Specification and Evaluation of Plasticizer Migration Simulants for Human Blood Products: A Delphi Study.

Potentially toxic plasticizers are commonly added to polyvinyl chloride medical devices for transfusion in order to improve their flexibility and workability. As the plasticizers are not chemically bonded to the PVC, they can be released into labile blood products (LBPs) during storage. Ideally, LBPs would be used in laboratory studies of plasticizer migration from the medical device. However, short supply (i.e., limited stocks of human blood in collection centres) has prompted the development of specific simulants for each type of LBP in the evaluation of new transfusion devices. We performed a Delphi study with a multidisciplinary panel of 24 experts. In the first (qualitative) phase, the panel developed consensus definitions of the specification criteria to be met by each migration simulant. Next, we reviewed the literature on techniques for simulating the migration of plasticizers into LBPs. A questionnaire was elaborated and sent out to the experts, and the replies were synthesized in order to obtain a consensus. The qualitative study established specifications for each biological matrix (whole blood, red blood cell concentrate, plasma, and platelet concentrate) and defined the criteria required for a suitable LBP simulant. Ten criteria were suggested: physical and chemical characteristics, opacity, form, stability, composition, ability to mimic a particular clinical situation, ease and safety of use, a simulant-plastic interaction correlated with blood, and compatibility with analytical methods. The questionnaire data revealed a consensus on the use of natural products (such as pig's blood) to mimic the four LBPs. Opinions diverged with regard to synthetic products. However, an isotonic solution and a rheological property modifier were considered to be of value in the design of synthetic simulants. Consensus reached by the Delphi group could be used as a database for the development of simulants used to assess the migration of plasticizers from PVC bags into LBPs.

Non-phthalate plasticizer DEHT preserves adequate blood component quality during storage in PVC blood bags.

BACKGROUND AND OBJECTIVES: Commercial blood bags are predominantly made of polyvinyl chloride (PVC) plasticized with di(2-ethylhexyl) phthalate (DEHP). DEHP is favourable for storage of red blood cells (RBC). Historically, removal of DEHP from blood bags has been linked to unacceptable haemolysis levels. Oncoming regulatory restrictions for DEHP due to toxicity concerns increase the urgency to replace DEHP without compromising RBC quality. Di(2-ethylhexyl) terephthalate (DEHT) is one suggested substitute. The aim of this study was to compare PVC-DEHT to PVC-DEHP blood bags using additive solutions saline-adenine-glucose-mannitol (SAGM) and phosphate-adenine-glucose-guanosine-saline-mannitol (PAGGSM), to determine whether DEHT can maintain acceptable component quality. MATERIALS AND METHODS: RBC concentrates (N = 64), platelet concentrates (N = 16) and fresh frozen plasma (N = 32) were produced from whole blood collected into either DEHT or DEHP plasticized systems. Using a pool-and-split study design, pairs of identical RBC content were created within each plasticizer arm and assigned either SAGM or PAGGSM. Storage effects were assessed weekly for 49 days (RBC), 7 days (platelets) and before/after freezing (plasma). RESULTS: Though haemolysis was slightly higher in DEHT, all study arms remained below half of the European limit 0·8%. K+ was lower in DEHT than in DEHP independent of additive solution. The metabolic parameters were not influenced by choice of plasticizer. Platelet activation/metabolism and plasma content were similarly preserved. CONCLUSION: Our study demonstrates that the plasticizer DEHT provides adequate blood component quality. We propose DEHT as a strong future candidate for replacement of DEHP in blood bags.

Fosfomycin and Staphylococcus aureus: transcriptomic approach to assess effect on biofilm, and fate of unattached cells.

