Vancomycin-resistant enterococci (VRE), particularly vancomycin-resistant Enterococcus faecium (VREfm), have emerged among the leading pathogens causing hospital-acquired infections worldwide. We aimed to examine whether there were newly introduced clones contributing to this increase and to assess the risk factors for mortality in patients with VREfm bacteraemia.

Between 2003 and 2010, all medical records of adult patients diagnosed with VREfm bacteraemia at a university hospital in Taiwan were reviewed. Antibiotic susceptibility, genotyping and multilocus sequence typing of the VREfm isolates were performed.

During the study period, the prevalence of non-duplicated blood VRE isolates increased significantly from 3.9% in 2003 to 18.9% in 2010 (P < 0.0001). One-hundred-and-forty-nine patients with VREfm bacteraemia were noted and 102 isolates of VREfm were available for microbiological characterization. All isolates were susceptible to daptomycin and linezolid. Sequence type (ST) 18 and ST414 were the two predominant emerging STs from 2009 to 2010, accounting for 29.7% and 25.0% of all isolates, respectively. Patients who received immunosuppressives, had a high Charlson comorbidity index or experienced septic shock had a significantly higher 14 day mortality rate. Patients who had bacteraemia caused by ST414 isolates and received appropriate antibiotics had a lower 14 day mortality rate.

The prevalence of the VRE that caused bacteraemia increased from 2003 to 2010. This increase might be attributed to the clonal spread of VREfm belonging to ST18 and ST414. The all-cause 14 day mortality rate was lower in patients with bacteraemia due to VREfm isolates that belonged to ST414.

Polyanionic polymers, including lipoteichoic acid and wall teichoic acid, are important determinants of the charged character of the staphylococcal cell wall. This study was designed to investigate the extent to which teichoic acid contributes to protection from anionic azo dyes and to identify barriers to drug penetration for development of new antibiotics for multidrug-resistant Staphylococcus aureus infection.

We studied antimicrobial activity of azo dyes against S. aureus strains with or without inhibition of teichoic acid in vitro and in vivo.

We observed that inhibition of wall teichoic acid expression resulted in an ~1000-fold increase in susceptibility to azo dyes such as Congo red, reducing its MIC from >1024 to <4 mg/L. Sensitization occurred when the first step in the wall teichoic acid pathway, catalysed by TarO, was inhibited either by mutation or by chemical inhibition. In contrast, genetic blockade of lipoteichoic acid biosynthesis did not confer Congo red susceptibility. Based on this finding, combination therapy was tested using the highly synergistic combination of Congo red plus tunicamycin at sub-MIC concentrations (to inhibit wall teichoic acid biosynthesis). The combination rescued Caenorhabditis elegans from a lethal challenge of S. aureus.

Our studies show that wall teichoic acid confers protection to S. aureus from anionic azo dyes and related compounds, and its inhibition raises the prospect of development of new combination therapies based on this inhibition.

The aim of this study was to investigate different hydrophobic gentamicin formulations [gentamicin-bis(2-ethylhexyl) sulfosuccinate (GEN-AOT), microstructured GEN-AOT (PCA GEN-AOT) and GEN-AOT-loaded poly(lactide-co-glycolide) acid (PLGA) nanoparticles (NPs)] in view of improving its therapeutic index against intracellular bacteria. The intracellular accumulation, subcellular distribution and intracellular activity of GEN-AOT and NPs in different monocytic–macrophagic cell lines were studied.

Human THP-1 and murine J774 phagocytic cells were incubated with GEN-AOT formulations at relevant extracellular concentrations [from 1x MIC to 18 mg/L (human C_max)], and their intracellular accumulation, subcellular distribution and toxicity were evaluated and compared with those of conventional unmodified gentamicin. Intracellular activity of the formulations was determined against bacteria showing different subcellular localizations, namely Staphylococcus aureus (phagolysosomes) and Listeria monocytogenes (cytosol).

