Missed pertussis diagnosis during co-infection with Bordetella holmesii.

The purpose of this study is to identify predictive factors associated with missed diagnosis of B. pertussis-B. holmesii co-infection by assessing the analytical performance of a commercially available multiplexed PCR assay and by building a prediction model based on clinical signs and symptoms for detecting co-infections. This is a retrospective study on the electronic health records of all clinical samples that tested positive to either B. pertussis or B. holmesii from January 2015 to January 2018 at Geneva University Hospitals. Multivariate logistic regression was used to build a model for co-infection prediction based on the electronic health record chart review. Limit of detection was determined for all targets of the commercial multiplexed PCR assay used on respiratory samples. A regression model, developed from clinical symptoms and signs, predicted B. pertussis and B. holmesii co-infection with an accuracy of 82.9% (95% CI 67.9-92.8%, p value = .012), for respiratory samples positive with any of the two tested Bordetella species. We found that the LOD of the PCR reaction targeting ptxS1 is higher than that reported by the manufacturer by a factor 10. The current testing strategy misses B. pertussis and B. holmesii co-infections by reporting only B. holmesii infections. Thus, we advocate to perform serological testing for detecting a response against pertussis toxin whenever a sample is found positive for B. holmesii. These findings are important, both from a clinical and epidemiological point of view, as the former impacts the choice of antimicrobial drugs and the latter biases surveillance data, by underestimating B. pertussis infections during co-infections.

Deciphering Multifactorial Resistance Phenotypes in Acinetobacter baumannii by Genomics and Targeted Label-free Proteomics.

Resistance to ß-lactams in Acinetobacter baumannii involves various mechanisms. To decipher them, whole genome sequencing (WGS) and real-time quantitative polymerase chain reaction (RT-qPCR) were complemented by mass spectrometry (MS) in selected reaction monitoring mode (SRM) in 39 clinical isolates. The targeted label-free proteomic approach enabled, in one hour and using a single method, the quantitative detection of 16 proteins associated with antibiotic resistance: eight acquired ß-lactamases (i.e. GES, NDM-1, OXA-23, OXA-24, OXA-58, PER, TEM-1, and VEB), two resident ß-lactamases (i.e. ADC and OXA-51-like) and six components of the two major efflux systems (i.e. AdeABC and AdeIJK). Results were normalized using "bacterial quantotypic peptides," i.e. peptide markers of the bacterial quantity, to obtain precise protein quantitation (on average 8.93% coefficient of variation for three biological replicates). This allowed to correlate the levels of resistance to ß-lactam with those of the production of acquired as well as resident ß-lactamases or of efflux systems. SRM detected enhanced ADC or OXA-51-like production and absence or increased efflux pump production. Precise protein quantitation was particularly valuable to detect resistance mechanisms mediated by regulated genes or by overexpression of chromosomal genes. Combination of WGS and MS, two orthogonal and complementary techniques, allows thereby interpretation of the resistance phenotypes at the molecular level.

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.

Development of Mass Spectrometry Imaging on skeletal muscle to characterize the local pro-inflammatory and pro-resolution lipid responses in a vaccination context.

Vaccine reactogenicity is well documented at the clinical level but the mechanism involved at the local or systemic level are still poorly understood. Muscular tissue where most vaccines are administered is the first place of interaction between the vaccine formulation and the host's immune cells. So far, this site of vaccine administration is not well documented from a mechanistic standpoint. The study of early molecular events at the injection site is crucial to understand the local response to vaccines. In this paper, we report a standardized workflow, from the injection of vaccine formulations in rabbit muscle, to the analysis by desorption electrospray ionization and histology staining to understand the role of lipids involved in the inflammation and its resolution on striated muscular tissue. The analysis of lipid mediators was optimized at the site of needle insertion to allow the spatial comparison of cellular infiltrates at the injection site. We showed that lipids were distributed across the spatial tissue morphology in a time-dependent manner. The MS imaging applied to vaccinology could pave the way to a better understanding of vaccine reactogenicity and mechanism of action.

Changes in Microbiota Profiles After Prolonged Frozen Storage of Stool Suspensions.

Introduction: Fecal microbiota transplantation (FMT) is recommended as safe and effective treatment for recurrent Clostridioides difficile infections. Freezing the FMT preparation simplifies the process, allowing a single stool sample to be used for multiple receivers and over an extended period of time. We aimed to assess the effect of long-term frozen storage on bacterial taxonomic profiles of a stool suspension prepared for FMT. Methods: DNA was extracted from a stool suspension before freezing and sequentially during the 18-month storage period at -80°C. Two different protocols were used for DNA extraction. The first relied on a classical mechanical and chemical cell disruption to extract both intra- and extracellular DNA; the second included specific pre-treatments aimed at removing free DNA and DNA from human and damaged bacterial cells. Taxonomic profiling of bacterial communities was performed by sequencing of V3-V4 16S rRNA gene amplicons. Results: Microbiota profiles obtained by whole DNA extraction procedure remained relatively stable during frozen storage. When DNA extraction procedure included specific pre-treatments, microbiota similarity between fresh and frozen samples progressively decreased with longer frozen storage times; notably, the abundance of Bacteroidetes decreased in a storage duration-dependent manner. The abundance of Firmicutes, the main butyrate producers in the colon, were not much affected by frozen storage for up to 1 year. Conclusion: Our data show that metataxonomic analysis of frozen stool suspensions subjected to specific pre-treatments prior to DNA extractions might provide an interesting indication of bacterial resistance to stress conditions and thus of chances of survival in FMT recipients.