Interest has been rekindled in the old antibiotic fosfomycin, partly because of its ability to penetrate biofilm. Using a transcriptomic approach, we investigated the modifications induced by fosfomycin in sessile cells of a clinical Staphylococcus aureus isolated from a device-associated infection. Cells still able to form biofilm after 4 h of incubation in the presence of subinhibitory concentrations of fosfomycin and cells from 24-h-old biofilm later submitted to fosfomycin had 6.77% and 9.41%, respectively, of differentially expressed genes compared with their antibiotic-free control. Fosfomycin induced mostly downregulation of genes assigned to nucleotide, amino acid and carbohydrate transport, and metabolism. Adhesins and capsular biosynthesis proteins encoding genes were downregulated in fosfomycin-grown biofilm, whereas the murein hydrolase regulator lgrA and a D-lactate dehydrogenase-encoding gene were upregulated. In fosfomycin-treated biofilm, the expression of genes encoding adhesins, the cell wall biosynthesis protein ScdA, and to a lesser extent the fosfomycin target MurA was also decreased. Unattached cells surrounding fosfomycin-grown biofilm showed greater ability to form aggregates than their counterparts obtained without fosfomycin. Reducing their global metabolism and lowering cell wall turnover would allow some S. aureus cells to grow in biofilm despite fosfomycin stress while promoting hyperadherent phenotype in the vicinity of the fosfomycin-treated biofilm.

Discrimination of Escherichia coli and Shigella spp. by Nuclear Magnetic Resonance Based Metabolomic Characterization of Culture Media.

Dysentery is a major health threat that dramatically impacts childhood morbidity and mortality in developing countries. Various pathogenic agents cause dysentery, such as Shigella spp. and Escherichia coli, which are very closely related if not identical species. Sensitive and precise detection and identification of the infectious agent is important to target the best therapeutic strategy, but the differential diagnosis of these two groups remains a challenge using conventional methods. Here, we present a nuclear magnetic resonance (NMR) based multivariate classification model employing bacterial metabolic footprints in postculture growth media with remarkable segregation capability, including the discrimination of lactose negative E. coli and Shigella spp. Our results confirm the potential of metabolomic markers in the field of bacterial identification for the distinction of even very closely related species.

Colistin Heteroresistance and Involvement of the PmrAB Regulatory System in Acinetobacter baumannii.

Multidrug-resistant Acinetobacter baumannii infection has recently emerged as a worldwide clinical problem, and colistin is increasingly being used as a last-resort therapy. Despite its favorable bacterial killing, resistance and heteroresistance (HR) to colistin have been described. The purpose of the present study was to investigate the role of the PmrAB regulatory pathway in laboratory-selected mutants representative of global epidemic strains. From three unrelated A. baumannii clinical strains (sequence types 2, 3, and 20), eight colistin-resistant mutants were selected. Half of the mutants showed HR to colistin according to the reference method (population analysis profiling), whereas the other half exhibited stable resistance. M12I mutation within pmrA and M308R, S144KLAGS, and P170L mutations for pmrB were associated with HR to colistin, while T235I, A226T, and P233S mutations within pmrB were associated with stable resistance. The transcript levels of the pmrCAB operon were upregulated in all the mutants. Compensatory mutations were explored for some mutants. A single mutant (T235I mutant) displayed a compensatory mutation through ISAba1 mobilization within the pmrB gene that was associated with the loss of colistin resistance. The mutant resistance phenotype associated with T235I was partially restored in a trans-complementation assay turning to HR. The level of colistin resistance was correlated with the level of expression of pmrC in the trans-complemented strains. This report shows the role of different mutations in the PmrAB regulatory pathway and warns of the development of colistin HR that could be present but not easily detected through routine testing.

Phenotypic and Genomic Characterization of AmpC-Producing Klebsiella pneumoniae From Korea.

The prevalence of multidrug-resistant gram-negative bacteria has continuously increased over the past few years; bacterial strains producing AmpC ß-lactamases and/or extended-spectrum ß-lactamases (ESBLs) are of particular concern. We combined high-resolution whole genome sequencing and phenotypic data to elucidate the mechanisms of resistance to cephamycin and ß-lactamase in Korean Klebsiella pneumoniae strains, in which no AmpC-encoding genes were detected by PCR. We identified several genes that alone or in combination can potentially explain the resistance phenotype. We showed that different mechanisms could explain the resistance phenotype, emphasizing the limitations of the PCR and the importance of distinguishing closely-related gene variants.

