Detection of Bacillus anthracis and Bacillus anthracis-like spores in soil from state of Rio de Janeiro, Brazil

BACKGROUND Bacillus anthracis is the aetiologic agent of anthrax, a re-emerging, septicaemic, haemorrhagic and lethal disease that affects humans, domestic ruminants and wildlife. Plasmids pXO1 and pXO2 are attributes that confer pathogenicity to B. anthracis strains. This bacterium was used as biological weapon in the World Wars and in the biological attack in the United States of America at 2001. B. anthracis is classified as a Tier 1 bioterrorism agent by the Centers for Diseases Control and Prevention. Anthrax is recognised as a re-emerging disease. Several studies concerning the dynamics of B. anthracis cycle in soil revealed that nonpathogenic B. anthracis strains due to lack of pXO2 plasmid are commonly found in some types of soil. OBJECTIVES This study aimed isolation and identification of B. anthracis spores in soil samples of the state of Rio de Janeiro, Brazil. METHODS Phenotypic and genotypic approaches were used to identify isolates including MALDI-TOF/MS, motility test, susceptibility to gamma phage and penicillin, survey for pag and cap genes as surrogates of pXO1 and pXO2 plasmids, respectively, and sequencing of 16SrRNA-encoding gene. Physicochemical analysis of the soil samples were carried out to describe soil characteristics. FINDINGS We observed the presence of one B. anthracis pXO1+ and pXO2- isolated from clay loam soil; one B. anthracis-like strain pXO1+ and pXO2-isolated from loamy sand; and 10 Bacillus spp. strains sensitive to phage-gamma that need better characterisation to define which their species were recovered from loamy sand. MAIN CONCLUSIONS This work showed promising results and it was the first study to report results from an active surveillance for B. anthracis in Brazil.

Plasmid Dissemination and Selection of a Multidrug-Resistant Klebsiella pneumoniae Strain during Transplant-Associated Antibiotic Therapy.

Antibiotics, which are used both to prevent and to treat infections, are a mainstay therapy for lifesaving procedures such as transplantation. For this reason, and many others, increased antibiotic resistance among human-associated pathogens, such as the carbapenem-resistant Enterobacteriaceae species, is of grave concern. In this study, we report on a hematopoietic stem cell transplant recipient in whom cultures detected the emergence of carbapenem resistance and spread across five strains of bacteria that persisted for over a year. Carbapenem resistance in Citrobacter freundii, Enterobacter cloacae, Klebsiella aerogenes, and Klebsiella pneumoniae was linked to a pair of plasmids, each carrying the Klebsiella pneumoniae carbapenemase gene (blaKPC). Surveillance cultures identified a carbapenem-susceptible strain of Citrobacter freundii that may have become resistant through horizontal gene transfer of these plasmids. Selection of a multidrug-resistant Klebsiella pneumoniae strain was also detected following combination antibiotic therapy. Here we report a plasmid carrying the blaKPC gene with broad host range that poses the additional threat of spreading to endogenous members of the human gut microbiome.IMPORTANCE Antibiotic-resistant bacteria are a serious threat to medically fragile patient populations. The spread of antibiotic resistance through plasmid-mediated mechanisms is of grave concern as it can lead to the conversion of endogenous patient-associated strains to difficult-to-treat pathogens.

Clonal diversity of Cutibacterium acnes (formerly Propionibacterium acnes) in prosthetic joint infections.

Prosthetic joint infections (PJIs) are rare but feared complications following joint replacement surgery. Cutibacterium acnes is a skin commensal that is best known for its role in acne vulgaris but can also cause invasive infections such as PJIs. Some phylotypes might be associated with specific diseases, and recently, a plasmid was detected that might harbour important virulence genes. In this study, we characterized C. acnes isolates from 63 patients with PJIs (n = 140 isolates) and from the skin of 56 healthy individuals (n = 56 isolates), using molecular methods to determine the phylotype and investigate the presence of the plasmid. Single-locus sequence typing and a polymerase chain reaction designed to detect the plasmid were performed on all 196 isolates. No statistically significant differences in sequence types were seen between the two study groups indicating that the C. acnes that causes PJIs originates from the patients own normal skin microbiota. Of the 27 patients with multiple tissue samples, 19 displayed the same sequence types among all their samples. Single-locus sequence typing identified different genotypes among consecutive C. acnes isolates from four patients with recurrent infections. The plasmid was found among 17 isolates distributed in both groups, indicating that it might not be a marker for virulence regarding PJIs. Patients presenting multiple sequence types in tissue samples may represent contamination or a true polyclonal infection due to C. acnes.

