Carbapenem-resistant Klebsiella pneumoniae: the role of plasmids in emergence, dissemination, and evolution of a major clinical challenge.

INTRODUCTION: Klebsiella pneumoniae is a major agent of healthcare-associated infections and a cause of some community-acquired infections, including severe bacteremic infections associated with metastatic abscesses in liver and other organs. Clinical relevance is compounded by its outstanding propensity to evolve antibiotic resistance. In particular, the emergence and dissemination of carbapenem resistance in K. pneumoniae has posed a major challenge due to the few residual treatment options, which have only recently been expanded by some new agents. The epidemiological success of carbapenem-resistant K. pneumoniae (CR-Kp) is mainly linked with clonal lineages that produce carbapenem-hydrolyzing enzymes (carbapenemases) encoded by plasmids. AREAS COVERED: Here, we provide an updated overview on the mechanisms underlying the emergence and dissemination of CR-Kp, focusing on the role that plasmids have played in this phenomenon and in the co-evolution of resistance and virulence in K. pneumoniae. EXPERT OPINION: CR-Kp have disseminated on a global scale, representing one of the most important contemporary public health issues. These strains are almost invariably associated with complex multi-drug resistance (MDR) phenotypes, which can also include recently approved antibiotics. The heterogeneity of the molecular bases responsible for these phenotypes poses significant hurdles for therapeutic and diagnostic purposes.

Multiple Brain Metastases Radiosurgery with CyberKnife Device: Dosimetric Comparison between Fixed/Iris and Multileaf Collimator Plans.

Purpose: In our institution, stereotactic radiosurgery of multiple brain metastases is performed with the CyberKnife® (CK) device, using fixed/Iris collimators. In this study, nineteen fixed/Iris plans were recalculated with the multileaf collimator (MLC), to assess if it is possible to produce plans with comparable dosimetric overall quality. Materials and Methods: For consistent comparisons, MLC plans were re-optimized and re-normalized in order to achieve the same minimum dose for the total planning target volume (PTVtot). Conformation number (CN), homogeneity index (HI) and dose gradient index (DGI) metrics were evaluated. The dose to the brain was evaluated as the volume receiving 12 Gy (V12) and as the integral dose (ID). The normal tissue complication probability (NTCP) for brain radionecrosis was calculated as a function of V12. Results: The reoptimized plans were reviewed by the radiation oncologist and were found clinically acceptable according to the The American Association of Physicists in Medicine (AAPM) Task Group-101 protocol. However, fixed/Iris plans provided significantly higher CN (+8.6%), HI (+2.2%), and DGI (+44.0%) values, and significantly lower ID values (-35.9%). For PTVtot less than the median value of 2.58cc, fixed/Iris plans provided significantly lower NTCP values. On the other side, MLC plans provided significantly lower treatment times (-18.4%), number of monitor units (-33.3%), beams (-46.0%) and nodes (-21.3%). Conclusions: CK-MLC plans for the stereotactic treatment of brain multi metastases could provide an important advantage in terms of treatment duration. However, to contain the increased risk for brain radionecrosis, it could be useful to calculate MLC plans only for patients with large PTVtot.

Effectiveness of Flattening-Filter-Free versus Flattened Beams in V79 and Glioblastoma Patient-Derived Stem-like Cells.

Literature data on the administration of conventional high-dose beams with (FF) or without flattening filters (FFF) show conflicting results on biological effects at the cellular level. To contribute to this field, we irradiated V79 Chinese hamster lung fibroblasts and two patient-derived glioblastoma stem-like cell lines (GSCs-named #1 and #83) using a clinical 10 MV accelerator with FF (at 4 Gy/min) and FFF (at two dose rates 4 and 24 Gy/min). Cell killing and DNA damage induction, determined using the γ-H2AX assay, and gene expression were studied. No significant differences in the early survival of V79 cells were observed as a function of dose rates and FF or FFF beams, while a trend of reduction in late survival was observed at the highest dose rate with the FFF beam. GSCs showed similar survival levels as a function of dose rates, both delivered in the FFF regimen. The amount of DNA damage measured for both dose rates after 2 h was much higher in line #1 than in line #83, with statistically significant differences between the two dose rates only in line #83. The gene expression analysis of the two GSC lines indicates gene signatures mimicking the prognosis of glioblastoma (GBM) patients derived from a public database. Overall, the results support the current use of FFF and highlight the possibility of identifying patients with candidate gene signatures that could benefit from irradiation with FFF beams at a high dose rate.

