Non-phenotypic tests to detect and characterize antibiotic resistance mechanisms in Enterobacteriaceae

In the past 2 decades, we have observed a rapid increase of infections due to multidrug-resistant Enterobacteriaceae. Regrettably, these isolates possess genes encoding for extended-spectrum β-lactamases (e.g., blaCTX-M, blaTEM, blaSHV) or plasmid-mediated AmpCs (e.g., blaCMY) that confer resistance to last-generation cephalosporins. Furthermore, other resistance traits against quinolones (e.g., mutations in gyrA and parC, qnr elements) and aminoglycosides (e.g., aminoglycosides modifying enzymes and 16S rRNA methylases) are also frequently co-associated. Even more concerning is the rapid increase of Enterobacteriaceae carrying genes conferring resistance to carbapenems (e.g., blaKPC, blaNDM). Therefore, the spread of these pathogens puts in peril our antibiotic options. Unfortunately, standard microbiological procedures require several days to isolate the responsible pathogen and to provide correct antimicrobial susceptibility test results. This delay impacts the rapid implementation of adequate antimicrobial treatment and infection control countermeasures. Thus, there is emerging interest in the early and more sensitive detection of resistance mechanisms. Modern non-phenotypic tests are promising in this respect, and hence, can influence both clinical outcome and healthcare costs. In this review, we present a summary of the most advanced methods (e.g., next-generation DNA sequencing, multiplex PCRs, real-time PCRs, microarrays, MALDI-TOF MS, and PCR/ESI MS) presently available for the rapid detection of antibiotic resistance genes in Enterobacteriaceae. Taking into account speed, manageability, accuracy, versatility, and costs, the possible settings of application (research, clinic, and epidemiology) of these methods and their superiority against standard phenotypic methods are discussed.

Evolutionary conservation of residues in vertebrate DNA polymerase N conferring low fidelity and bypass activity.

POLN is a nuclear A-family DNA polymerase encoded in vertebrate genomes. POLN has unusual fidelity and DNA lesion bypass properties, including strong strand displacement activity, low fidelity favoring incorporation of T for template G and accurate translesion synthesis past a 5S-thymine glycol (5S-Tg). We searched for conserved features of the polymerase domain that distinguish it from prokaryotic pol I-type DNA polymerases. A Lys residue (679 in human POLN) of particular interest was identified in the conserved 'O-helix' of motif 4 in the fingers sub-domain. The corresponding residue is one of the most important for controlling fidelity of prokaryotic pol I and is a nonpolar Ala or Thr in those enzymes. Kinetic measurements show that K679A or K679T POLN mutant DNA polymerases have full activity on nondamaged templates, but poorly incorporate T opposite template G and do not bypass 5S-Tg efficiently. We also found that a conserved Tyr residue in the same motif not only affects sensitivity to dideoxynucleotides, but also greatly influences enzyme activity, fidelity and bypass. Protein sequence alignment reveals that POLN has three specific insertions in the DNA polymerase domain. The results demonstrate that residues have been strictly retained during evolution that confer unique bypass and fidelity properties on POLN.

ABERRATIONS OF A PUTATIVE TUMOR SUPPRESSOR GENE SEL1L IN PANCREATIC DUCTAL ADENOCARCINOMA

Introduction: Pancreatic cancer is the fourth leading cause of cancer-related death among males and females in the United States. Sel-1-like (SEL1L) is a putative tumor suppressor gene that is downregulated in a significant proportion of human pancreatic ductal adenocarcinoma (PDAC). It was hypothesized that SEL1L expression could be down-modulated by somatic mutation, loss of heterozygosity (LOH), CpG island hypermethylation and/or aberrantly expressed microRNAs (miRNAs). Material and methods: In 42 PDAC tumors, the SEL1L coding region was amplified using reverse transcription polymerase chain reaction (RT-PCR), and analyzed by agarose gel electrophoresis and sequenced to search for mutations. Using fluorescent fragment analysis, two intragenic microsatellites in the SEL1L gene region were examined to detect LOH in a total of 73 pairs of PDAC tumors and normal-appearing adjacent tissues. Bisulfite DNA sequencing was performed to determine the methylation status of the SEL1L promoter in 41 PDAC tumors and 6 PDAC cell lines. Using real-time quantitative PCR, the expression levels of SEL1L mRNA and 7 aberrantly upregulated miRNAs that potentially target SEL1L were assessed in 42 PDAC tumor and normal pairs. Statistical methods were applied to evaluate the correlation between SEL1L mRNA and the miRNAs. Further the interaction was determined by functional analysis using a molecular biological approach. Results: No mutations were detected in the SEL1L coding region. More than 50% of the samples displayed abnormally alternate or aberrant spliced transcripts of SEL1L. About 14.5% of the tumors displayed LOH at the CAR/CAL microsatellite locus and 10.7% at the RepIN20 microsatellite locus. However, the presence of LOH did not show significant association with SEL1L downregulation. No methylation was observed in the SEL1L promoter. Statistical analysis showed that SEL1L mRNA expression levels significantly and inversely correlated with the expression of hsa-mir-143, hsa-mir-155, and hsa-mir-223. Functional analysis indicated that hsa-mir-155 acted as a suppressor of SEL1L in PL18 and MDAPanc3 PDAC cell lines. Discussion: Evidence from these studies suggested that SEL1L was possibly downregulated by aberrantly upregulated miRNAs in PDAC. Future studies should be directed towards developing a better understanding of the mechanisms for generation of aberrant SEL1L transcripts, and further analysis of miRNAs that may downregulate SEL1L.