DoE to improve supercoiled p53-pDNA purification by O-phospho-l-tyrosine chromatography.

P53 is implicated in various cellular functions and several studies have shown that transfection of cancer cells with wild-type p53-expressing plasmids could directly drive cells into growth arrest and/or apoptosis. In the present work, the 6.07 kbp pcDNA3-FLAG-p53 plasmid, which encodes the p53 tumor suppressor, was produced and recovered from a recombinant cell culture of Escherichia coli DH5α. Following plasmid biosynthesis, the O-phospho-l-tyrosine chromatographic matrix was explored to purify the supercoiled p53-encoding plasmid. In order to quickly determine the optimal chromatographic performance and to obtain the required purity degree, maximizing the recovery yield of the supercoiled plasmid DNA, the Composite Central Face design was applied. The model revealed to be statistically significant (p-value < 0.05), with coefficient of determination of 0.9434 for the recovery yield and 0.9581 for purity and the central point was successfully validated. After the chromatographic process optimization by using the design of experiments tool, 49.7% of the supercoiled p53-encoding plasmid was recovered with 98.2% of purity, when a decreasing ammonium sulphate gradient was applied. The dynamic binding capacity of the O-phospho-l-tyrosine agarose column was 0.35 ±â€¯0.02 mg pDNA/mL matrix at 50% of the breakthrough. Finally, the purified sample was analysed to assess the content of endotoxins, proteins and genomic DNA, showing that all these impurity levels were below the recommendations of the regulatory agencies.

Polyphasic taxonomic analysis of Parasedimentitalea marina gen. nov., sp. nov., a psychrotolerant bacterium isolated from deep sea water of the New Britain Trench.

A novel Rhodobacteraceae bacterium, strain W43T, was isolated from a deep-sea water sample from the New Britain Trench. Strain W43T exhibited the highest 16S rRNA gene sequence similarity of 96.5% to Sedimentitalea nanhaiensis DSM 24252T, Phaeobacter gallaeciensis DSM 26640T, Phaeobacter inhibens DSM 16374T, and Phaeobacter porticola P97T. Phylogenetic analysis of the 16S rRNA gene and phylogenomic analysis of the genome showed that strain W43T formed an independent monophyletic branch within the family Rhodobacteraceae. Strain W43T was Gram-stain-negative, rod-shaped, and grew optimally at 16-20°C, pH 6.5-7.0 and 2% (w/v) NaCl. The principal fatty acids were C18:1ω7c/C18:1ω6c, major respiratory quinone was ubiquinone-10 and predominant polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The 5 080 916 bp long genome, comprising a circular chromosome and four plasmids, exhibits a G + C content of 55.9 mol%. The combined phenotypic, chemotaxonomic and molecular data show that strain W43T represents a novel species of a novel genus, for which the name Parasedimentitalea marina gen. nov. sp. nov. is proposed (type strain W43T = MCCC 1K03532T = KCTC 62635T).

Characterization of uropathogenic ESBL-producing Escherichia coli isolated from hospitalized patients in western Algeria.

