Improvement of transport condition of swabs for group B streptococcal (GBS) screening

peer reviewedBACKGROUND For the screening-based strategy for prevention of perinatal GBS disease, CDC Guidelines as many others recommend use of appropriate transport media (Amies, Stuart, e.g.) and processing of specimen as soon as possible within 1 to 4 days. False negative cultures occur for several causes including lost of GBS viability during transport. Could Lim broth, recommended for the selective enrichment, and Granada tubes be used as transport media for swab? Simulating conditions of routine practice, Lim broth and Granada tubes, were evaluated in vitro as transport media. METHODS Tubes of 3 brands of Lim broth (Becton Dickinson, bioMérieux, Copan) and Granada tubes (bioMérieux) were inoculated with low inocula of 10-100 CFU of GBS. Each type of tubes was incubated at 4°C, room T° (RT) and 35°C. GBS were enumerated from each tube by subculture on blood agar after 1, 2, 3 and 4 days of storage at the different T°. All tests were processed in triplicates with 3 strains of GBS belonging to serotype Ia, III and V. RESULTS No difference of survival was observed between the 3 strains. T° had significant impact on GBS recovery for each type of tubes. At 4°C the viability was hardly sustained along the 4 days. At RT and 35°C, an increase >6 log of the inocula was observed. The increase of GBS density was sustained at least 4 days for the 3 brands of Lim broth. For the Granada broth, such increase was also observed but at day 3 for tubes incubated at 35°C, viability decreased and for some tubes, GBS subcultures were negative at day 3 or 4. CONCLUSION To improve sensitivity of GBS screening cultures, Lim broth could be recommended as a strong transport media and the advisable storage condition would be RT to 35°C up to 4 days. In this way, initiating selective enrichment culture at the time of collection of specimen would provide higher sensitivity even for low density of colonization. Transport at 4°C should be avoided in favour with RT to 35°C. Studies in clinical setting are expected. For Granada tubes, storage at RT was fine but improvement seemed restricted in time at 35°C as there was a loss of viability after 3 days. For Granada tubes, extended evaluation and delimitation of use are needed

Prospective Two-Center Comparison of Three Chromogenic Agars for Methicillin-Resistant Staphylococcus aureus Screening in Hospitalized Patients

Three chromogenic media, chromID MRSA SMART (SMART), chromID MRSA first generation (chromID), and Brilliance MRSA (OX2), were evaluated for methicillin-resistant Staphylococcus aureus (MRSA) screening using 1,220 samples. The sensitivity at 24 h was significantly better with the SMART agar (66.4%) than that with chromID agar (50.5%). Enrichment and incubation until 48 h are still needed for an optimal yield

Evaluation of the automated BD Phoenix CPO Detect test for detection and classification of carbapenemases in Gram negatives.

We evaluated the performance of the automated BD Phoenix CPO Detect test (BD-CPO test) for detection and Ambler classification of carbapenemases in Enterobacteriaceae, P.aeruginosa and A.baumannii complex. A collection of 287 Gram-negative clinical isolates, with a reduced susceptibility to at least one carbapenem including 184 carbapenemase-producing organisms (CPO) and 103 non-CPO, was tested. The BD-CPO test showed an overall sensitivity of 89.7% and specificity of 83.5% for carbapenemase detection. 1/7 of class A, 82.9% of class B, and 89.8% of class D carbapenemases were correctly classified. Poor detection sensitivity of 68.9% and specificity of 62.1% in P.aeruginosa was observed. However, combination with ceftazidime/avibactam susceptibility, provided by this panel, increased the performances for P.aeruginosa. The integration of an automated carbapenemase detection and classification in routine susceptibility panels would save time and help for therapeutic management. Further developments are needed to improve the accuracy of the BD-CPO test

The Persister Character of Clinical Isolates of Staphylococcus aureus Contributes to Faster Evolution to Resistance and Higher Survival in THP-1 Monocytes: A Study With Moxifloxacin

Staphylococcus aureus may cause relapsing infections. We previously showed that S. aureus SH1000 surviving intracellularly to bactericidal antibiotics are persisters. Here, we used 54 non-duplicate clinical isolates to assess links between persistence, resistance evolution, and intracellular survival, using moxifloxacin throughout as test bactericidal antibiotic. The relative persister fraction (RPF: percentage of inoculum surviving to 100× MIC moxifloxacin in stationary phase culture for each isolate relative to ATCC 25923) was determined to categorize isolates with low (≤10) or high (>10) RPF. Evolution to resistance (moxifloxacin MIC ≥ 0.5 mg/L) was triggered by serial passages at 0.5× MIC (with daily concentration readjustments). Intracellular moxifloxacin maximal efficacy (Emax) was determined by 24 h concentration-response experiments [pharmacodynamic model (Hill-Langmuir)] with infected THP-1 monocytes exposed to moxifloxacin (0.01 to 100× MIC) after phagocytosis. Division of intracellular survivors was followed by green fluorescence protein dilution (FACS). Most (30/36) moxifloxacin-susceptible isolates showed low RPF but all moxifloxacin-resistant (n = 18) isolates harbored high RPF. Evolution to resistance of susceptible isolates was faster for those with high vs. low RPF (with SOS response and topoisomerase-encoding genes overexpression). Intracellularly, moxifloxacin Emax was decreased (less negative) for isolates with high vs. low RPF, independently from resistance. Moxifloxacin intracellular survivors were non-dividing. The data demonstrate and quantitate persisters in clinical isolates of S. aureus, and show that this phenotype accelerates resistance evolution and is associated with intracellular survival in spite of high antibiotic concentrations. Isolates with high RPF may represent a possible cause of treatment failure not directly related to resistance in patients receiving active antibiotics

European external quality assessments for identification, molecular typing and characterization of Staphylococcus aureus

Objectives: We present the results of two European external quality assessments (EQAs) conducted in 2014 and 2016 under the auspices of the Study Group on Staphylococci and Staphylococcal Infections of ESCMID. The objective was to assess the performance of participating centres in characterizing Staphylococcus aureus using their standard in-house phenotypic and genotypic protocols. Methods: A total of 11 well-characterized blindly coded S. aureus (n = 9), Staphylococcus argenteus (n = 1) and Staphylococcus capitis (n = 1) strains were distributed to participants for analysis. Species identification, MIC determination, antimicrobial susceptibility testing, antimicrobial resistance and toxin gene detection and molecular typing including spa typing, SCCmec typing and MLST were performed. Results: Thirteen laboratories from 12 European countries participated in one EQA or both EQAs. Despite considerable diversity in the methods employed, good concordance (90%-100%) with expected results was obtained. Discrepancies were observed for: (i) identification of the S. argenteus strain; (ii) phenotypic detection of low-level resistance to oxacillin in the mecC-positive strain; (iii) phenotypic detection of the inducible MLSB strain; and (iv) WGS-based detection of some resistance and toxin genes. Conclusions: Overall, good concordance (90%-100%) with expected results was observed. In some instances, the accurate detection of resistance and toxin genes from WGS data proved problematic, highlighting the need for validated and internationally agreed-on bioinformatics pipelines before such techniques are implemented routinely by microbiology laboratories. We strongly recommend all national reference laboratories and laboratories acting as referral centres to participate in such EQA initiatives