Molecular and Kinetic Characterization of MOX-9, a Plasmid-Mediated Enzyme Representative of a Novel Sublineage of MOX-Type Class C ß-Lactamases.

The MOX lineage of ß-lactamases includes a group of molecular class C enzymes (AmpCs) encoded by genes mobilized from the chromosomes of Aeromonas spp. to plasmids. MOX-9, previously identified as a plasmid-encoded enzyme from a Citrobacter freundii isolate, belongs to a novel sublineage of MOX enzymes, derived from the resident Aeromonas media AmpC. The blaMOX-9 gene was found to be carried on a transposon, named Tn7469, likely responsible for its mobilization to plasmidic context. MOX-9 was overexpressed in Escherichia coli, purified, and subjected to biochemical characterization. Kinetic analysis showed a relatively narrow-spectrum profile with strong preference for cephalosporin substrates, with some differences compared with MOX-1 and MOX-2. MOX-9 was not inhibited by clavulanate and sulbactam, while both tazobactam and avibactam acted as inhibitors in the micromolar range.

Phosphatidylcholine Liposomes Down-Modulate CD4 Expression Reducing HIV Entry in Human Type-1 Macrophages.

A strategy adopted to combat human immunodeficiency virus type-1 (HIV-1) infection is based on interfering with virus entry into target cells. In this study, we found that phosphatidylcholine (PC) liposomes reduced the expression of the CD4 receptor in human primary type-1 macrophages but not in CD4+ T cells. The down-regulation was specific to CD4, as any effect was not observed in CCR5 membrane expression. Moreover, the reduction of membrane CD4 expression required the Ca2+-independent protein kinase C (PKC), which in turn mediated serine phosphorylation in the intracytoplasmic tail of the CD4 receptor. Serine phosphorylation of CD4 was also associated with its internalization and degradation in acidic compartments. Finally, the observed CD4 downregulation induced by PC liposomes in human primary macrophages reduced the entry of both single-cycle replication and replication competent R5 tropic HIV-1. Altogether, these results show that PC liposomes reduce HIV entry in human macrophages and may impact HIV pathogenesis by lowering the viral reservoir.

Fighting MDR-Klebsiella pneumoniae Infections by a Combined Host- and Pathogen-Directed Therapeutic Approach.

Klebsiella pneumoniae is an opportunistic pathogen that is very difficult to treat mainly due to its high propensity to acquire complex resistance traits. Notably, multidrug resistance (MDR)-Klebsiella pneumoniae (KP) infections are responsible for 22%-72% of mortality among hospitalized and immunocompromised patients. Although treatments with new drugs or with combined antibiotic therapies have some degree of success, there is still the urgency to investigate and develop an efficient approach against MDR-KP infections. In this study, we have evaluated, in an in vitro model of human macrophages, the efficacy of a combined treatment consisting of apoptotic body-like liposomes loaded with phosphatidylinositol 5-phosphate (ABL/PI5P) and φBO1E, a lytic phage specific for the major high-risk clone of KPC-positive MDR-KP. Results show that ABL/PI5P did not affect in a direct manner KKBO-1 viability, being able to reduce only the intracellular KKBO-1 bacterial load. As expected, φBO1E was effective mainly on reducing extracellular bacilli. Importantly, the combination of both treatments resulted in a simultaneous reduction of both intracellular and extracellular bacilli. Moreover, the combined treatment of KKBO-1-infected cells reduced proinflammatory TNF-α and IL-1ß cytokines and increased anti-inflammatory TGF-ß cytokine production. Overall, our data support the therapeutic value of a combined host- and pathogen-directed therapy as a promising approach, alternative to single treatments, to simultaneously target intracellular and extracellular pathogens and improve the clinical management of patients infected with MDR pathogens such as MDR-KP.

Application of Bacteriophages for Human Health: An Old Approach against Contemporary "Bad Bugs".

The breadth of the antimicrobial resistance (AMR) problem exposes humankind to serious threats, which could lead, in the near future, to a worrisome raising of mortality and morbidity rates due to infections by "bad bugs" [...].

Combined Host- and Pathogen-Directed Therapy for the Control of Mycobacterium abscessus Infection.