INTRODUCTION: The aim of this study is to assess the prevalence and molecular characterization of uropathogenic Extended spectrum ß-lactamases (ESBLs) producing Escherichia coli. METHODOLOGY: During 3 years, all hospitalized patients at the University-affiliated hospital of Tlemcen and presenting urinary tract infections caused by E. coli were considered as potential study participants. These E. coli isolates were examined phenotypically for ESBL production. ESBL strains were subjected to antimicrobial susceptibility testing and were investigated for the presence of plasmid mediated quinolone resistance genes, 16SrRNA methylase genes and virulence genes by using conventional PCR and DNA sequencing. The molecular characterization of ESBL strains was established by phylogenetic grouping method and ERIC-PCR. RESULTS: The overall prevalence of ESBL was 32.5%. The blaCTX-M-15 was the most frequently detected in ESBL isolates, followed by blaCTX-M-14, blaCTX-M-28, blaCTX-M-1 and blaSHV-12 respectively. The plasmid-mediated quinolone resistance genes were detected in the 15 ESBL strains with the aac(6')-Ib-cr gene was the most detected followed by qnrB1 and qnrA1 gene respectively. Among the 22 ESBL isolates resistant to gentamicin and amikacin, the 16SrRNA methylase genes were detected in 4 isolates. The sfa and pap virulent genes were founds in 26% and 22% of isolates receptively. The genotyping analysis of all strains revealed that almost were belonged to phylogenetic groups A1 and A0 and fourteen distinct clones. CONCLUSION: The emergence of uropathogenic ESBL isolates and the high rate of blaCTX-M are alarming in Algeria. Strict measure must be required to control the further spread of these strains in Algerian hospitals.

Relação ancestral e pan-resistoma plasmidial de Escherichia coli produtora de CTX-M-8 e MCR-1 na interface humana, animal e ambiental / Ancestral relationship and plasmid pan-resistome of CTX-M-8- and MCR-1-producing Escherichia coli in human-animal-environmental interface

Linhagens de Escherichia coli produtoras de ß-lactamase de espectro estendido (ESßL) do tipo CTX-M são endêmicas no Brasil, sendo prevalentes em casos de infecções hospitalares e ambulatoriais. Atualmente, cepas produtoras de CTX-M têm sido recuperadas de ambientes urbanos, animais de companhia ou de produção e de alimentos de origem animal, inclusive afetando o agronegócio, o que aponta uma possível rota de disseminação em diferentes ecossistemas. Recentemente, nesta espécie, foi descoberto um novo gene, chamado de mcr-1, que confere resistência transferível à colistina, um dos últimos antibióticos eficazes para o tratamento de infecções causadas por bactérias produtoras de ESBL e carbapenemases. Deste modo, o presente estudo tem como objetivo elucidar os aspectos sobre a caracterização e a relação de plasmídeos que carregam genes do tipo blaCTX-M-8 e mcr- 1 em cepas de E. coli isoladas de seres humanos, animais, ambiente aquático e alimentos, no Brasil. Neste estudo são apresentados os resultados da análise plasmidial de 25 cepas de E. coli, das quais nove apresentaram o genótipo blaCTX-M-8/IncI1, 11 apresentaram o genótipo mcr-1/IncX4 e cinco apresentaram ambos os genótipos blaCTX-M-8/IncI1 e mcr-1/IncX4. Dos resultados, podemos observar que plasmídeos IncI1 (blaCTX-M-8) e IncX4 (mcr-1) estão circulando no Brasil desde o ano de 2009 entre diferentes clones (STs) de E. coli e em diferentes ambientes e hospedeiros. Os plasmídeos IncI1 foram conjugativos e pertencentes ao ST113, exceto o plasmídeo recuperado de um isolado humano, que foi pertencente ao ST131. Os plasmídeos IncI1 apresentaram sua arquitetura conservada, com a presença de genes de replicação, transferência e estabilidade. A partir do alinhamento, os plasmídeos IncI1 apresentaram 94-99% de similaridade genética entre eles. Dentre os plasmídeos IncX4, independente da fonte de isolamento, todos permaneceram com sua arquitetura altamente conservada. Entretanto, apenas dois plasmídeos (um encontrado em uma cepa de animal e outro encontrado em uma cepa de ambiente aquático) apresentaram uma IS1294, truncando o gene de mobilização. Na análise comparativa, todos os plasmídeos IncX4 apresentaram similaridade genética de 95-99,9% entre eles. No alinhamento de plasmídeos IncX4 brasileiros contra plasmídeos de outras regiões geográficas, foi observada similaridade genética > 99,9%, o que confirma a estabilidade e conservação desses plasmídeos. Neste estudo foram reportados dados inéditos da primeira identificação do gene mcr-1 em diferentes ecossistemas no Brasil, assim como a nova variante mcr-5.3. A análise filogenética dos plasmídeos IncI1 e IncX4, destacam que ambos compartilham uma arquitetura conservada, e a evolução é atribuída à aquisição de genes de resistência. Adicionalmente, um novo vetor de disseminação do gene mcr-1 no Brasil foi identificado - o plasmídeo IncHI2. Os resultados desse estudo demonstram o grave problema da resistência bacteriana dentro do conceito One-health e que, com o avanço de ferramentas moleculares, a identificação e a resolução desse problema poderá estar cada vez mais próxima de ser elucidada