TP53 as a Biomarker in Head and Neck Squamous Cell Carcinoma

Currently, there are no molecular biomarkers that guide treatment decisions for patients with head and neck squamous cell carcinoma (HNSCC). Several retrospective studies have evaluated TP53 in HNSCC, and results have suggested that specific mutations are associated with poor outcome. However, there exists heterogeneity among these studies in the site and stage of disease of the patients reviewed, the treatments rendered, and methods of evaluating TP53 mutation. Thus, it remains unclear as to which patients and in which clinical settings TP53 mutation is most useful in predicting treatment failure. In the current study, we reviewed the records of a cohort of patients with advanced, resectable HNSCC who received surgery and post-operative radiation (PORT) and had DNA isolated from fresh tumor tissue obtained at the time of surgery. TP53 mutations were identified using Sanger sequencing of exons 2-11 and the associated splice regions of the TP53 gene. We have found that the group of patients with either non-disruptive or disruptive TP53 mutations had decreased overall survival, disease-free survival, and an increased rate of distant metastasis. When examined as an independent factor, disruptive mutation was strongly associated with the development of distant metastasis. As a second aim of this project, we performed a pilot study examining the utility of the AmpliChip® p53 test as a practical method for TP53 sequencing in the clinical setting. AmpliChip® testing and Sanger sequencing was performed on a separate cohort of patients with HNSCC. Our study demonstrated the ablity of the AmpliChip® to call TP53 mutation from a single formalin-fixed paraffin-embedded slide. The results from AmpliChip® testing were identical with the Sanger method in 11 of 19 cases, with a higher rate of mutation calls using the AmpliChip® test. TP53 mutation is a potential prognostic biomarker among patients with advanced, resectable HNSCC treated with surgery and PORT. Whether this subgroup of patients could benefit from the addition of concurrent or induction chemotherapy remains to be evaluated in prospective clinical trials. Our pilot study of the p53 AmpliChip® suggests this could be a practical and reliable method of TP53 analysis in the clinical setting.

Genetic and physical analysis of the bacteriophage P22 tail protein

A plasmid based genetic system was developed for the tail protein of the Salmonella typhimurium bacteriophage P22 and used to isolate and characterize tail protein mutants. The tail protein is a trimeric structural protein of the phage and an endorhamnosidase whose activity is essential for infection. The gene for the tail protein has previously been cloned into a plasmid expression vector and sequenced. A plate complementation assay for tail protein produced from the cloned gene was developed and used to isolate 27 tail protein mutants following mutagenesis of the cloned gene. These mutations were mapped into 12 deletion intervals using deletions which were made on plasmids in vitro and crossed onto P22. The base substitutions were determined by DNA sequencing. The majority of mutants had missense or nonsense mutations in the protein coding portion of the gene; however four of the mutants were in the putative transcription terminator. The oligomeric state of tail protein from the 15 missense mutants was investigated using SDS and nondenaturing polyacrylamide gel electrophoresis of cell lysates. Wild-type tail protein retains its trimeric structure in SDS gels at room temperature. Two of the mutant proteins also migrated as trimers in SDS gels, yet one of these had a considerably faster mobility than wild-type trimer. Its migration was the same as wild-type in a nondenaturing gel, so it is thought to be a trimer which is partially denatured by SDS. Four of the mutants produced proteins which migrate at the position of a monomer in an SDS gel but cannot be seen on a nondenaturing gel. These proteins are thought to be either monomers or soluble aggregates which cannot enter the nondenaturing gel. The remainder of mutants produce protein which is degraded. The mutant tail protein which had normal trimeric mobility on SDS and nondenaturing gels was purified. This protein has essentially wild-type ability to attach to phage capsids, but its endorhamnosidase activity is only 4% of wild-type. ^