GEN-AOT formulations accumulated 2-fold (GEN-AOT) to 8-fold (GEN-AOT NPs) more than gentamicin in phagocytic cells, with a predominant subcellular localization in the soluble fraction (cytosol) and with no significant cellular toxicity. NP formulations allowed gentamicin to exert its intracellular activity after shorter incubation times and/or at lower concentrations. With an extracellular concentration of 10x MIC, a 1 log₁₀ decrease in S. aureus intracellular inoculum was obtained after 12 h instead of 24 h for NPs versus free gentamicin, and a static effect was observed against L. monocytogenes at 24 h with NPs, while free gentamicin was ineffective.

GEN-AOT formulations yielded a high cellular accumulation, especially in the cytosol, which resulted in improved efficacy against both intracellular S. aureus and L. monocytogenes.

The goal of this study was to investigate the epidemiological characteristics and the surrounding genetic structure of bla_NDM-1 in non-baumannii Acinetobacter spp. in China.

Non-baumannii Acinetobacter spp. were collected from 28 provinces in China and were screened for the presence of bla_NDM-1 using PCR. The following four methods were used to classify the Acinetobacter isolates: the Vitek 2 system, 16S-23S rRNA gene intergenic spacer sequencing, amplified rDNA restriction analysis and partial rpoB sequence analysis. An S1-PFGE assay and Southern blot hybridization were performed to determine the plasmid location of bla_NDM-1. The transferability of bla_NDM-1-harbouring plasmids was confirmed by conjugation experiments and electrotransformation. The surrounding genetic structure of the bla_NDM-1 gene was analysed using a restriction endonuclease-based cloning approach and primer walking.

Among 726 non-baumannii Acinetobacter spp., nine isolates collected from six different provinces and assigned to seven different Acinetobacter spp. contained the bla_NDM-1 gene. None of these isolates was directly infectious to the patients or demonstrated an epidemiological importation from abroad. These bla_NDM-1 genes were located on plasmids that could be transferred to Escherichia coli J53 by conjugation and Acinetobacter baylyi ADP1 by electrotransformation. Seven of the nine strains shared a common genetic structure in which bla_NDM-1 was flanked by two copies of ISAba125.

The clinical challenge posed by bla_NDM-1 is currently minimal in China; however, more attention should be devoted to monitoring the dissemination of this gene due to its potential transferability via the ISAba125-associated transposon.

Triazole resistance in Aspergillus fumigatus due to a single azole resistance mechanism (TR/L98H) is increasingly reported in European countries. Data from patients with cystic fibrosis (CF) are limited. Our study aimed to investigate the prevalence and molecular mechanisms of azole resistance in A. fumigatus in a cohort of patients with CF.

Eighty-five A. fumigatus isolates from 50 CF patients, collected between January 2010 and April 2011, were retrospectively analysed for azole resistance using agar plates containing 4 mg/L itraconazole. MICs of itraconazole, voriconazole and posaconazole were determined according to EUCAST methodology for each isolate able to grow on this medium. Species identification was performed by sequencing of the β-tubulin gene. Sequencing analysis of the cyp51A gene and its promoter region was conducted.

Nine isolates (four patients, 8% prevalence) were able to grow on itraconazole-containing agar plates. Itraconazole resistance was confirmed by EUCAST methodology (MICs >2 mg/L). All isolates had mutations in the cyp51A gene at residues previously involved in azole resistance: L98H (n = 5), M220T (n = 4) and G54R (n = 1). One patient had three genetically distinct azole-resistant isolates identified during the study. The isolates with L98H that were recovered from three patients (6% prevalence) also had the 34 bp tandem repeat in the promoter region of cyp51A (TR/L98H) and displayed multiazole resistance.

We report an 8% prevalence of itraconazole resistance in CF patients in our centre, mostly driven by TR/L98H (6%). Our data confirm that TR/L98H occurs in France and can be highly prevalent in CF patients.

