some of the genes were missing. The third set was distantly related and appeared to be present on the chromosomes of all At. caldus strains irrespective of whether they had been exposed to elevated levels of arsenic.

The complete sequences of plasmids pB2 and pB3 provide evidence for a recent ancestor of the IncP-1β group without any accessory genes

The nucleotide sequences of the broad-host-range antibiotic resistance plasmids pB2 (61 kb) and pB3 (56 kb), which were isolated from a wastewater treatment plant, were determined and analysed. Both have a nearly identical IncP-1beta backbone, which diverged early from the sequenced IncP-1beta plasmids R751, pB10, pJP4, pADP1 and pUO1. In contrast to the latter plasmids, the pB2 and pB3 backbone does not seem to have undergone any deletions. The complete partition gene parA is located downstream of the mating pair formation (trb) module. A 14.4 kb or 19.0 kb mobile genetic element is present between traC and parA of pB3 and pB2, respectively. This region is typical for insertions in IncP-1beta plasmids, but the insertion site is unique. Both elements differ only by a duplication in pB2 of a tetA(C)-tetR-tnpA(IS26) fragment. The 5 bp target site duplication and the 26 bp inverted repeats flanking the mobile genetic elements are still intact, indicating that the insertion occurred recently. The element consists of three nested transposable elements: (i) a relict of a Tn402-like transposon with a gene for a new class D beta-lactamase (bla(NPS-2)); (ii) within that, another Tn402-like element with a class 1 integron harbouring the gene cassettes cmlA1 for a chloramphenicol efflux protein and aadA2 encoding a streptomycin/spectinomycin adenylyltransferase, and a copy of IS6100; (iii) into the integrase gene intI1 a tetracycline resistance module tetA(C)-tetR flanked by copies of IS26 is inserted. Interestingly, in contrast to all other IncP-1beta plasmids analysed so far, the oriV region between trfA and klcA is not interrupted by accessory genes, and there is no indication that previously inserted accessory genes have subsequently been deleted. The genes kluAB are also missing in that region and should thus be considered acquired genes. These findings, together with the fact that IncP-1beta plasmids acquired accessory elements at various positions in the backbone, suggest that IncP-1beta plasmids without any accessory genes exist in microbial communities. They must occasionally acquire accessory genes by transposition events, resulting in those plasmids that have been found based on selectable phenotypic traits.

Antibiotic multiresistance plasmid pRSB101 isolated from a wastewater treatment plant is related to plasmids residing in phytopathogenic bacteria and carries eight different resistance determinants including a multidrug transport system

Ten different antibiotic resistance plasmids conferring high-level erythromycin resistance were isolated from an activated sludge bacterial community of a wastewater treatment plant by applying a transformation-based approach. One of these plasmids, designated pRSB101, mediates resistance to tetracycline, erythromycin, roxythromycin, sulfonamides, cephalosporins, spectinomycin, streptomycin, trimethoprim, nalidixic acid and low concentrations of norfloxacin. Plasmid pRSB101 was completely sequenced and annotated. Its size is 47 829 bp. Conserved synteny exists between the pRSB101 replication/partition (rep/par) module and the pXAC33-replicon from the phytopathogen Xanthomonas axonopodis pv. citri. The second pRSB101 backbone module encodes a three-Mob-protein type mobilization (mob) system with homology to that of IncQ-like plasmids. Plasmid pRSB101 is mobilizable with the help of the IncP-1alpha plasmid RP4 providing transfer functions in trans. A 20 kb resistance region on pRSB101 is located within an integron-containing Tn402-like transposon. The variable region of the class 1 integron carries the genes dhfr1 for a dihydrofolate reductase, aadA2 for a spectinomycin/streptomycin adenylyltransferase and bla(TLA-2) for a so far unknown Ambler class A extended spectrum beta-lactamase. The integron-specific 3'-segment (qacEDelta1-sul1-orf5Delta) is connected to a macrolide resistance operon consisting of the genes mph(A) (macrolide 2'-phosphotransferase I), mrx (hydrophobic protein of unknown function) and mphR(A) (regulatory protein). Finally, a putative mobile element with the tetracycline resistance genes tetA (tetracycline efflux pump) and tetR was identified upstream of the Tn402-specific transposase gene tniA. The second 'genetic load' region on pRSB101 harbours four distinct mobile genetic elements, another integron belonging to a new class and footprints of two more transposable elements. A tripartite multidrug (MDR) transporter consisting of an ATP-binding-cassette (ABC)-type ATPase and permease, and an efflux membrane fusion protein (MFP) of the RND-family is encoded between the replication/partition and the mobilization module. Homologues of the macrolide resistance genes mph(A), mrx and mphR(A) were detected on eight other erythromycin resistance-plasmids isolated from activated sludge bacteria. Plasmid pRSB101-like repA amplicons were also obtained from plasmid-DNA preparations of the final effluents of the wastewater treatment plant indicating that pRSB101-like plasmids are released with the final effluents into the environment.

