Christine Lammens

Development of a Multiplex PCR for the Detection of asa1, gelE, cylA, esp, and hyl Genes in Enterococci and Survey for Virulence Determinants among European Hospital Isolates of Enterococcus faecium

ABSTRACT A multiplex PCR for the simultaneous detection of five virulence genes (asa1, gelE, cylA, esp, and hyl) in enterococci was developed. The presence of these genes was investigated in 153 clinical and 118 fecal Enterococcus faecium isolates from inpatients at an increased risk of developing infections (such as patients in intensive care units and hematology wards) from 13 hospitals in eight European countries. Of the 271 E. faecium isolates, 135 were vancomycin resistant E. faecium (VREF) isolates and 136 were vancomycin susceptible E. faecium (VSEF) isolates. Susceptibilities to ampicillin, gentamicin, streptomycin, vancomycin, teicoplanin, ramoplanin, quinupristin-dalfopristin, and linezolid were tested by the microdilution method. Overall, the prevalence of esp was significantly higher (P = 0.03) in clinical VREF isolates (92%) than in fecal VREF isolates (73%). In Italy, the prevalence of esp was significantly higher (P = 0.02) in VREF isolates (91%) than in VSEF isolates (68%), whereas in the United Kingdom, hyl was significantly more prevalent (P = 0.01) in VREF isolates (71%) than in VSEF isolates (29%). No significant differences were found for the other countries. Pulsed-field gel electrophoresis was used to check the clonality among the strains tested and showed the spread of two center-specific (esp-positive) VREF clones in Italy and one center-specific (hyl-positive) clone in the United Kingdom. These clones were resistant to ampicillin, gentamicin, and streptomycin. The multiplex PCR reported in this study is a convenient and rapid method for the simultaneous detection of the virulence genes asa1, gelE, cylA, esp, and hyl in enterococci. Molecular analysis showed the intrahospital spread of esp-positive VREF clones (in Italy) and hyl-positive VREF clones (in the United Kingdom); the role of hyl remains to be elucidated.

Effect of azithromycin and clarithromycin therapy on pharyngeal carriage of macrolide-resistant streptococci in healthy volunteers: a randomised, double-blind, placebo-controlled study

BACKGROUND Resistance to antibiotics is a major public-health problem, and studies that link antibiotic use and resistance have shown an association but not a causal effect. We used the macrolides azithromycin and clarithromycin to investigate the direct effect of antibiotic exposure on resistance in the oral streptococcal flora of healthy volunteers. METHODS Volunteers were treated with azithromycin (n=74), clarithromycin (74), or placebo (76) in a randomised, double-blind trial. Pharyngeal swabs were obtained before and after administration of study treatment through 180 days. The proportion of streptococci that were macrolide resistant was assessed and the molecular basis of any change in resistance investigated. Analyses were done on an intent-to-treat basis. This study is registered with ClinicalTrials.gov, number NCT00354952. FINDINGS The number of dropouts (n=20) was much the same in all groups until day 42; dropouts increased substantially at day 180 (105). Both macrolides significantly increased the proportion of macrolide-resistant streptococci compared with the placebo at all points studied, peaking at day 8 in the clarithromycin group (mean increase 50.0%, 95% CI 41.7-58.2; p<0.0001) and at day 4 in the azithromycin group (53.4%, 43.4-63.5; p<0.0001). The proportion of macrolide-resistant streptococci was higher after azithromycin treatment than after clarithromycin use, with the largest difference between the two groups at day 28 (17.4% difference, 9.2-25.6; p<0.0001). Use of clarithromycin, but not of azithromycin, selected for the erm(B) gene, which confers high-level macrolide resistance. INTERPRETATION This study shows that, notwithstanding the different outcomes of resistance selection, macrolide use is the single most important driver of the emergence of macrolide resistance in vivo. Physicians prescribing antibiotics should take into account the striking ecological side-effects of such antibiotics.