Mycobacterium abscessus is the etiological agent of severe pulmonary infections in vulnerable patients, such as those with cystic fibrosis (CF), where it represents a relevant cause of morbidity and mortality. Treatment of pulmonary infections caused by M. abscessus remains extremely difficult, as this species is resistant to most classes of antibiotics, including macrolides, aminoglycosides, rifamycins, tetracyclines, and ß-lactams. Here, we show that apoptotic body like liposomes loaded with phosphatidylinositol 5-phosphate (ABL/PI5P) enhance the antimycobacterial response, both in macrophages from healthy donors exposed to pharmacological inhibition of cystic fibrosis transmembrane conductance regulator (CFTR) and in macrophages from CF patients, by enhancing phagosome acidification and reactive oxygen species (ROS) production. The treatment with liposomes of wild-type as well as CF mice, intratracheally infected with M. abscessus, resulted in about a 2-log reduction of pulmonary mycobacterial burden and a significant reduction of macrophages and neutrophils in bronchoalveolar lavage fluid (BALF). Finally, the combination treatment with ABL/PI5P and amikacin, to specifically target intracellular and extracellular bacilli, resulted in a further significant reduction of both pulmonary mycobacterial burden and inflammatory response in comparison with the single treatments. These results offer the conceptual basis for a novel therapeutic regimen based on antibiotic and bioactive liposomes, used as a combined host- and pathogen-directed therapeutic strategy, aimed at the control of M. abscessus infection, and of related immunopathogenic responses, for which therapeutic options are still limited. IMPORTANCE Mycobacterium abscessus is an opportunistic pathogen intrinsically resistant to many antibiotics, frequently linked to chronic pulmonary infections, and representing a relevant cause of morbidity and mortality, especially in immunocompromised patients, such as those affected by cystic fibrosis. M. abscessus-caused pulmonary infection treatment is extremely difficult due to its high toxicity and long-lasting regimen with life-impairing side effects and the scarce availability of new antibiotics approved for human use. In this context, there is an urgent need for the development of an alternative therapeutic strategy that aims at improving the current management of patients affected by chronic M. abscessus infections. Our data support the therapeutic value of a combined host- and pathogen-directed therapy as a promising approach, as an alternative to single treatments, to simultaneously target intracellular and extracellular pathogens and improve the clinical management of patients infected with multidrug-resistant pathogens such as M. abscessus.

Phosphatidylserine Liposomes Reduce Inflammatory Response, Mycobacterial Viability, and HIV Replication in Coinfected Human Macrophages.

Chronic immune activation is the key pathogenetic event of Mycobacterium tuberculosis-human immunodeficiency virus (HIV) coinfection. We assessed the therapeutic value of phosphatidylserine-liposome (PS-L) in an in vitro model of M. tuberculosis-HIV coinfection. PS-L reduced nuclear factor-κB activation and the downstream production of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and IL-6 in bacille Calmette-Guérin-infected macrophages and of TNF-α and IL-1ß in M. tuberculosis-infected and M. tuberculosis-HIV-coinfected macrophages. Importantly, a significant reduction of intracellular M. tuberculosis viability and HIV replication were also observed. These results support the further exploitation of PS-L as host-directed therapy for M. tuberculosis-HIV coinfection.

Evidence of Another Anthropic Impact on Iguana delicatissima from the Lesser Antilles: The Presence of Antibiotic Resistant Enterobacteria.

The improper use of antibiotics by humans may promote the dissemination of resistance in wildlife. The persistence and spread of acquired antibiotic resistance and human-associated bacteria in the environment, while representing a threat to wildlife, can also be exploited as a tool to monitor the extent of human impact, particularly on endangered animal species. Hence, we investigated both the associated enterobacterial species and the presence of acquired resistance traits in the cloacal microbiota of the critically endangered lesser Antillean iguana (Iguana delicatissima), by comparing two separate populations living in similar climatic conditions but exposed to different anthropic pressures. A combination of techniques, including direct plating, DNA sequencing and antimicrobial susceptibility testing allowed us to characterize the dominant enterobacterial populations, the antibiotic resistant strains and their profiles. A higher frequency of Escherichia coli was found in the samples from the more anthropized site, where multi-drug resistant strains were also isolated. These results confirm how human-associated bacteria as well as their antibiotic-resistance determinants may be transferred to wildlife, which, in turn, may act as a reservoir of antibiotic resistance.

Phage Resistance Is Associated with Decreased Virulence in KPC-Producing Klebsiella pneumoniae of the Clonal Group 258 Clade II Lineage.