Transcription factor SEF: Purification and functional characterization

Cytokine-induced transcription of the serum amyloid A3 (SAA3) gene promoter requires a transcriptional enhancer that contains three functional elements: two C/EBP-binding sites and a third site that interacts with a constitutively expressed transcription factor, SAA3 enhancer factor (SEF). Deletion or site-specific mutations in the SEF-binding site drastically reduced SAA3 promoter activity, strongly suggesting that SEF is important in SAA3 promoter function. To further elucidate its role in the regulation of the SAA3 gene, we purified SEF from HeLa cell nuclear extracts to near homogeneity by using conventional liquid chromatography and DNA-affinity chromatography. Ultraviolet cross-linking and Southwestern experiments indicated that SEF consisted of a single polypeptide with an apparent molecular mass of 65 kDa. Protein sequencing, oligonucleotide competition and antibody supershift experiments identified SEF as transcription factor LBP-1c/CP2/LSF. Cotransfection of SEF expression plasmid with SAA3-luciferase reporter resulted in 3- to 5-fold activation of SAA3 promoter. Interestingly, when SEF-transfected cells were treated with either conditioned medium (CM) or interleukin (IL) 1, the SAA3 promoter was synergistically activated in a dose-dependent manner. Furthermore, when SEF-binding site was mutated, the response of SAA3 promoter to IL-1 or CM stimulation was abolished or drastically decreased, suggesting that SEF may functionally cooperate with an IL-1-inducible transcription factor. Indeed, our functional studies showed that NFκB is a key transcription factor that mediates the IL-1-induced expression of SAA3 gene, and that SEF can synergize with NFκBp65 to activate SAA3 promoter. By coimmunoprecipitation experiments, we found that SEF could specifically interact with NFκBp65, and that the association of these two factors was enhanced upon IL-1 and CM stimulation. This suggests that the molecular basis for the functional synergy between SEF and NFκB may be due to the ability of SEF to physically interact with NPκB. In addition to its interaction with SEF, NFκB-dependent activation also requires the weak κB site in the C element and its interaction with C/EBP. Besides its role in regulating SAA3 gene expression, we provide evidence that SEF could also bind in a sequence-specific manner to the promoters of α2-macroglobulin, Aα fibrinogen, and 6–16 genes and to an intronic enhancer of the human Wilm's tumor 1 gene, suggesting a functional role in the regulation of these genes. By coimmunoprecipitation experiments, we determined that SEF could specifically associate with both Stat3 and Stat2 upon cytokine stimulation. To examine the functional roles of such interactions, we evaluated the effects of SEF on the transcriptional regulation of two reporter genes: Aα fibrinogen and 6–16, which are IL-6- and interferon-α-responsive, respectively. Our results showed that cotransfection of SEF expression plasmid can activate the expression of Aα fibrinogen gene and 6–16 gene. Moreover, SEF can dramatically enhance the interferon-α-induced expression of 6–16 gene and IL-6-induced expression of Aα fibrinogen gene, suggesting that SEF may functionally cooperate with ISGF3 and Stat3 to mediate interferon-α and IL-6 signaling. ^ Our findings that SEF can interact with multiple cytokine-inducible transcription factors to mediate the expression of target genes open a new avenue of investigation of cooperative transcriptional regulation of gene expression, and should further our understanding of differential gene expression in response to a specific stimulus. In summary, our data provide evidence that SEF can mediate the signaling of different cytokines by interacting with various cytokine-inducible transcription factors. ^

Occurrence and Genetic Characteristics of Third-Generation Cephalosporin-Resistant Escherichia coli in Swiss Retail Meat