Identification of a novel plasmid-mediated colistin-resistance gene, mcr-2, in Escherichia coli, Belgium, June 2016

We identified a novel plasmid-mediated colistin-resistance gene in porcine and bovine colistin-resistant Escherichia coli that did not contain mcr-1. The gene, termed mcr-2, a 1,617 bp phosphoethanolamine transferase harboured on an IncX4 plasmid, has 76.7% nucleotide identity to mcr-1. Prevalence of mcr-2 in porcine colistin-resistant E. coli (11/53) in Belgium was higher than that of mcr-1 (7/53). These data call for an immediate introduction of mcr-2 screening in ongoing molecular epidemiological surveillance of colistin-resistant Gram-negative pathogens.

Evaluation of Arbitrarily Primed PCR Analysis and Pulsed-Field Gel Electrophoresis of Large Genomic DNA Fragments for Identification of Enterococci Important in Human Medicine

The increasing problems encountered with enterococcal nosocomial infections and the intrinsic and acquired resistance of the enterococci to different antimicrobial compounds highlight the need for a rapid identification technique. Enterococcus faecalis is readily identified by biochemical tests, but species differentiation within the Enterococcus faecium and Enterococcus gallinarum species groups is less well established. In the present study, 66 strains representing the most prevalent human enterococci were used to develop a PCR-based species-specific identification protocol. Whole-cell protein analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used as a reference method for species identification. In addition, the genomic SmaI macro-restriction fragment distribution of all of the strains was examined by pulsed-field gel electrophoresis (PFGE). Oligonucleotide D11344-primed PCR was as discriminative as whole-cell protein analysis and resulted in more easily interpreted band patterns. This PCR-based technique allowed identification of clinical isolates by visual examination of the DNA profiles obtained. The inability of both methods to discriminate between Enterococcus casseliflavus and Enterococcus flavescens brought into question the species status of E. flavescens. PFGE did not result in species-discriminative DNA bands or band patterns, but proved to be superior for interpretation of interstrain relationships.

Multiplex PCR for Simultaneous Detection of Macrolide and Tetracycline Resistance Determinants in Streptococci

ABSTRACT Resistance to macrolides and tetracyclines is increasing among streptococci and co-occurs as their resistance determinants are carried on the same mobile element. We developed a multiplex PCR to facilitate simultaneous and specific detection of resistance determinants for both macrolides [erm(A), erm(B), and mef(A/E)] and tetracyclines [tet(M), tet(O), tet(K), and tet(L)] in streptococci.

Use of serum C reactive protein and procalcitonin concentrations in addition to symptoms and signs to predict pneumonia in patients presenting to primary care with acute cough: diagnostic study

Objectives To quantify the diagnostic accuracy of selected inflammatory markers in addition to symptoms and signs for predicting pneumonia and to derive a diagnostic tool. Design Diagnostic study performed between 2007 and 2010. Participants had their history taken, underwent physical examination and measurement of C reactive protein (CRP) and procalcitonin in venous blood on the day they first consulted, and underwent chest radiography within seven days. Setting Primary care centres in 12 European countries. Participants Adults presenting with acute cough. Main outcome measures Pneumonia as determined by radiologists, who were blind to all other information when they judged chest radiographs. Results Of 3106 eligible patients, 286 were excluded because of missing or inadequate chest radiographs, leaving 2820 patients (mean age 50, 40% men) of whom 140 (5%) had pneumonia. Re-assessment of a subset of 1675 chest radiographs showed agreement in 94% (κ 0.45, 95% confidence interval 0.36 to 0.54). Six published “symptoms and signs models” varied in their discrimination (area under receiver operating characteristics curve (ROC) ranged from 0.55 (95% confidence interval 0.50 to 0.61) to 0.71 (0.66 to 0.76)). The optimal combination of clinical prediction items derived from our patients included absence of runny nose and presence of breathlessness, crackles and diminished breath sounds on auscultation, tachycardia, and fever, with an ROC area of 0.70 (0.65 to 0.75). Addition of CRP at the optimal cut off of >30 mg/L increased the ROC area to 0.77 (0.73 to 0.81) and improved the diagnostic classification (net reclassification improvement 28%). In the 1556 patients classified according to symptoms, signs, and CRP >30 mg/L as “low risk” (<2.5%) for pneumonia, the prevalence of pneumonia was 2%. In the 132 patients classified as “high risk” (>20%), the prevalence of pneumonia was 31%. The positive likelihood ratio of low, intermediate, and high risk for pneumonia was 0.4, 1.2, and 8.6 respectively. Measurement of procalcitonin added no relevant additional diagnostic information. A simplified diagnostic score based on symptoms, signs, and CRP >30 mg/L resulted in proportions of pneumonia of 0.7%, 3.8%, and 18.2% in the low, intermediate, and high risk group respectively. Conclusions A clinical rule based on symptoms and signs to predict pneumonia in patients presenting to primary care with acute cough performed best in patients with mild or severe clinical presentation. Addition of CRP concentration at the optimal cut off of >30 mg/L improved diagnostic information, but measurement of procalcitonin concentration did not add clinically relevant information in this group.