Phage therapy is now reconsidered with interest in the treatment of bacterial infections. A major piece of information for this application is the definition of the molecular targets exploited by phages to infect bacteria. Here, the genetic basis of resistance to the lytic phage φBO1E by its susceptible host Klebsiella pneumoniae KKBO-1 has been investigated. KKBO-1 phage-resistant mutants were obtained by infection at high multiplicity. One mutant, designated BO-FR-1, was selected for subsequent experiments, including virulence assessment in a Galleria mellonella infection model and characterization by whole-genome sequencing. Infection with BO-FR-1 was associated with a significantly lower mortality when compared to that of the parental strain. The BO-FR-1 genome differed from KKBO-1 by a single nonsense mutation into the wbaP gene, which encodes a glycosyltransferase involved in the first step of the biosynthesis of the capsular polysaccharide (CPS). Phage susceptibility was restored when BO-FR-1 was complemented with the constitutive wbaP gene. Our results demonstrated that φBO1E infects KKBO-1 targeting the bacterial CPS. Interestingly, BO-FR-1 was less virulent than the parental strain, suggesting that in the context of the interplay among phage, bacterial pathogen and host, the emergence of phage resistance may be beneficial for the host.

Liposomes Loaded With Phosphatidylinositol 5-Phosphate Improve the Antimicrobial Response to Pseudomonas aeruginosa in Impaired Macrophages From Cystic Fibrosis Patients and Limit Airway Inflammatory Response.

Despite intensive antimicrobial and anti-inflammatory therapies, cystic fibrosis (CF) patients are subjected to chronic infections due to opportunistic pathogens, including multidrug resistant (MDR) Pseudomonas aeruginosa. Macrophages from CF patients show many evidences of reduced phagocytosis in terms of internalization capability, phagosome maturation, and intracellular bacterial killing. In this study, we investigated if apoptotic body-like liposomes (ABLs) loaded with phosphatidylinositol 5-phosphate (PI5P), known to regulate actin dynamics and vesicular trafficking, could restore phagocytic machinery while limiting inflammatory response in in vitro and in vivo models of MDR P. aeruginosa infection. Our results show that the in vitro treatment with ABL carrying PI5P (ABL/PI5P) enhances bacterial uptake, ROS production, phagosome acidification, and intracellular bacterial killing in human monocyte-derived macrophages (MDMs) with pharmacologically inhibited cystic fibrosis transmembrane conductance regulator channel (CFTR), and improve uptake and intracellular killing of MDR P. aeruginosa in CF macrophages with impaired bactericidal activity. Moreover, ABL/PI5P stimulation of CFTR-inhibited MDM infected with MDR P. aeruginosa significantly reduces NF-κB activation and the production of TNF-α, IL-1ß, and IL-6, while increasing IL-10 and TGF-ß levels. The therapeutic efficacy of ABL/PI5P given by pulmonary administration was evaluated in a murine model of chronic infection with MDR P. aeruginosa. The treatment with ABL/PI5P significantly reduces pulmonary neutrophil infiltrate and the levels of KC and MCP-2 cytokines in the lungs, without affecting pulmonary bacterial load. Altogether, these results show that the ABL/PI5P treatment may represent a promising host-directed therapeutic approach to improve the impaired phagocytosis and to limit the potentially tissue-damaging inflammatory response in CF.

Characterization of vB_StuS_MMDA13, a Newly Discovered Bacteriophage Infecting the Agar-Degrading Species Sphingomonas turrisvirgatae.

Members of Sphingomonas genus have gained a notable interest for their use in a wide range of biotechnological applications, ranging from bioremediation to the production of valuable compounds of industrial interest. To date, knowledge on phages targeting Sphingomonas spp. are still scarce. Here, we describe and characterize a lytic bacteriophage, named vB_StuS_MMDA13, able to infect the Sphingomonas turrisvirgatae MCT13 type strain. Physiological characterization demonstrated that vB_StuS_MMDA13 has a narrow host range, a long latency period, a low burst size, and it is overall stable to both temperature and pH variations. The phage has a double-stranded DNA genome of 63,743 bp, with 89 open reading frames arranged in two opposite arms separated by a 1186 bp non-coding region and shows a very low global similarity to any other known phages. Interestingly, vB_StuS_MMDA13 is endowed with an original nucleotide modification biosynthetic gene cluster, which greatly differs from those of its most closely related phages of the Nipunavirus genus. vB_StuS_MMDA13 is the first characterized lytic bacteriophage of the Siphoviridae family infecting members of the Sphingomonas genus.

Abundance of Colistin-Resistant, OXA-23- and ArmA-Producing Acinetobacter baumannii Belonging to International Clone 2 in Greece.