Enhanced detection of carbapenemase-producing Enterobacteriaceae by an optimized phenol red assay.

Screening for the detection of carbapenemase-producing bacteria still encounters issues related to workflow, limit of detection, or qualitative interpretation. We developed a spectrophotometry-based version of the Carba NP phenol red assay (Nordmann et al., 2012) in a microtiter plate format, compatible with low bacterial cell counts. We were able to detect highly active carbapenemases such as KPC and IMP in 30min. A wider range of carbapenemases including OXA-48 were detected using higher inocula, still being competitive compared with currently available phenol red assays. Validation experiments of our test with a panel of 81 Enterobacteriaceae showed good performance with 93% of sensitivity and 92% of specificity. The compatibility of our routine-friendly protocol with automation offers great perspectives for high throughput screening in outbreak situations and/or in big laboratories.

Identification of mycobacterium spp. and nocardia spp. from solid and liquid cultures by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).

Identification of microorganisms by MALDI-TOF MS has been widely accepted in clinical microbiology. However, for Mycobacterium spp. and Nocardia spp. such identification has not yet reached the optimal level of routine testing. Here we describe the development of an identification tool for 49 and 15 species of Mycobacterium spp. and Nocardia spp., respectively. During database construction, a number of ambiguous reference identifications were revealed and corrected via molecular analyses. Eventually, more than 2000 individual mass spectra acquired from 494 strains were included in a reference database and subjected to bio-statistical analyses. This led to correct species identification and correct combination of species into several complexes or groups, such as the Mycobacterium tuberculosis complex. With the Advanced Spectrum Classifier algorithm, class-specific bin weights were determined and tested by cross-validation experiments with good results. When challenged with independent isolates, overall identification performance was 90% for identification of Mycobacterium spp. and 88% for Nocardia spp. However, for a number of Mycobacterium sp. isolates, no identification could be achieved and in most cases, this could be attributed to the production of polymers that masked the species-specific protein peak patterns. For the species where >20 isolates were tested, correct identification reached 95% or higher. With the current spectral database, the identification of Mycobacterium spp. and Nocardia spp. by MALDI-TOF MS can be performed in routine clinical diagnostics although in some complicated cases verification by sequencing remains mandatory.

Genome Sequence of a Clinical Staphylococcus aureus Strain from a Prosthetic Joint Infection.

Here, we report the genome sequence ofStaphylococcus aureusLYO-S2, an isolate with sequence type (ST) 45 that was isolated in 2001 from a prosthetic joint infection.

A Chlorhexidine- Agar Plate Culture Medium Protocol to Complement Standard Broth Culture of Mycobacterium tuberculosis.

The culture of Mycobacterium tuberculosis using parallel inoculation of a solid culture medium and a liquid broth provides the gold standard for the diagnosis of tuberculosis. Here, we evaluated a chlorhexidine decontamination-MOD9 solid medium protocol versus the standard NALC-NaOH-Bactec 960 MGIT protocol for the diagnosis of pulmonary tuberculosis by culture. Three-hundred clinical specimens comprising 193 sputa, 30 bronchial aspirates, 10 broncho-alveolar lavages, 47 stools, and 20 urines were prospectively submitted for the routine diagnosis of tuberculosis. The contamination rates were 5/300 (1.7%) using the MOD9 protocol and 17/300 (5.7%) with the Bactec protocol, respectively (P < 0.05, Fisher exact test). Of a total of 50 Mycobacterium isolates (48 M. tuberculosis and two Mycobacterium abscessus) were cultured. Out of these 50, 48 (96%) isolates were found using the MOD9 protocol versus 35 (70%) when using the Bactec protocol (P < 0.05, Fisher exact test). The time to positivity was 10.1 ± 3.9 days versus 14.7 ± 7.3 days, respectively, (P < 0.05, Student's t-test). These data confirmed the usefulness of parallel inoculation of a solid culture medium with broth for the recovery of M. tuberculosis in agreement with current recommendations. More specifically, chlorhexidine decontamination and inoculation of the MOD9 solid medium could be proposed to complement the standard Bactec 960 MGIT broth protocol.

