Rapid and high-throughput evaluation of diverse configurations of engineered lysins using the VersaTile technique

Bacteriophage-encoded lysins are an emerging class of antibacterial enzymes based on peptidoglycan degradation. The modular composition of lysins is a hallmark feature enabling optimization of antibacterial and pharmacological properties by engineering of lysin candidates based on lysin and non-lysin modules. In this regard, the recent introduction of the VersaTile technique allows the rapid construction of large modular lysin libraries based on a premade repository of building blocks. In this study, we perform a high-throughput construction and screening of five combinatorial lysin libraries with different configurations, targeting Klebsiella pneumoniae. An elaborate analysis of the activity distribution of 940 variants and sequencing data of 74 top hits inhibiting the growth of Klebsiella pneumoniae could be associated with specific design rules. Specific outer membrane permeabilizing peptides (OMPs) and enzymatically active domains (EADs) are significantly overrepresented among the top hits, while cell wall binding domains (CBDs) are equally represented. Especially libraries with the configuration (OMP–linker–CBD–EAD) and the inverse configuration (CBD–EAD–linker–OMP) yield the most active variants, with discernible clusters of variants that emerge above the remaining variants. The approach implemented here provides a blueprint for discovery campaigns of engineered lysins starting from libraries with different configurations and compositions

Comparison of procedures for RNA-extraction from peripheral blood mononuclear cells

RNA quality and quantity are important factors for ensuring the accuracy of gene expression analysis and other RNA-based downstream applications. Thus far, only a limited number of methodological studies have compared sample storage and RNA extraction procedures for human cells. We compared three commercially available RNA extraction kits, i.e., (NucliSENS) easyMAG, RNeasy (Mini Kit) and RiboPure (RNA Purification Kit–blood). In addition, additional conditions, such as storage medium and storage temperature of human peripheral blood mononuclear cells were evaluated, i.e., 4 °C for RNAlater or -80 °C for QIAzol and for the respective cognate lysis buffers; easyMAG, RNeasy or RiboPure. RNA was extracted from aliquots that had been stored for one day (Run 1) or 83 days (Run 2). After DNase treatment, quantity and quality of RNA were assessed by means of a NanoDrop spectrophotometer, 2100 Bioanalyzer and RT-qPCR for the ACTB reference gene. We observed that high-quality RNA can be obtained using RNeasy and RiboPure, regardless of the storage medium, whereas samples stored in RNAlater resulted in the least amount of RNA extracted. In addition, RiboPure combined with storage of samples in its cognate lysis buffer yielded twice as much RNA as all other procedures. These results were supported by RT-qPCR and by the reproducibility observed for two independent extraction runs

Development of a qPCR platform for quantification of the five bacteriophages within bacteriophage cocktail 2 (BFC2)

To determine phage titers accurately, reproducibly and in a non-laborious and cost-effective manner, we describe the development of a qPCR platform for molecular quantification of five phages present in bacteriophage cocktail 2 (BFC2). We compared the performance of this molecular approach, with regard to quantification and reproducibility, with the standard culture-based double agar overlay method (DAO). We demonstrated that quantification of each of the five phages in BFC2 was possible by means of qPCR, without prior DNA extraction, but yields were significantly higher in comparison to DAO. Although DAO is assumed to provide an indication of the number of infective phage particles, whereas qPCR only provides information on the number of phage genomes, the difference in yield (qPCR/DAO ratio) was observed to be phage-dependent and appeared rather constant for all phages when analyzing different (freshly prepared) stocks of these phages. While DAO is necessary to determine sensitivity of clinical strains against phages in clinical applications, qPCR might be a valid alternative for rapid and reproducible quantification of freshly prepared stocks, after initial establishment of a correction factor towards DAO

A case of phage therapy against pandrug-resistant Achromobacter xylosoxidans in a 12-year-old lung-transplanted cystic fibrosis patient

Bacteriophages are a promising therapeutic strategy among cystic fibrosis and lung-transplanted patients, considering the high frequency of colonization/infection caused by pandrug-resistant bacteria. However, little clinical data are available regarding the use of phages for infections with Achromobacter xylosoxidans. A 12-year-old lung-transplanted cystic fibrosis patient received two rounds of phage therapy because of persistent lung infection with pandrug-resistant A. xylosoxidans. Clinical tolerance was perfect, but initial bronchoalveolar lavage (BAL) still grew A. xylosoxidans. The patient's respiratory condition slowly improved and oxygen therapy was stopped. Low-grade airway colonization by A. xylosoxidans persisted for months before samples turned negative. No re-colonisation occurred more than two years after phage therapy was performed and imipenem treatment was stopped. Whole genome sequencing indicated that the eight A. xylosoxidans isolates, collected during phage therapy, belonged to four delineated strains, whereby one had a stop mutation in a gene for a phage receptor. The dynamics of lung colonisation were documented by means of strain-specific qPCRs on different BALs. We report the first case of phage therapy for A. xylosoxidans lung infection in a lung-transplanted patient. The dynamics of airway colonization was more complex than deduced from bacterial culture, involving phage susceptible as well as phage resistant strains