Characterization and localization of drug resistance determinants in multidrug-resistant, integron-carrying Salmonella enterica serotype Typhimurium strains.

The genetic background of the antimicrobial resistance of 10 selected multiresistant Salmonella serotype Typhimurium (S. Typhimurium) strains (including the emerging monophasic variant [4,5,12:i:- ]) was investigated. All strains shared class 1 integrons (with seven types of variable regions) and belonged to different lineages (L1-L6) according to their phage types, DNA polymorphisms by XbaI-pulsed-field gel electrophoresis (PFGE), integrons, and/or resistance patterns. The strains were screened for the presence and localization (chromosomal or plasmid) of 32 DNA sequences representing integron-, Tn21-like transposon-, resistance-, and virulence-plasmid genes. Strains belonging to lineage L1 (definitive phage type DT104) carried the 90-kb Salmonella virulence plasmid together with the complete or partial chromosomally located Salmonella Genomic Island 1 (SGI1). All strains belonging to the other five lineages carried their resistance determinants on various resistance plasmids. Two of these strains showed complex plasmid profiles, which included a 95 kb virulence plasmid together with two or four resistance plasmids. Two strains carried a resistance plasmid that lacked the virulence-plasmid-encoding sequences. The remaining two strains carried two different hybrid virulence-resistance plasmids. Twenty-three of the DNA sequences could be assigned to distinct XbaI genomic restriction patterns (PFGE profiles). In this way, the influence of the resistance and virulence plasmids on the PFGE profiles was determined, and several groups of resistance genes could be identified. The data obtained represent a useful epidemiological tool for tracing the emergence and distribution of multiresistant S. Typhimurium worldwide.

A transposon-located arsenic resistance mechanism from a strain of Acidithiobacillus caldus isolated from commercial, arsenopyrite biooxidation tanks

The moderately thermophilic, sulfur-oxidizing bacterium Acidithiobacillus caldus is an important role player in continuous-flow biooxidation processes associated with biomining. Biooxidation of arsenopyrite concentrates by a combination of Leptospirillum and At. caldus results in the release of soluble arsenic into the surrounding environment. Arsenate and arsenite are toxic to most organisms, yet At. caldus is able to thrive in this harsh environment. The purpose of this study was to identify and investigate arsenic resistance genes in At. caldus and compare its ars operon with those previously studied in other bacteria. A gene bank of a South African At. caldus strain #6 was constructed and transformed into an Escherichia coli arsenic-sensitive mutant strain to identify clones containing At. caldus arsenic resistance genes. A clone with an insert of 6.4 kb was selected and after two cycles of chromosome-walking a further 5.6 kb of DNA upstream of the original clone was isolated. This region was sequenced and found to have an unusual ars operon with a functional arsB gene, two arsD-like and two arsA-like genes. The arsenic resistance mechanism appears to be located on a Tn 21-like transposon and includes transposase and resolvase-like genes with the entire assemblage being flanked by Tn 21-like 38 bp inverted repeats (IR). Southern hybridization experiments using the arsB gene as a probe against total DNA from six different At. caldus strains indicated that the three South African strains which had been previously exposed to arsenic gave a positive hybridization signal, while three other At. caldus strains (KU, BC13 and C-SH12) did not. Southern hybridization of pulse field gels indicated that the arsB gene was chromosomally encoded. Genes encoding for NADH oxidase-like and IMP dehydrogenase-like enzymes were situated between the arsA and arsB genes. PCR and Southern hybridization experiments revealed that this unusual arrangement was not an artefact of cloning but that the 2.1 kb NADH oxidase-like and IMP dehydrogenase-like gene containing region was present between the arsA and arsB genes in all three South African strains. When transformed into the E. coli arsenic-sensitive mutant, the transposon-located system conferred increased resistance to arsenite, but not to arsenate. This indicated that the NADH oxidase-like gene did not function as an arsenate reductase in E. coli. It is possible that increased exposure to arsenic placed selection pressure on the three South African strains to acquire a Tn 21-like transposon containing an ars operon to enhance their arsenic resistance.

Genetic characterization of a novel metallo-beta-lactamase gene, blaIMP-13, harboured by a novel Tn5051-type transposon disseminating carbapenemase genes in Europe: report from the SENTRY worldwide antimicrobial surveillance programme.