Carbapenem resistant Acinetobacter baumannii (CRAB) represents one of the most challenging pathogens in clinical settings. Colistin is routinely used for treatment of infections by this pathogen, but increasing colistin resistance has been reported. We obtained 122 CRAB isolates from nine Greek hospitals between 2015 and 2017, and those colistin resistant (ColR; N = 40, 32.8%) were whole genome sequenced, also by including two colistin susceptible (ColS) isolates for comparison. All ColR isolates were characterized by a previously described mutation, PmrBA226V, which was associated with low-level colistin resistance. Some isolates were characterized by additional mutations in PmrB (E140V or L178F) or PmrA (K172I or D10N), first described here, and higher colistin minimum inhibitory concentrations (MICs), up to 64 mg/L. Mass spectrometry analysis of lipid A showed the presence of a phosphoethanolamine (pEtN) moiety on lipid A, likely resulting from the PmrA/B-induced pmrC overexpression. Interestingly, also the two ColS isolates had the same lipid A modification, suggesting that not all lipid A modifications lead to colistin resistance or that other factors could contribute to the resistance phenotype. Most of the isolates (N = 37, 92.5%) belonged to the globally distributed international clone (IC) 2 and comprised four different sequence types (STs) as defined by using the Oxford scheme (ST 425, 208, 451, and 436). Three isolates belonged to IC1 and ST1567. All the genomes harbored an intrinsic bla OXA-51 group carbapenemase gene, where bla OXA-66 and bla OXA-69 were associated with IC2 and IC1, respectively. Carbapenem resistance was due to the most commonly reported acquired carbapenemase gene bla OXA-23, with ISAba1 located upstream of the gene and likely increasing its expression. The armA gene, associated with high-level resistance to aminoglycosides, was detected in 87.5% of isolates. Collectively, these results revealed a convergent evolution of different clonal lineages toward the same colistin resistance mechanism, thus limiting the effective therapeutic options for the treatment of CRAB infections.

Determination of the capsular polysaccharide structure of the Klebsiella pneumoniae ST512 representative strain KPB-1 and assignments of the glycosyltransferases functions.

Klebsiella pneumoniae strain KPB-1 was isolated in early 2011 from the pleural fluid of an inpatient admitted at an Italian hospital. It was characterized to produce the KPC-3 carbapenemase and to belong to sequence type 512, a derivative of sequence type 258 clade II characterized by the cps-2 gene cluster. The K-antigen of K. pneumoniae KPB-1 was purified and its structure determined by using GLC-MS of appropriate carbohydrate derivatives and 1D and 2D NMR spectroscopy of the native polysaccharide. All the collected data demonstrated the following repeating unit for the K. pneumoniae KPB-1 capsular polysaccharide: The reactions catalysed by each glycosyltransferase in the cps-2 gene cluster were assigned on the basis of structural homology with other Klebsiella K antigens.

Genomic Epidemiology of Carbapenem- and Colistin-Resistant Klebsiella pneumoniae Isolates From Serbia: Predominance of ST101 Strains Carrying a Novel OXA-48 Plasmid.

Klebsiella pneumoniae is a major cause of severe healthcare-associated infections and often shows MDR phenotypes. Carbapenem resistance is frequent, and colistin represents a key molecule to treat infections caused by such isolates. Here we evaluated the antimicrobial resistance (AMR) mechanisms and the genomic epidemiology of clinical K. pneumoniae isolates from Serbia. Consecutive non-replicate K. pneumoniae clinical isolates (n = 2,298) were collected from seven hospitals located in five Serbian cities and tested for carbapenem resistance by disk diffusion. Isolates resistant to at least one carbapenem (n = 426) were further tested for colistin resistance with Etest or Vitek2. Broth microdilution (BMD) was performed to confirm the colistin resistance phenotype, and colistin-resistant isolates (N = 45, 10.6%) were characterized by Vitek2 and whole genome sequencing. Three different clonal groups (CGs) were observed: CG101 (ST101, N = 38), CG258 (ST437, N = 4; ST340, N = 1; ST258, N = 1) and CG17 (ST336, N = 1). mcr genes, encoding for acquired colistin resistance, were not observed, while all the genomes presented mutations previously associated with colistin resistance. In particular, all strains had a mutated MgrB, with MgrBC28S being the prevalent mutation and associated with ST101. Isolates belonging to ST101 harbored the carbapenemase OXA-48, which is generally encoded by an IncL/M plasmid that was no detected in our isolates. MinION sequencing was performed on a representative ST101 strain, and the obtained long reads were assembled together with the Illumina high quality reads to decipher the bla OXA- 48 genetic background. The bla OXA- 48 gene was located in a novel IncFIA-IncR hybrid plasmid, also containing the extended spectrum ß-lactamase-encoding gene bla CTX-M-15 and several other AMR genes. Non-ST101 isolates presented different MgrB alterations (C28S, C28Y, K2∗, K3∗, Q30∗, adenine deletion leading to frameshift and premature termination, IS5-mediated inactivation) and expressed different carbapenemases: OXA-48 (ST437 and ST336), NDM-1 (ST437 and ST340) and KPC-2 (ST258). Our study reports the clonal expansion of the newly emerging ST101 clone in Serbia. This high-risk clone appears adept at acquiring resistance, and efforts should be made to contain the spread of such clone.