CTX-M-type extended-spectrum-ß-lactamase (ESßL)-producing-Escherichia coli are endemic in Brazil and are prevalent in cases of nosocomial and ambulatory infections. Currently, CTXM-producing strains have been recovered from urban environments, companion/production animals and animal source foods, which indicates a possible route of dissemination in different ecosystems. Recently, in this species, a new gene, called mcr-1, has been discovered, conferring transferable resistance to colistin, one of the last effective antibiotics for the treatment of infections caused by ESBL- and carbapenemases -producing bacteria. Thus, the present study aims to elucidate unknown aspects of the pan-resistome and ancestral relationship of plasmids carrying blaCTX-M-8 and mcr-1 genes in strains of E. coli isolated from humans, animals, aquatic environment and food, in Brazil. In this study, we present results from the plasmidial analysis of 25 E. coli strains, from which nine presented the blaCTX-M-8/IncI1 genotype, 11 presented the mcr-1/IncX4, and five presented both blaCTX-M-8/IncI1 and mcr-1/IncX4 genotypes. Among these results, we can observe that IncI1 (blaCTX-M-8) and IncX4 (mcr-1) plasmids are circulating in Brazil since 2009, between different E. coli clones (STs) and different hosts and environments. IncI1 plasmids were conjugative and assigned to ST113, with exception of a plasmid recovered from a human isolate, which was assigned to ST131. IncI1 plasmids presented conserved architecture, with the presence of genes of replication, transference, and stability. From the alignment analysis, IncI1 plasmids presented 94-99% genetic similarity among them. Among the IncX4 plasmids, regardless the isolation source, their architecture remained highly conserved. However, only two plasmids (one detected in an animal's strain and another detected in an aquatic environment's strain) presented an IS1294, truncating the mobilization gene. In the comparative analysis, all IncX4 plasmids presented 95-99,9% genetic similarity among them. In the alignment of Brazilian IncX4 plasmids against plasmids from other geographic regions, >99.9% genetic similarity was observed, confirming the stability and conservation of these plasmids. In this study, unprecedented data from the first identification of the mcr-1 gene in different ecosystems in Brazil, as well as the new variant, mcr-5.3. Additionally, it was identified a new dissemination vector of the mcr-1 gene in Brazil - the IncHI2 plasmid. Phylogenetic analysis of IncI1and IncX4 plasmids highlight that both share a conserved backbone, and evolution is attributed to the acquisition of clinically relevant antimicrobial resistance genes. The results from this study demonstrate the serious problem of the bacterial resistance within the "One-Health" concept and that, with the advance of molecular tools, identification and resolution of this problem may be increasingly closer to being elucidate

Impedimetric gene assay for BCR/ABL transcripts in plasmids of patients with chronic myeloid leukemia.