Prevalence and genetic relatedness were determined for third-generation cephalosporin-resistant Escherichia coli (3GC-R-Ec) detected in Swiss beef, veal, pork, and poultry retail meat. Samples from meat-packing plants (MPPs) processing 70% of the slaughtered animals in Switzerland were purchased at different intervals between April and June 2013 and analyzed. Sixty-nine 3GC-R-Ec isolates were obtained and characterized by microarray, PCR/DNA sequencing, Multi Locus Sequence Typing (MLST), and plasmid replicon typing. Plasmids of selected strains were transformed by electroporation into E. coli TOP10 cells and analyzed by plasmid MLST. The prevalence of 3GC-R-Ec was 73.3% in chicken and 2% in beef meat. No 3GC-R-Ec were found in pork and veal. Overall, the blaCTX-M-1 (79.4%), blaCMY-2 (17.6%), blaCMY-4 (1.5%), and blaSHV-12 (1.5%) β-lactamase genes were detected, as well as other genes conferring resistance to chloramphenicol (cmlA1-like), sulfonamides (sul), tetracycline (tet), and trimethoprim (dfrA). The 3GC-R-Ec from chicken meat often harbored virulence genes associated with avian pathogens. Plasmid incompatibility (Inc) groups IncI1, IncFIB, IncFII, and IncB/O were the most frequent. A high rate of clonality (e.g., ST1304, ST38, and ST93) among isolates from the same MPPs suggests that strains persist at the plant and spread to meat at the carcass-processing stage. Additionally, the presence of the blaCTX-M-1 gene on an IncI1 plasmid sequence type 3 (IncI1/pST3) in genetically diverse strains indicates interstrain spread of an epidemic plasmid. The blaCMY-2 and blaCMY-4 genes were located on IncB/O plasmids. This study represents the first comprehensive assessment of 3GC-R-Ec in meat in Switzerland. It demonstrates the need for monitoring contaminants and for the adaptation of the Hazard Analysis and Critical Control Point concept to avoid the spread of multidrug-resistant bacteria through the food chain.

Exploring Hoogsteen and reversed-Hoogsteen duplex and triplex formation with tricyclo-DNA purine sequences

The duplex- and triplex-formation properties of the tricyclo-DNA purine decamer 5'p-gagaaggaaa-3' as a single strand or as part of a hairpin duplex with corresponding parallel and antiparallel pyrimidine DNA and RNA complements, as well as with antiparallel purine DNA and RNA complements, were investigated by UV melting curve analysis, circular dichroism spectroscopy, and gel mobility shift experiments. It was found that tricyclo-DNA forms very stable duplexes with the pyrimidine RNA and DNA complements not only in the Watson-Crick pairing mode, but also in the Hoogsteen one. Below pH 6.0, the tc-DNA/DNA and tc-DNA/RNA Hoogsteen duplexes were found to be more stable than the corresponding Watson-Crick DNA duplexes. Triplexes of the hairpin structure with parallel pyrimidine complements revealed even stronger Hoogsteen pairing relative to the duplexes, presumably due to structural preorganization phenomena. Triplex formation with antiparallel pyrimidine and purine third strands (reversed-Hoogsteen motif) could not be observed and seem to be unstable

Low dose irinotecan improves advanced lupus nephritis in mice potentially by changing DNA relaxation and anti–double-stranded DNA binding

Objective Albeit clear advances in the treatment of SLE, many patients still present with refractory lupus nephritis requiring new treatment strategies for this disease. Here we determined whether reduced doses of the topoisomerase I inhibitor irinotecan, which is known as chemotherapeutic agent, were able to suppress SLE in NZB/W F1 mice. We further evaluated the potential mechanism how irinotecan influenced the course of SLE. Methods NZB/W F1 mice were treated with low dose irinotecan either from week 24 of age or from established glomerulonephritis defined by a proteinuria ≥grade 3+. Binding of anti-dsDNA antibodies was measured by ELISA; and DNA relaxation was visualized by gel electrophoresis. Results Significantly reduced irinotecan dosages improved lupus nephritis and prolonged survival in NZB/W F1 mice. The lowest dose successfully used for the treatment of established murine lupus nephritis was more than 50 times lower than the dose usually applied for chemotherapy in humans. As a mechanism, low dose irinotecan reduced B cell activity; however, the levels of B cell activity in irinotecan-treated mice were similar to those in Balb/c mice of the same age suggesting that irinotecan did not induce a clear immunosuppression. In addition, incubation of double-stranded (ds) DNA with topoisomerase I increased binding of murine and human anti-dsDNA antibodies showing for the first time that relaxed DNA is more susceptible to anti-dsDNA antibody binding. This effect was reversed by addition of the topoisomerase I inhibitor camptothecin. Conclusion Our results propose topoisomerase I inhibitors as a novel and targeted therapy for SLE. © 2014 American College of Rheumatology.