Progress in proteomics for clinical microbiology: MALDI-TOF MS for microbial species identification and more.

Although classical proteomic approaches are still used regularly in routine clinical diagnostic procedures, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) MS has recently moved into diagnostic microbiology laboratories. MALDI-TOF MS is currently replacing phenotypic microbial identification. Many laboratories now use MALDI-TOF MS for its high efficiency, both from a diagnostic and a cost-per-analysis point of view. The US FDA has now cleared two of the commercially available systems for in vitro diagnostics. This will further spark development of MS applications in antimicrobial susceptibility testing and epidemiology. This review summarizes the state of affairs of MALDI-TOF MS in clinical microbiology; however, this is an active field of research subject to rapid evolution. We emphasize assessment of the clinical relevance and studies focusing on data obtained through comparative analyses of different MALDI-TOF MS instrumentation and multicenter validation studies. The future of MALDI-TOF MS, including antimicrobial susceptibility testing and epidemiological typing, is also highlighted.

Rapid Bacterial Identification, Resistance, Virulence and Type Profiling using Selected Reaction Monitoring Mass Spectrometry.

Mass spectrometry (MS) in Selected Reaction Monitoring (SRM) mode is proposed for in-depth characterisation of microorganisms in a multiplexed analysis. Within 60-80 minutes, the SRM method performs microbial identification (I), antibiotic-resistance detection (R), virulence assessment (V) and it provides epidemiological typing information (T). This SRM application is illustrated by the analysis of the human pathogen Staphylococcus aureus, demonstrating its promise for rapid characterisation of bacteria from positive blood cultures of sepsis patients.

Effects of antibiotics on biofilm and unattached cells of a clinical Staphylococcus aureus isolate from bone and joint infection.

Treatment of orthopaedic infections remains challenging owing to the inability of antibiotics to eradicate biofilms and prevent their regrowth. The present study characterized the effects of 12 antibiotics on in vitro biofilm formed by a representative strain of meticillin-susceptible Staphylococcus aureus (MSSA) isolated from a bone infection. Determination of the minimum biofilm eradication concentrations indicated that in vitro eradication of 24 h-old biofilms required concentrations up to 51,200 times higher than MICs. The influence of the same panel of antibiotics was also investigated on biofilm formation at concentrations including the breakpoints, by numbering viable cells in the suspensions (individual cells) and the biofilm biomass. Except for fusidic acid, the presence of antibiotics during the initial steps of biofilm formation resulted in significant decreases in the number of sessile viable bacteria at the highest concentrations tested. Ceftarolin, daptomycin, fosfomycin, gentamicin, ofloxacin, rifampicin and vancomycin were the most effective drugs. Confocal microscopy analysis indicated that daptomycin was more efficient at bacteria lysis than gentamicin and vancomycin. However, viable individual cells were still detectable in the assays performed with ceftarolin, fosfomycin, ofloxacin, rifampicin and vancomycin at concentrations for which no sessile cells were detected. Although none of the molecules tested was effective at classical therapeutic concentrations against 24 h-old MSSA biofilms, all except fusidic acid were able to impair biofilm formation at concentrations near the breakpoints. However, presence of viable individual unattached cells could imply a significant risk of microbial dissemination and increased risk of infections.

Label-free SRM-based relative quantification of antibiotic resistance mechanisms in Pseudomonas aeruginosa clinical isolates.