A label-free impedimetric biosensor was developed for determination of BCR/ABL transcripts. Specific DNA primers were covalently immobilized on a gold electrode modified with carboxylated multiwalled carbon nanotubes (cMWCNTs) and zinc oxide nanoparticles (ZnO-NPs). Aggregation of the ZnO-NPs is prevented by the introduction of an amino-modified silica coating, which also allows a subsequent covalent linkage to cMWCNTs. The impedimetric biosensor was typically operated at a working voltage of +10 mV vs. Ag/AgCl, in a frequency range from 100 mHz to 100 kHz. Studies on the surface morphology and electrochemical properties of the electrode demonstrated improved bioactivity. Amperometric currents and impedimetric parameters, such as charge transfer resistance, varied significantly throughout the construction of the biosensor. The hybridization process was also evidenced by changes in the topography of the surface after exposure to samples containing BCR/ABL. The gene sensor has a linear concentration range for the target gene of 6.94 aM to 694 fM with a limit of detection as low as 0.039 aM. Also, the biosensor is selective and reproducible with a standard deviation of 4.1%. Three replicates for each experimental condition were used. Hence, it is perceived to be a viable tool for early-stage diagnosis of the BCR/ABL fusion gene and monitoring of major molecular remission in clinical samples. Graphical abstract Schematic of a highly sensitive hybridization assay for the BCR/ABL fusion gene. It is based on ZnO nanoparticle functionalized with 3-(aminopropyl)triethoxysilane.

Cooperative Toehold: A Mechanism To Activate DNA Strand Displacement and Construct Biosensors.

Toehold-mediated DNA strand displacement has proven powerful in the construction of various DNA circuits, DNA machines, and biosensors. So far, many new toehold activation mechanisms have been developed to achieve programmed DNA strand displacement behaviors. However, almost all those toeholds are inflexible via either a covalently attached manner or a complementary hybridization strategy, which limit the versatility of DNA devices. To solve this problem, we developed a new toehold, named "cooperative toehold", to activate DNA strand displacement. On the basis of a base stacking mechanism, the cooperative toehold is comprised of two moieties with completely independent DNA sequences between each other. The cooperative toehold enabled one to continuously tune the rate of DNA strand displacement, as well as more sophisticated strand displacement reactions. The cooperative toehold has also been employed as a universal signal translator for biosensors to qualitatively determine RNA and ATP. Moreover, as a novel specific PCR monitoring system, cooperative toehold-mediated DNA strand displacement can detect the pUC18 plasmid in genomic DNA samples with an aM detection limit.

Arginine homopeptides for plasmid DNA purification using monolithic supports.

Purification of plasmid DNA targeting therapeutic applications still presents many challenges, namely on supports and specific ligand development. Monolithic supports have emerged as interesting approaches for purifying pDNA due to its excellent mass transfer properties and higher binding capacity values. Moreover, arginine ligands were already described to establish specific and preferential interactions with pDNA. Additionally, some studies revealed the ability of arginine based cationic peptides to condense plasmid DNA, which increased lengthening can result in strongest interactions with higher binding capacities for chromatographic purposes of large molecules such as pDNA. In this work, arginine homopeptides were immobilized in monolithic supports and their performance was evaluated and compared with a single arginine monolithic column regarding supercoiled (sc) plasmid DNA purification. Specific interactions of arginine based peptides with several nucleic acids present in a clarified Escherichia coli lysate sample showed potential for the sc pDNA purification. Effectively, the immobilization of the arginine homopeptides became more functional compared with the single arginine amino acid, showing higher binding capacities, which was also reflected in the intensity of the interactions. The combination of structural versatilities of monoliths with the specificity of arginine peptides raised as a promising strategy for sc pDNA purification.

Optimizing host cell physiology and stress avoidance for the production of recombinant human tumour necrosis factor α in Escherichia coli.

As high-level recombinant protein production (RPP) exerts a massive stress on the production host, an extensive literature on RPP optimization focuses on separating the growth phase from RPP production once sufficient biomass has been obtained. The aim of the current investigation was to optimize the benefits of the relatively neglected alternative strategy to achieve high-level RPP during growth by minimizing stress on the host. High yields of the biopharmaceutical recombinant human tumour necrosis factor alpha (rhTNFα) were obtained by fed-batch fermentation relevant to industrial production based upon parameters that most severely affected RPP in preliminary laboratory scale batch cultures. Decreasing the inducer concentration and growth temperature, but increasing the production period, were far more effective for increasing RPP yields than changing the growth phase at which production was induced. High yields of up to 5 g l-1 of rhTNFα were obtained with minimal plasmid loss, even in synthetic media that lack animal-derived components and are therefore fully compliant with regulatory requirements. Most of the product was soluble and biologically active. In summary, stress minimization was shown to be an effective way to optimize the production of rhTNFα. Data generated in shake-flask experiments allowed the design of intensified bioreactor cultures in which RPP and growth could be balanced, leading to higher yield of both rhTNFα and biomass than with previous fermentations. An additional benefit of this approach is avoidance of lysis during harvesting and downstream processing.