Targeting the Gut Microbiota to Treat Cachexia.

Cachexia occurs in many chronic diseases and is associated with increased morbidity and mortality. It is treated by nutritional support but often with limited effectiveness, leading to the search of other therapeutic strategies. The modulation of gut microbiota, whether through pro-, pre-, syn- or antibiotics or fecal transplantation, is attracting ever-growing interest in the field of obesity, but could also be an interesting and innovative alternative for treating cachexia. This article reviews the evidence linking the features of malnutrition, as defined by the Global Leadership Initiative on Malnutrition [low body mass index (BMI), unintentional body weight loss, low muscle mass, low appetite, and systemic inflammation] and the gut microbiota in human adults with cachexia-associated diseases, and shows the limitations of the present research in that field with suggestions for future directions.

Automated Incubation and Digital Image Analysis of Chromogenic Media Using Copan WASPLab Enables Rapid Detection of Vancomycin-Resistant Enterococcus.

Objective: The aim of the present study was to assess whether the WASPLab automation enables faster detection of vancomycin-resistant Enterococcus (VRE) on chromogenic VRE-specific plates by shortening the incubation time. Methods: Ninety different VRE culture negative rectal ESwab specimens were spiked with various concentrations (ranging from 3 × 102 to 3 × 107 CFU/ml) of 10 Enterococcus faecium strains (vancomycin MICs ranging from 32 to >256 mg/l), 3 E. faecium VanB strains (vancomycin MICs: 4, 8, and 16 mg/l), and 2 E. faecium VanB strains displaying vancomycin heteroresistance (vancomycin MICs: 64 and 96 mg/l). Results: Besides the two strains exhibiting vancomycin heteroresistance, all the other 13 VRE strains included in this study were detected as early as 24 h on the WASPLab even if the inoculum was low (3 × 103 CFU/ml). When the vancomycin MICs were high, all strains were detected as early as at 18 h. However, 30 h was a conservative time point for finalizing the analysis of chromogenic cultures. Conclusion: These results suggested that the WASPLab automated incubation could allow decreasing the initial incubation time to 18 h, followed by an intermediate time at 24 h and a final incubation period of 30 h for VRE culture screening, to deliver rapid results without affecting the analytical sensitivity.

Next-Generation Sequencing for the Diagnosis of Challenging Culture-Negative Endocarditis.

Diagnosis of culture-negative infective endocarditis usually implies indirect pathogen identification by serologic or molecular techniques. Clinical metagenomics, relying on next-generation sequencing (NGS) is an emerging approach that allows pathogen identification in challenging situations, as evidenced by a clinical case. We sequenced the DNA extracted from the surgically-removed frozen valve tissue from a patient with suspected infective endocarditis with negative blood and valve cultures. Mapping of the sequence reads against reference genomic sequences, a 16S rRNA gene database and clade-specific marker genes suggested an infection caused by Cardiobacterium hominis.

Mass spectrometry methods for predicting antibiotic resistance.

Developing elaborate techniques for clinical applications can be a complicated process. Whole-cell MALDI-TOF MS revolutionized reliable microorganism identification in clinical microbiology laboratories and is now replacing phenotypic microbial identification. This technique is a generic, accurate, rapid, and cost-effective growth-based method. Antibiotic resistance keeps emerging in environmental and clinical microorganisms, leading to clinical therapeutic challenges, especially for Gram-negative bacteria. Antimicrobial susceptibility testing is used to reliably predict antimicrobial success in treating infection, but it is inherently limited by the need to isolate and grow cultures, delaying the application of appropriate therapies. Antibiotic resistance prediction by growth-independent methods is expected to reduce the turnaround time. Recently, the potential of next-generation sequencing and microarrays in predicting microbial resistance has been demonstrated, and this review evaluates the potential of MS in this field. First, technological advances are described, and the possibility of predicting antibiotic resistance by MS is then illustrated for three prototypical human pathogens: Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Clearly, MS methods can identify antimicrobial resistance mediated by horizontal gene transfers or by mutations that affect the quantity of a gene product, whereas antimicrobial resistance mediated by target mutations remains difficult to detect.

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.

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.