Both acquired and intrinsic mechanisms play a crucial role in Pseudomonas aeruginosa antibiotic resistance. Many clinically relevant resistance mechanisms result from changes in gene expression, namely multidrug efflux pump overproduction, AmpC ß-lactamase induction or derepression, and inactivation or repression of the carbapenem-specific porin OprD. Changes in gene expression are usually assessed using reverse-transcription quantitative real-time PCR (RT-qPCR) assays. Here, we evaluated label-free Selected Reaction Monitoring (SRM)-based mass spectrometry to directly quantify proteins involved in antibiotic resistance. We evaluated the label-free SRM using a defined set of P. aeruginosa isolates with known resistance mechanisms and compared it with RT-qPCR. Referring to efflux systems, we found a more robust relative quantification of antibiotic resistance mechanisms by SRM than RT-qPCR. The SRM-based approach was applied to a set of clinical P. aeruginosa isolates to detect antibiotic resistance proteins. This multiplexed SRM-based approach is a rapid and reliable method for the simultaneous detection and quantification of resistance mechanisms and we demonstrate its relevance for antibiotic resistance prediction.

The ongoing revolution of MALDI-TOF mass spectrometry for microbiology reaches tropical Africa.

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) represents a revolution in routine pathogen identification in clinical microbiology laboratories. A MALDI-TOF MS was introduced to tropical Africa in the clinical microbiology laboratory of the Hôpital Principal de Dakar (Senegal) and used for routine pathogen identification. Using MS, 2,429 bacteria and fungi isolated from patients were directly assayed, leading to the identification of 2,082 bacteria (85.7%) and 206 fungi (8.5%) at the species level, 109 bacteria (4.5%) at the genus level, and 16 bacteria (0.75%) at the family level. Sixteen isolates remained unidentified (0.75%). Escherichia coli was the most prevalent species (25.8%) followed by Klebsiella pneumoniae (14.8%), Streptococcus agalactiae (6.2%), Acinetobacter baumannii (6.1%), Pseudomonas aeruginosa (5.9%), and Staphylococcus aureus (5.9%). MALDI-TOF MS has also enabled the detection of rare bacteria and fungi. MALDI-TOF MS is a powerful tool for the identification of bacterial and fungal species involved in infectious diseases in tropical Africa.

Meropenem/colistin synergy testing for multidrug-resistant Acinetobacter baumannii strains by a two-dimensional gradient technique applicable in routine microbiology.

OBJECTIVES: Precise assessment of potential therapeutic synergy, antagonism or indifference between antimicrobial agents currently depends on time-consuming and hard-to-standardize in vitro chequerboard titration methods. We here present a method based on a novel two-dimensional antibiotic gradient technique named Xact™. METHODS: We used a test comprising a combination of perpendicular gradients of meropenem and colistin in a single quadrant. We compared test outcomes with those obtained with classical chequerboard microbroth dilution testing in a study involving 27 unique strains of multidrug-resistant Acinetobacter baumannii from diverse origins. RESULTS: We were able to demonstrate 92% concordance between the new technology and classical chequerboard titration using the A. baumannii collection. Two strains could not be analysed by Xact™ due to their out-of-range MIC of meropenem (>128 mg/L). CONCLUSIONS: The new test was shown to be diagnostically useful, easy to implement and less labour intensive than the classical method.

Three-dimensional characterization of bacterial microcolonies on solid agar-based culture media.

For the last century, in vitro diagnostic process in microbiology has mainly relied on the growth of bacteria on the surface of a solid agar medium. Nevertheless, few studies focused in the past on the dynamics of microcolonies growth on agar surface before 8 to 10h of incubation. In this article, chromatic confocal microscopy has been applied to characterize the early development of a bacterial colony. This technology relies on a differential focusing depth of the white light. It allows one to fully measure the tridimensional shape of microcolonies more quickly than classical confocal microscopy but with the same spatial resolution. Placing the device in an incubator, the method was able to individually track colonies growing on an agar plate, and to follow the evolution of their surface or volume. Using an appropriate statistical modeling framework, for a given microorganism, the doubling time has been estimated for each individual colony, as well as its variability between colonies, both within and between agar plates. A proof of concept led on four bacterial strains of four distinct species demonstrated the feasibility and the interest of the approach. It showed in particular that doubling times derived from early tri-dimensional measurements on microcolonies differed from classical measurements in micro-dilutions based on optical diffusion. Such a precise characterization of the tri-dimensional shape of microcolonies in their late-lag to early-exponential phase could be beneficial in terms of in vitro diagnostics. Indeed, real-time monitoring of the biomass available in a colony could allow to run well established microbial identification workflows like, for instance, MALDI-TOF mass-spectrometry, as soon as a sufficient quantity of material is available, thereby reducing the time needed to provide a diagnostic. Moreover, as done for pre-identification of macro-colonies, morphological indicators such as three-dimensional growth profiles derived from microcolonies could be used to perform a first pre-identification step, but in a shorten time.