Protocol for Efficient Generation and Characterization of Adeno-Associated Viral Vectors.

Adeno-associated virus vectors are a powerful tool for gene transfer approaches. We have established a simple and fast plasmid-based production system for achieving high adeno-associated virus titers within 6 working days. The same procedure can be used for all serotypes and thus allows direct comparability of different serotypes. In this protocol we describe a step-by-step procedure that results in well-characterized vectors suitable for both in vitro approaches and preclinical studies.

Infectious Agents in Bovine Red Meat and Milk and Their Potential Role in Cancer and Other Chronic Diseases.

Red meat and dairy products have frequently been suggested to represent risk factors for certain cancers, chronic neurodegenerative diseases, and autoimmune and cardiovascular disorders. This review summarizes the evidence and investigates the possible involvement of infectious factors in these diseases. The isolation of small circular single-stranded DNA molecules from serum and dairy products of Eurasian Aurochs (Bos taurus)-derived cattle, obviously persisting as episomes in infected cells, provides the basis for further investigations. Gene expression of these agents in human cells has been demonstrated, and frequent infection of humans is implicated by the detection of antibodies in a high percentage of healthy individuals. Epidemiological observations suggest their relationship to the development multiple sclerosis, to heterophile antibodies, and to N-glycolylneuraminic acid (Neu5Gc) containing cell surface receptors.

Nanoparticle Tracking Analysis for Determination of Hydrodynamic Diameter, Concentration, and Zeta-Potential of Polyplex Nanoparticles.

Nanoparticle tracking analysis (NTA) is a recently developed nanoparticle characterization technique that offers certain advantages over dynamic light scattering for characterizing polyplex nanoparticles in particular. Dynamic light scattering results in intensity-weighted average measurements of nanoparticle characteristics. In contrast, NTA directly tracks individual particles, enabling concentration measurements as well as the direct determination of number-weighted particle size and zeta-potential. A direct number-weighted assessment of nanoparticle characteristics is particularly useful for polydisperse samples of particles, including many varieties of gene delivery particles that can be prone to aggregation. Here, we describe the synthesis of poly(beta-amino ester)/deoxyribonucleic acid (PBAE/DNA) polyplex nanoparticles and their characterization using NTA to determine hydrodynamic diameter, zeta-potential, and concentration. Additionally, we detail methods of labeling nucleic acids with fluorophores to assess only those polyplex nanoparticles containing plasmids via NTA. Polymeric gene delivery of exogenous plasmid DNA has great potential for treating a wide variety of diseases by inducing cells to express a gene of interest.

Klebsiella pneumoniae in Singapore: Hypervirulent Infections and the Carbapenemase Threat.

Klebsiella pneumoniae remains a major pathogen responsible for localized infections such as cystitis and pneumonia, and disseminated infections that may result in severe sepsis and death. Invasive disease such as liver abscesses and endogenous endophthalmitis are associated with capsular serotypes K1 and K2. These infections require a prolonged course of antimicrobial treatment which has evolved over the years from inpatient treatment to outpatient parenteral antibiotic therapy. The emergence of plasmid-mediated resistance began with extended-spectrum ß-lactamases (ESBLs) and AmpC ß-lactamases. This was followed by carbapenemase genes and now plasmid transmissible colistin resistance (mcr), thus limiting viable treatment options. Plasmid-mediated carbapenemase production in Singapore was first reported in 1996. Carbapenemase production has since become the predominant mechanism of carbapenem resistance and incidence rates continue to increase over time. Although carbapenemases can occur in all Enterobacteriaceae, K. pneumoniae are the most common carrier of carbapenemase genes. Alternative treatment options are urgently required before the simplest infections, let alone invasive infections are left potentially untreatable. Clinical management requires guidance from robust laboratory testing methods to optimize patient outcomes. We explore past and present trends in treatment of K. pneumoniae infections, and discuss future treatment options and gaps in knowledge for further study.