The lytic bacteriophage vB_EfaH_EF1TV, a new member of the Herelleviridae family, disrupts biofilm produced by Enterococcus faecalis clinical strains.

OBJECTIVES: The aim of this study is to characterize a new bacteriophage able to infect Enterococcus faecalis, and to evaluate its ability to disrupt biofilm. METHODS: The vB_EfaH_EF1TV (EF1TV) host-range was determined by spot test and efficiency of plating using a collection of 15E. faecalis clinical strains. The phage genome was sequenced with a next generation sequencing approach. Anti-biofilm activity was tested by crystal violet method and confocal laser scanning microscopy. Phage-resistant mutants were selected and sequenced to investigate receptors exploited by phage for infection. RESULTS: EF1TV is a newly discoveredE. faecalis phage which belongs to the Herelleviridae family. EF1TV, whose genome is 98% identical to φEF24C, is characterized by a linear dsDNA genome of 143,507 bp with direct terminal repeats of 1,911 bp. The phage is able to infect E. faecalis and shows also the ability to degrade biofilm produced by strains of this species. The results were confirmed by confocal laser scanning microscopy analyzing the biofilm reduction in the same optical field before and after phage infection. CONCLUSIONS: The EF1TV phage shows promising features such as an obligatory lytic nature, an anti-biofilm activity and the absence of integration-related proteins, antibiotic resistance determinants and virulence factors, and therefore could be a promising tool for therapeutic applications.

The Urgent Need for Novel Antimicrobial Agents and Strategies to Fight Antibiotic Resistance.

Antibiotic resistance in bacterial pathogens has currently reached very high and alarming levels [...].

Characterization of Tn6349, a novel mosaic transposon carrying poxtA, cfr and other resistance determinants, inserted in the chromosome of an ST5-MRSA-II strain of clinical origin.

OBJECTIVES: To characterize the genetic element carrying the poxtA oxazolidinone resistance gene found in the poxtA index strain Staphylococcus aureus AOUC-0915 isolated from a cystic fibrosis patient. METHODS: The genetic context of poxtA was investigated by bioinformatics analysis of WGS data of strain AOUC-0915, followed by PCR and confirmatory Sanger sequencing for repetitive regions. Conjugation and electrotransformation experiments were carried out to assess horizontal transferability using S. aureus and Enterococcus faecalis recipients. Production of phage particles was evaluated by PCR using DNA preparations obtained after phage induction. Excision of the transposon carrying poxtA was evaluated by inverse PCR experiments for detection of circular intermediates. RESULTS: poxtA was found to be associated with a 48 kb composite transposon of original structure, named Tn6349, inserted into a φN315-like prophage. The transposon was bounded by two IS1216 insertion sequences, carried several resistance genes [erm(B), cfr, poxtA and fexB] and exhibited a mosaic structure made by a derivative of plasmid pE35048-oc (previously described in an Enterococcus faecium clinical isolate) and Tn6657, a novel composite transposon carrying the poxtA and fexB genes. Excision ability of Tn6349 as a circular intermediate was demonstrated. Transferability of Tn6349 or modules thereof to S. aureus or E. faecalis by either conjugation or electrotransformation was not detected. Induction of the φN315-like prophage carrying Tn6349 was not observed. CONCLUSIONS: This study describes the structure of Tn6349, a novel composite transposon carrying several resistance determinants to anti-ribosomal drugs, including cfr and poxtA, from an oxazolidinone-resistant MRSA strain. Analysis of Tn6349 revealed a modular structure that could favour the mobilization of its resistance determinants.