SNPs for parentage testing and traceability in globally diverse breeds of sheep

DNA-based parentage determination accelerates genetic improvement in sheep by increasing pedigree accuracy. Single nucleotide polymorphism (SNP) markers can be used for determining parentage and to provide unique molecular identifiers for tracing sheep products to their source. However, the utility of a particular "parentage SNP" varies by breed depending on its minor allele frequency (MAF) and its sequence context. Our aims were to identify parentage SNPs with exceptional qualities for use in globally diverse breeds and to develop a subset for use in North American sheep. Starting with genotypes from 2,915 sheep and 74 breed groups provided by the International Sheep Genomics Consortium (ISGC), we analyzed 47,693 autosomal SNPs by multiple criteria and selected 163 with desirable properties for parentage testing. On average, each of the 163 SNPs was highly informative (MAF≥0.3) in 48±5 breed groups. Nearby polymorphisms that could otherwise confound genetic testing were identified by whole genome and Sanger sequencing of 166 sheep from 54 breed groups. A genetic test with 109 of the 163 parentage SNPs was developed for matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. The scoring rates and accuracies for these 109 SNPs were greater than 99% in a panel of North American sheep. In a blinded set of 96 families (sire, dam, and non-identical twin lambs), each parent of every lamb was identified without using the other parent's genotype. In 74 ISGC breed groups, the median estimates for probability of a coincidental match between two animals (PI), and the fraction of potential adults excluded from parentage (PE) were 1.1×10(-39) and 0.999987, respectively, for the 109 SNPs combined. The availability of a well-characterized set of 163 parentage SNPs facilitates the development of high-throughput genetic technologies for implementing accurate and economical parentage testing and traceability in many of the world's sheep breeds.

DNA hypomethylation and oxidative stress-mediated increase in genomic instability in equine sarcoid-derived fibroblasts

It is widely accepted that equine sarcoid disease, the most common skin associated neoplasm in equids, is induced by bovine papillomavirus (BPV-1). Although BPV-1 DNA has been found in almost all examined sarcoids so far, its detailed impact on the horse's host cell metabolism is largely unknown. We used equine fibroblast cell lines originating from sarcoid biopsies to study BPV-1-associated changes on DNA methylation status and oxidative stress parameters. Sarcoid-derived fibroblasts manifested increased proliferation in vitro, transcriptional rDNA activity (NORs expression) and DNA hypomethylation compared to control cells. Cells isolated from equine sarcoids suffered from oxidative stress: the expression of antioxidant enzymes was decreased and the superoxide production was increased. Moreover, increased ploidy, oxidative DNA damage and micronuclei formation was monitored in sarcoid cells. We postulate that both altered DNA methylation status and redox milieu may affect genomic stability in BPV-1-infected cells and in turn contribute to sarcoid pathology.

DNA-based diagnosis of rare diseases in veterinary medicine: a 4.4 kb deletion of ITGB4 is associated with epidermolysis bullosa in Charolais cattle.

BACKGROUND Rare diseases in livestock animals are traditionally poorly diagnosed. Other than clinical description and pathological examination, the underlying causes have, for the most part, remained unknown. A single case of congenital skin fragility in cattle was observed, necropsy, histological and ultrastructural examinations were carried out and whole genome sequencing was utilized to identify the causative mutation. RESULTS A single purebred female Charolais calf with severe skin lesions was delivered full-term and died spontaneously after birth. The clinical and pathological findings exactly matched the gross description given by previous reports on epitheliogenesis imperfecta and epidermolysis bullosa (EB) in cattle. Histological and ultrastructural changes were consistent with EB junctionalis (EBJ). Genetic analysis revealed a previously unpublished ITGB4 loss-of-function mutation; the affected calf was homozygous for a 4.4 kb deletion involving exons 17 to 22, and the dam carried a single copy of the deletion indicating recessive inheritance. The homozygous mutant genotype did not occur in healthy controls of various breeds but some heterozygous carriers were found among Charolais cattle belonging to the affected herd. The mutant allele was absent in a representative sample of unrelated sires of the German Charolais population. CONCLUSION This is the first time in which a recessively inherited ITGB4 associated EBJ has been reported in cattle. The identification of heterozygous carriers is of importance in avoiding the transmission of this defect in future. Current DNA sequencing methods offer a powerful tool for understanding the genetic background of rare diseases in domestic animals having a reference genome sequence available.