Carbapenem-resistant Gram-negative bacilli are reported increasingly and represent an emerging public health concern. Laboratory detection of extended-spectrum β-lactamase (ESBL), plasmid-mediated cephalosporinase (pAmpC) and carbapenemase producers remains a challenge for microbiology laboratories and is important to avoid clinical failure due to inappropriate antimicrobial therapy and to prevent nosocomial outbreaks. We evaluated a novel microarray, the ‘Check-MDR CT103 array’ test (Check-Points, Wageningen, The Netherlands), that employs highly specific DNA markers to identify the β-lactamase genes of ESBLs (TEM, SHV and CTX-M, and discriminates between ESBL and non-ESBL TEM and SHV variants), of pAmpC (CMY-2-like, DHA, FOX, ACC-1, ACT/MIR and CMY-1-like/MOX) and of carbapenemases (KPC, OXA-48, VIM, IMP and NDM).

One-hundred-and-eighty-seven well-characterized Gram-negative bacilli isolates possessing different bla genes were tested. Total DNAs were extracted using a Qiagen DNA mini kit. The ‘Check-MDR CT103 array’ was used as recommended by the manufacturer.

The system correctly identified representatives of the three ESBL gene families tested, including differentiation between non-ESBL and ESBL TEM and SHV variants. All bla_CTX-M genes were classified into the appropriate family group (i.e. CTX-M-1 group, CTX-M-2 group, CTX-M-9 group and CTX-M-8/25/26 group). In addition, the clinically relevant plasmid-encoded cephalosporinase and carbapenemase genes were also reliably detected. Specificities and sensitivities of 100% were recorded for most bla genes.

The ‘Check-MDR CT103 array’ is a powerful high-throughput tool for rapid identification of ESBL, pAmpC and carbapenemase producers in culture. Because of its rapid performance, this platform is a valuable tool for epidemiological or infection control studies.

Kanamycin is an important second-line drug used to treat multidrug-resistant (MDR) tuberculosis (TB). Molecular analysis of the rrs gene seems to be not enough to identify every case of kanamycin resistance. In the present study we evaluated the incidence of eis mutations in kanamycin-resistant Mycobacterium tuberculosis isolates.

We analysed 70 MDR M. tuberculosis clinical isolates. All isolates were screened for rrs and eis mutations using single-strand conformation polymorphism and sequencing. Phenotypic drug susceptibility testing was performed using Bactec MGIT 960 and the absolute concentration method on Lowenstein–Jensen medium.

eis mutations were found in 10 isolates. The most prevalent mutations were the A1401G substitution in the rrs gene and the C14T substitution in the eis promoter region.

Our study shows that the eis promoter region is a useful molecular marker of kanamycin resistance in the Moscow region. Complex analysis of rrs and eis mutations will significantly reduce the time to diagnose kanamycin resistance in TB patients, compared with phenotypic drug resistance testing.

The aim of this study was to assess 25-hydroxyvitamin D (vitamin D) status in an HIV-infected adult population and to define HIV- and antiretroviral-related factors associated with vitamin D deficiency.

Using data from a prospective cohort of HIV-infected adult patients followed in five French centres (Dat'AIDS cohort), we evaluated the prevalence of vitamin D deficiency/insufficiency (<30 ng/mL). A multiple linear regression model was used to examine risk factors for vitamin D deficiency (≤10 ng/mL).

Vitamin D deficiency/insufficiency was observed in 86.7% of the 2994 patients, including 55.6% with vitamin D insufficiency and 31.1% with vitamin D deficiency. In multivariate analysis, factors associated with vitamin D deficiency were current smoking [adjusted OR (aOR) 1.55], estimated glomerular filtration rate ≥90 mL/min/1.73 m² (aOR 1.51), vitamin D measurement not performed in summer (aOR 0.27), CD4 <350 cells/mm³ (aOR 1.37 for CD4 200 to <350 and 1.62 for CD4 <200 cells/mm³) and antiretroviral therapy (aOR 2.61). Gender, body mass index, age, coinfection and previous AIDS were not associated factors. In the antiretroviral-treated population (n = 2660), besides the same factors found in the whole population, efavirenz was the only drug to be significantly associated with deficiency, with an aOR of 1.89 (95% CI 1.45–2.47).

Vitamin D deficiency is frequent in this HIV-infected population. Patients on antiretroviral therapy are at higher risk of vitamin D deficiency than antiretroviral-naive patients, with an increased risk in patients receiving efavirenz. No effect of the other antiretrovirals, including the latest (etravirine, darunavir, raltegravir), was found.