Recent Advances in Rapid Antimicrobial Susceptibility Testing.

BACKGROUND: Antimicrobial susceptibility testing (AST) is classically performed using growth-based techniques that essentially require viable bacterial matter to become visible to the naked eye or a sophisticated densitometer. CONTENT: Technologies based on the measurement of bacterial density in suspension have evolved marginally in accuracy and rapidity over the 20th century, but assays expanded for new combinations of bacteria and antimicrobials have been automated, and made amenable to high-throughput turn-around. Over the past 25 years, elevated AST rapidity has been provided by nucleic acid-mediated amplification technologies, proteomic and other "omic" methodologies, and the use of next-generation sequencing. In rare cases, AST at the level of single-cell visualization was developed. This has not yet led to major changes in routine high-throughput clinical microbiological detection of antimicrobial resistance. SUMMARY: We here present a review of the new generation of methods and describe what is still urgently needed for their implementation in day-to-day management of the treatment of infectious diseases.

Host-Pathogen Adhesion as the Basis of Innovative Diagnostics for Emerging Pathogens.

Infectious diseases are an existential health threat, potentiated by emerging and re-emerging viruses and increasing bacterial antibiotic resistance. Targeted treatment of infectious diseases requires precision diagnostics, especially in cases where broad-range therapeutics such as antibiotics fail. There is thus an increasing need for new approaches to develop sensitive and specific in vitro diagnostic (IVD) tests. Basic science and translational research are needed to identify key microbial molecules as diagnostic targets, to identify relevant host counterparts, and to use this knowledge in developing or improving IVD. In this regard, an overlooked feature is the capacity of pathogens to adhere specifically to host cells and tissues. The molecular entities relevant for pathogen-surface interaction are the so-called adhesins. Adhesins vary from protein compounds to (poly-)saccharides or lipid structures that interact with eukaryotic host cell matrix molecules and receptors. Such interactions co-define the specificity and sensitivity of a diagnostic test. Currently, adhesin-receptor binding is typically used in the pre-analytical phase of IVD tests, focusing on pathogen enrichment. Further exploration of adhesin-ligand interaction, supported by present high-throughput "omics" technologies, might stimulate a new generation of broadly applicable pathogen detection and characterization tools. This review describes recent results of novel structure-defining technologies allowing for detailed molecular analysis of adhesins, their receptors and complexes. Since the host ligands evolve slowly, the corresponding adhesin interaction is under selective pressure to maintain a constant receptor binding domain. IVD should exploit such conserved binding sites and, in particular, use the human ligand to enrich the pathogen. We provide an inventory of methods based on adhesion factors and pathogen attachment mechanisms, which can also be of relevance to currently emerging pathogens, including SARS-CoV-2, the causative agent of COVID-19.

Automated antimicrobial susceptibility testing of slow-growing Pseudomonas aeruginosa strains in the presence of tetrazolium salt WST-1.

Slow growing, mucoid isolates of Pseudomonas aeruginosa require adaptation of the protocol used for automated antimicrobial susceptibility testing (AST). In the present study we used a water soluble tetrazolium salt WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) in combination with menadione for possibly improving AST of slow growing and biofilm-forming P. aeruginosa isolates from cystic fibrosis (CF) patients. WST-1 and menadione addition ensures sensitive detection of microbial growth increase in the presence of antibiotics that may remain undetected with the automated VITEK® 2 method. We observed that 32.8% of P. aeruginosa isolates from CF and bronchiectasis patients produced an elevated absorbance signal intensity thereby increasing the sensitivity while maintaining the accuracy of VITEK 2. Our study merits future investigation with other slow growing pathogenic bacterial species.

Phenotypic and Genomic Variability of Serial Peri-Lung Transplantation Pseudomonas aeruginosa Isolates From Cystic Fibrosis Patients.

Cystic fibrosis (CF) represents one of the major genetic and chronic lung diseases affecting Caucasians of European descent. Patients with CF suffer from recurring infections that lead to further damage of the lungs. Pulmonary infection due to Pseudomonas aeruginosa is most prevalent, further increasing CF-related mortality. The present study describes the phenotypic and genotypic variations among 36 P. aeruginosa isolates obtained serially from a non-CF and five CF patients before, during and after lung transplantation (LTx). The classical and genomic investigation of these isolates revealed a common mucoid phenotype and only subtle differences in the genomes. Isolates originating from an individual patient shared ≥98.7% average nucleotide identity (ANI). However, when considering isolates from different patients, substantial variations in terms of sequence type (ST), virulence factors and antimicrobial resistance (AMR) genes were observed. Whole genome multi-locus sequence typing (MLST) confirmed the presence of unique STs per patient regardless of the time from LTx. It was supported by the monophyletic clustering found in the genome-wide phylogeny. The antibiogram shows that ≥91.6% of the isolates were susceptible to amikacin, colistin and tobramycin. For other antibiotics from the panel, isolates frequently showed resistance. Alternatively, a comparative analysis of the 36 P. aeruginosa isolates with 672 strains isolated from diverse ecologies demonstrated clustering of the CF isolates according to the LTx patients from whom they were isolated. We observed that despite LTx and associated measures, all patients remained persistently colonized with similar isolates. The present study shows how whole genome sequencing (WGS) along with phenotypic analysis can help us understand the evolution of P. aeruginosa over time especially its antibiotic resistance.

Differences and overlaps between Phd studies in diagnostic microbiology in industrial and academic settings.

Industrial and academic needs for innovation and fundamental research are essential and not widely different. Depending on the industrial setting, research and development (R&D) activities may be more focused on the developmental aspects given the need to ultimately sell useful products. However, one of the biggest differences between academic and industrial R&D will usually be the funding model applied and the priority setting between innovative research and product development. Generalizing, companies usually opt for development using customer- and consumer-derived funds whereas university research is driven by open innovation, mostly funded by taxpayer's money. Obviously, both approaches require scientific rigor and quality, dedication and perseverance and obtaining a PhD degree can be achieved in both settings. The formal differences between the two settings need to be realized and students should make an educated choice prior to the start of PhD-level research activities. Intrinsic differences in scientific approaches between the two categories of employers are not often discussed in great detail. We will here document our experience in this field and provide insights into the need for purely fundamental research, industrial R&D and current mixed models at the level of European funding of research. The field of diagnostics in clinical bacteriology and infectious diseases will serve as a source of reference.