In 2001, as part of the SENTRY worldwide antimicrobial surveillance programme, Pseudomonas aeruginosa 86-14571A was isolated at a hospital in Rome from a cancer patient with a bloodstream infection. The isolate was resistant to all antibiotics except amikacin, and displayed an imipenem MIC of 64 mg/L that decreased to 8 mg/L in the presence of EDTA. The resistance determinant was investigated. The resistant determinant was cloned in Escherichia coli using a shotgun cloning approach. Sequence analysis revealed the presence of a novel IMP-type metallo-beta-lactamase (MBL) gene, blaIMP-13. This encoded a protein displaying most identity to IMP variants: 93% and 92.3% identity, respectively, to IMP-8 and IMP-2 (previously identified in Italy). The protein had 19 amino acid changes from IMP-2 and 17 amino acid changes from IMP-8. The blaIMP-13 gene was found as a gene cassette in the first position of a class 1 integron. A 25 bp inverted repeat sequence IRi was identified 174 bp upstream of the class I integrase, which suggests that the integron is found on a Tn402-like transposon, or defective transposon derivative. This element, in turn, is located in the transposition locus (tnp region) of a Tn21 subfamily transposon that showed most identity to Tn5051, a transposon recently identified from a strain of Pseudomonas putida isolated in New York. Interestingly, the insertion point of the Tn402-like transposon and the sequence of the Tn5051-like genes were identical to those of the genetic element harbouring blaVIM-2 recently identified in Poland. The resistance determinant of P. aeruginosa 86-14571A is a novel IMP-type MBL carried on a composite transposon responsible for wide geographical dissemination of MBL genes in Europe.

Phenotypic and molecular characterization of tetracycline- and erythromycin-resistant strains of Streptococcus pneumoniae.

Sixty-five clinical isolates of Streptococcus pneumoniae, all collected in Italy between 1999 and 2002 and resistant to both tetracycline (MIC, >or=8 microg/ml) and erythromycin (MIC, >or=1 microg/ml), were investigated. Of these strains, 11% were penicillin resistant and 23% were penicillin intermediate. With the use of the erythromycin-clindamycin-rokitamycin triple-disk test, 14 strains were assigned to the constitutive (cMLS) phenotype of macrolide resistance, 44 were assigned to the partially inducible (iMcLS) phenotype, 1 was assigned to the inducible (iMLS) phenotype, and 6 were assigned to the efflux-mediated (M) phenotype. In PCR assays, 64 of the 65 strains were positive for the tetracycline resistance gene tet(M), the exception being the one M isolate susceptible to kanamycin, whereas tet(K), tet(L), and tet(O) were never found. All cMLS, iMcLS, and iMLS isolates had the erythromycin resistance gene erm(B), and all M phenotype isolates had the mef(A) or mef(E) gene. No isolate had the erm(A) gene. The int-Tn gene, encoding the integrase of the Tn916-Tn1545 family of conjugative transposons, was detected in 62 of the 65 test strains. Typing assays showed the strains to be to a great extent unrelated. Of 16 different serotypes detected, the most numerous were 23F (n = 13), 19A (n = 10), 19F (n = 9), 6B (n = 8), and 14 (n = 6). Of 49 different pulsed-field gel electrophoresis types identified, the majority (n = 39) were represented by a single isolate, while the most numerous type included five isolates. By high-resolution restriction analysis of PCR amplicons with four endonucleases, the tet(M) loci from the 64 tet(M)-positive pneumococci were classified into seven distinct restriction types. Overall, a Tn1545-like transposon could reasonably account for tetracycline and erythromycin resistance in the vast majority of the pneumococci of cMLS, iMcLS, and iMLS phenotypes, whereas a Tn916-like transposon could account for tetracycline resistance in most M phenotype strains.

Complete characterisation of Tn 5530 from Burkholderia cepacia strain 2a (pIJB1) and studies of 2,4-dichlorophenoxyacetate uptake by the organism

The complete genetic characterisation of Tn 5530 in Burkholderia cepacia strain 2a (pIJB1) has been accomplished, indicating that it is a Tn 3-like transposon with a complex structure bearing operons for the catabolism of 2,4-dichlorophenoxyacetate (2,4-D) and malonate. Tn 5530 is terminated at both ends by the IS 1071::IS 1471 element and the 2,4-D- and malonate-dissimilatory operons are separated by a region encoding a putA and lrp gene and a gene encoding a chloride channel protein. The chloride channel protein may have a role in the expulsion of chloride ions liberated by the dissimilation of 2,4-D. In addition, a putative transposase with a high level of sequence similarity to those of plasmid pGH1 from Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. glycinea, and a transcription factor similar to those of the TetR family with low but significant levels of sequence similarity to those identified in a number of other organisms was observed. The entire Tn 5530 sequence length, including the IS 1071::IS 1471 elements, was found to be 40,956 bp, and pIJB1 was replicon-typed and otherwise characterised as being of the IncP-1β subgroup, bearing merA and merD genes conferring resistance to mercuric chloride. The rate of uptake of 2,4-D by B. cepacia strain 2a was observed to proceed more readily at acid pH, suggesting involvement of the undissociated form of the compound. Uptake did not show saturation kinetics, was concentration-dependent, and appeared to occur in two stages; an initial accumulation followed by a linear second phase. Uptake could be inhibited by sodium azide but not by arsenate, N, N ′-dicyclohexylcarbodi-imide (DCCD) or carbonylcyanide m-chlorophenyl-hydrazone (CCCP) suggesting that it is not energy-dependent.