Comparison of matrix-assisted laser desorption ionization-time of flight mass spectrometry and molecular biology techniques for identification of Culicoides (Diptera: ceratopogonidae) biting midges in senegal.

Biting midges of the genus Culicoides are implicated as vectors for a wide variety of pathogens. The morphological identification of these arthropods may be difficult because of a lack of detailed investigation of taxonomy for this species in Africa. However, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) profiling is efficient for arthropod identification at the species level. This study established a spectrum database of Culicoides spp. from Senegal using MALDI-TOF. Identification of Culicoides insects to the species level before mass spectrometry was performed on the basis of morphological characters. MALDI-TOF MS reference spectra were determined for 437 field-caught Culicoides of 10 species. The protein profiles of all tested Culicoides revealed several peaks with mass ranges of 2 to 20 kDa. In a validation study, 72 Culicoides specimens in the target species were correctly identified at the species level with a similarity of 95 to 99.9%. Four Culicoides protein profiles were misidentified. Nevertheless, six SuperSpectra (C. imicola, C. enderleini, C. oxystoma, C. kingi, C. magnus, and C. fulvithorax) were created. Abdomens of midges were used to amplify and sequence a portion of the mitochondrial cytochrome oxidase I gene (COI). The results obtained using the MALDI-TOF MS method were consistent with the morphological identification and similar to the genetic identification. Protein profiling using MALDI-TOF is an efficient approach for the identification of Culicoides spp., and it is economically advantageous for approaches that require detailed and quantitative information of vector species that are collected in field. The database of African Culicoides MS spectra created is the first database in Africa. The COI sequences of five Culicoides species that were previously noncharacterized using molecular methods were deposited in GenBank.

Comparison of two approaches for the classification of 16S rRNA gene sequences.

The use of 16S rRNA gene sequences for microbial identification in clinical microbiology is accepted widely, and requires databases and algorithms. We compared a new research database containing curated 16S rRNA gene sequences in combination with the lca (lowest common ancestor) algorithm (RDB-LCA) to a commercially available 16S rDNA Centroid approach. We used 1025 bacterial isolates characterized by biochemistry, matrix-assisted laser desorption/ionization time-of-flight MS and 16S rDNA sequencing. Nearly 80 % of isolates were identified unambiguously at the species level by both classification platforms used. The remaining isolates were mostly identified correctly at the genus level due to the limited resolution of 16S rDNA sequencing. Discrepancies between both 16S rDNA platforms were due to differences in database content and the algorithm used, and could amount to up to 10.5 %. Up to 1.4 % of the analyses were found to be inconclusive. It is important to realize that despite the overall good performance of the pipelines for analysis, some inconclusive results remain that require additional in-depth analysis performed using supplementary methods.

Challenges in the culture-independent analysis of oral and respiratory samples from intubated patients.

The spread of microorganisms in hospitals is an important public health threat, and yet few studies have assessed how human microbial communities (microbiota) evolve in the hospital setting. Studies conducted so far have mainly focused on a limited number of bacterial species, mostly pathogenic ones and primarily during outbreaks. We explored the bacterial community diversity of the microbiota from oral and respiratory samples of intubated patients hospitalized in the intensive care unit and we discuss the technical challenges that may arise while using culture-independent approaches to study these types of samples.