Nucleic-Acid Analogs with Restricted Conformational Flexibility in the Sugar-Phosphate Backbone (‘Bicyclo-DNA’). Part 5. Synthesis, Characterization, and Pairing Properties of Oligo-αlpha-D-(bicyclodeoxynucleotides) of the Bases Adenine and Thymine (αlpha-Bicyclo-DNA)

10.1002/hlca.19950780816.abs A conformational analysis of the (3′S,5′R)-2′-deoxy-3′,5′-ethano-α-D-ribonucleosides (a-D-bicyclodeoxynucleosides) based on the X-ray analysis of N4-benzoyl-α-D-(bicyclodeoxycytidine) 6 and on 1H-NMR analysis of the α-D-bicyclodeoxynucleoside derivatives 1-7 reveals a rigid sugar structure with the furanose units in the l′-exo/2′-endo conformation and the secondary OH groups on the carbocyclic ring in the pseudoequatorial orientation. Oligonucleotides consisting of α-D-bicyclothymidine and α-D-bicyclodeoxyadenosine were successfully synthesized from the corresponding nucleosides by phosphoramidite methodology on a DNA synthesizer. An evaluation of their pairing properties with complementary natural RNA and DNA by means of UV/melting curves and CD spectroscopy show the following characteristics: i) α-bcd(A10) and α-bcd(T10) (α = short form of α-D)efficiently form complexes with complementary natural DNA and RNA. The stability of these hybrids is comparable or slightly lower as those with natural β-d(A10) or β-d(T10)( β = short form ofβ-D). ii) The strand orientation in α-bicyclo-DNA/β-DNA duplexes is parallel as was deduced from UV/melting curves of decamers with nonsymmetric base sequences. iii) CD Spectroscopy shows significant structural differences between α-bicyclo-DNA/β-DNA duplexes compared to α-DNA/β-DNA duplexes. Furthermore, α-bicyclo-DNA is ca. 100-fold more resistant to the enzyme snake-venom phosphodiesterase with respect to β-DNA and about equally resistant as α-DNA.

The eukaryotic way to defend and edit genomes by sRNA-targeted DNA deletion

While there is currently burgeoning interest in the application of the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) to genome editing, it is perhaps not widely appreciated that this is the second discovery of a small RNA (sRNA)-targeted DNA-deletion system. The first sRNA-targeted DNA-deletion system to be discovered, which we call IES/Ias (internal eliminated sequence/IES-associated genes) to contrast with CRISPR/Cas, is found in ciliates, and, like CRISPR/Cas, is thought to serve as a form of immune defense against invasive DNAs. The manner in which the ciliate IES/Ias system functions is distinct from that of the CRISPR/Cas system in archaea and bacteria, and arose independently through a synthesis of RNA interference-derived and DNA-specific molecular components. Despite the major differences between CRISPR/Cas and IES/Ias, both systems face similar conceptual challenges in targeting invasive DNAs. In this review, we focus on the discovery, effects, function, and evolutionary consequences of the IES/Ias system.

Chromosomally and extrachromosomally mediated high-level gentamicin resistance in Streptococcus agalactiae

Streptococcus agalactiae (group B Streptococcus, GBS) is a leading cause of sepsis in neonates. The rate of invasive GBS disease in non-pregnant adults also continues to climb. Aminoglycosides alone have little or no effect on GBS, but synergistic killing with penicillin has been shown in vitro. High-level gentamicin resistance (HLGR) in GBS isolates, however, leads to loss of a synergistic effect. We therefore performed a multicentre study to determine the frequency of HLGR GBS isolates and to elucidate the molecular mechanisms leading to gentamicin resistance. From eight centres in four countries, 1128 invasive and colonizing GBS isolates were pooled and investigated for the presence of HLGR. We identified two strains that displayed HLGR (BSU1203 and BSU452), both of which carried the aacA-aphD gene, typically conferring HLGR. Though, only one strain (BSU1203) also carried the previously described chromosomal gentamicin resistance transposon, designated Tn3706. In the other strain (BSU452), plasmid purification and subsequent DNA sequencing resulted in the detection of plasmid pIP501 carrying a remnant of a Tn3 family transposon. Its ability to confer HLGR was proven by transfer into an Enterococcus faecalis isolate. Conversely, loss of HLGR was documented after curing both GBS BSU452 and the transformed E. faecalis strain from the plasmid. This is the first report showing a plasmid mediated HLGR in GBS. Thus, in our clinical GBS isolates HLGR is mediated both chromosomally and extrachromosomally.