Colistin resistance gene mcr-1 harboured on a multidrug resistant plasmid

Reference: Malhotra Surbhi, Xavier Basil Britto, Das Anupam, Lammens Christine, Butaye Patrick, Goossens Herman.Colistin resistance gene mcr-1 harboured on a multidrug resistant plasmid The lancet infectious diseases ISSN 1473-3099 16:3(2016), p. 283-284 Full text (Publishers DOI): http://dx.doi.org/doi:10.1016/S1473-3099(16)00012-8 To cite this reference: http://hdl.handle.net/10067/1312920151162165141

Colistin-resistant Escherichia coli harbouring mcr-1 isolated from food animals in Hanoi, Vietnam

286 www.thelancet.com/infection Vol 16 March 2016 consistent with it being nested within an ISApl1 composite transposon (figure). IS1294 uses a one-ended, rolling circle transposition mechanism capable of mobilising adjacent sequences. We postulate that mcr-1 was originally introduced into the ISApl1 element by IS1294, which was subsequently lost. We have identifi ed mcr-1 in a human faecal E coli isolate in Cambodia taken 2 years before the human isolates were collected in the study by Liu and colleagues, and which is associated with diff erent genetic structures than those reported by Liu and colleagues. IS1294-mediated transposition of the gene into an ISApl1 composite transposon may have represented the original import mechanism of mcr-1 into Enterobacteriaceae. Large, established databases of whole genome sequences represent a rich repository to investigate the historical presence of novel resistance mechanisms.

A Sensitive Assay for Virus Discovery in Respiratory Clinical Samples

In 5–40% of respiratory infections in children, the diagnostics remain negative, suggesting that the patients might be infected with a yet unknown pathogen. Virus discovery cDNA-AFLP (VIDISCA) is a virus discovery method based on recognition of restriction enzyme cleavage sites, ligation of adaptors and subsequent amplification by PCR. However, direct discovery of unknown pathogens in nasopharyngeal swabs is difficult due to the high concentration of ribosomal RNA (rRNA) that acts as competitor. In the current study we optimized VIDISCA by adjusting the reverse transcription enzymes and decreasing rRNA amplification in the reverse transcription, using hexamer oligonucleotides that do not anneal to rRNA. Residual cDNA synthesis on rRNA templates was further reduced with oligonucleotides that anneal to rRNA but can not be extended due to 3′-dideoxy-C6-modification. With these modifications >90% reduction of rRNA amplification was established. Further improvement of the VIDISCA sensitivity was obtained by high throughput sequencing (VIDISCA-454). Eighteen nasopharyngeal swabs were analysed, all containing known respiratory viruses. We could identify the proper virus in the majority of samples tested (11/18). The median load in the VIDISCA-454 positive samples was 7.2 E5 viral genome copies/ml (ranging from 1.4 E3–7.7 E6). Our results show that optimization of VIDISCA and subsequent high-throughput-sequencing enhances sensitivity drastically and provides the opportunity to perform virus discovery directly in patient material.

An outbreak of Burkholderia cepacia with septicemia on a cardiology ward

During a 3-day period, eight patients developed septicemia with Burkholderia cepacia. Heparin injection was found to be a risk factor. Heparin was diluted with dextrose solution, which was aspirated from a 1-L bag. B cepacia, genotypically identical to the blood isolates, was isolated from this bag.