IncFIIk plasmid harbouring an amplification of 16S rRNA methyltransferase-encoding gene rmtH associated with mobile element ISCR2.

OBJECTIVES: To investigate the resistance mechanisms and genetic support underlying the high resistance level of the Klebsiella pneumoniae strain CMUL78 to aminoglycoside and ß-lactam antibiotics. METHODS: Antibiotic susceptibility was assessed by the disc diffusion method and MICs were determined by the microdilution method. Antibiotic resistance genes and their genetic environment were characterized by PCR and Sanger sequencing. Plasmid contents were analysed in the clinical strain and transconjugants obtained by mating-out assays. Complete plasmid sequencing was performed with PacBio and Illumina technology. RESULTS: Strain CMUL78 co-produced the 16S rRNA methyltransferase (RMTase) RmtH, carbapenemase OXA-48 and ESBL SHV-12. The rmtH- and blaSHV-12-encoding genes were harboured by a novel ∼115 kb IncFIIk plasmid designated pRmtH, and blaOXA-48 by a ∼62 kb IncL/M plasmid related to pOXA-48a. pRmtH plasmid possessed seven different stability modules, one of which is a novel hybrid toxin-antitoxin system. Interestingly, pRmtH plasmid harboured a 4-fold amplification of an rmtH-ISCR2 unit arranged in tandem and inserted within a novel IS26-based composite transposon designated Tn6329. CONCLUSIONS: This is the first known report of the 16S RMTase-encoding gene rmtH in a plasmid. The rmtH-ISCR2 unit was inserted in a composite transposon as a 4-fold tandem repeat, a scarcely reported organization.

Genotypic and phenotypic characterization of methicillin-resistant Staphylococcus aureus (MRSA) clones with high-level mupirocin resistance.

A high proportion of methicillin-resistant Staphylococcus aureus isolates recovered in one year period showed high-level mupirocin-resistance (HLMUPR-MRSA) in our environment (27.2%). HLMUPR-MRSA isolates were mainly collected from skin and soft tissue samples, and diabetes was the main related comorbidity condition. These isolates were more frequently found in vascular surgery. HLMUPR-MRSA was more resistant to aminoglycosides than mupirocin-susceptible MRSA, linked to the presence of bifunctional and/or nucleotidyltransferase enzymes with/without macrolide resistance associated with the msr(A) gene. Most of HLMUPR-MRSA isolates belonged to ST125/t067. Nine IS257-ileS2 amplification patterns (p3 was the most frequent) were observed in HLMUPR-MRSA isolates, suggesting the presence of several mupirocin-resistance-carrying plasmids in our environment and promoting the emergence of mupirocin resistance. The presence of the same IS257-ileS2 amplification pattern p3 in 65% of HLMUPR-MRSA, all of them ST125/t067, suggests a clonal spread in our hospital and community environment which could explain the high prevalence of HLMUPR-MRSA during the study period. An outbreak situation or an increase in mupirocin consumption was not observed.

Development and Validation of a Quantitative PCR Method for Equid Herpesvirus-2 Diagnostics in Respiratory Fluids.

The protocol describes a quantitative RT-PCR method for the detection and quantification of EHV-2 in equine respiratory fluids according to the NF U47-600 norm. After the development and first validation step, two distinct characterization steps were performed according to the AFNOR norm: (a) characterization of the qRT-PCR assay alone and (b) characterization of the whole analytical method. The validation of the whole analytical method included the portrayal of all steps between the extraction of nucleic acids and the final PCR analysis. Validation of the whole method is very important for virus detection by qRT-PCR in order to get an accurate determination of the viral genome load. Since the extraction step is the primary source of loss of biological material, it may be considered the main source of error of quantification between one protocol and another. For this reason, the AFNOR norm NF-U-47-600 recommends including the range of plasmid dilution before the extraction step. In addition, the limits of quantification depend on the source from which the virus is extracted. Viral genome load results, which are expressed in international units (IU), are easier to use in order to compare results between different laboratories. This new method of characterization of qRT-PCR should facilitate the harmonization of data presentation and interpretation between laboratories.