Evaluation of the Stability of Bacteriophages in Different Solutions Suitable for the Production of Magistral Preparations in Belgium

In Belgium, the incorporation of phages into magistral preparations for human application has been permitted since 2018. The stability of such preparations is of high importance to guarantee quality and efficacy throughout treatments. We evaluated the ability to preserve infectivity of four different phages active against three different bacterial species in five different buffer and infusion solutions commonly used in medicine and biotechnological manufacturing processes, at two different concentrations (9 and 7 log pfu/mL), stored at 4 °C. DPBS without Ca2+ and Mg2+ was found to be the best option, compared to the other solutions. Suspensions with phage concentrations of 7 log pfu/mL were unsuited as their activity dropped below the effective therapeutic dose (6-9 log pfu/mL), even after one week of storage at 4 °C. Strong variability between phages was observed, with Acinetobacter baumannii phage Acibel004 being stable in four out of five different solutions. We also studied the long term storage of lyophilized staphylococcal phage ISP, and found that the titer could be preserved during a period of almost 8 years when sucrose and trehalose were used as stabilizers. After rehydration of the lyophilized ISP phage in saline, the phage solutions remained stable at 4 °C during a period of 126 days

Rapid and high-throughput evaluation of diverse configura-tions of engineered lysins using the VersaTile technique

The emerging and spreading antibiotic resistance in bacteria is a severe and global problem and poses a worrying threat to our health care. Hence, the demand for alternative antibiotics is increasingly urgent. One of the most promising novel, alternative classes of antibiotics are lysins or bacteriophage-encoded peptidoglycan hydrolases, also called enzybiotics. A first lysin has recently successfully passed a superiority clinical phase II trial and got a Breakthrough Therapy designation by the FDA. Here, I present a high-throughput engineering and selection experiment for enzybiotics targeting Gram-negative Klebsiella species. These pathogens are a frequent cause of nosocomial infections that are often associated with a fatal outcome. The most prevalent species is Klebsiella pneumoniae, which belongs to group of carbapenem-resistant Enterobacteriaceae (CRE). With VersaTile technology, a quick and efficient method for combinatorial shuffling, we constructed five libraries of shuffled lysins, totaling to more than 400,000 different modular variants. Each library had a different configuration, comprising an outer membrane permeabilizing peptide, cell wall binding domain, enzymatically active domain and a possible linker, each time with a different order and composition. We analysed 940 variants from these libraries with a microtiter plate based growth inhibition assay targeting Klebsiella pneumoniae and Klebsiella oxytoca under different conditions. We found different outcomes depending on the lysin configuration and composition. The large number of constructed variants, facilitated by VersaTile technology, allows to explore the practically infinite sequence space of shuffled variants, to an unprecedented extent

Validation of High Resolution Melting Analysis (HRM) of the Amplified ITS2 Region for the Detection and Identification of Yeasts from Clinical Samples: Comparison with Culture and MALDI-TOF Based Identification.

Candida species are known as opportunistic pathogens, and a possible cause of invasive infections. Because of their species-specific antimycotic resistance patterns, reliable techniques for their detection, quantification and identification are needed. We validated a DNA amplification method for direct detection of Candida spp. from clinical samples, namely the ITS2-High Resolution Melting Analysis (direct method), by comparing it with a culture and MALDI-TOF Mass Spectrometry based method (indirect method) to establish the presence of Candida species in three different types of clinical samples.A total of 347 clinical samples, i.e. throat swabs, rectal swabs and vaginal swabs, were collected from the gynaecology/obstetrics, intensive care and haematology wards at the Ghent University Hospital, Belgium. For the direct method, ITS2-HRM was preceded by NucliSENS easyMAG DNA extraction, directly on the clinical samples. For the indirect method, clinical samples were cultured on Candida ID and individual colonies were identified by MALDI-TOF.For 83.9% of the samples there was complete concordance between both techniques, i.e. the same Candida species were detected in 31.1% of the samples or no Candida species were detected in 52.8% of the samples. In 16.1% of the clinical samples, discrepant results were obtained, of which only 6.01% were considered as major discrepancies. Discrepancies occurred mostly when overall numbers of Candida cells in the samples were low and/or when multiple species were present in the sample.Most of the discrepancies could be decided in the advantage of the direct method. This is due to samples in which no yeast could be cultured whereas low amounts could be detected by the direct method and to samples in which high quantities of Candida robusta according to ITS2-HRM were missed by culture on Candida ID agar. It remains to be decided whether the diagnostic advantages of the direct method compensate for its disadvantages

Detailed overview of the discrepancies, divided into following categories: Category A: direct method positive, but indirect method negative (n = 38); Category B: direct method more species than indirect method (n = 6); Category C: indirect method positive, but direct method negative (n = 2); Category D: indirect method more species than direct method (n = 9); and Category E: aspecific detection of nonfungal species by indirect method (n = 3).

<p>Legend: ALB: <i>Candida albicans</i>; DUB: <i>Candida dubliniensis</i>; GLA: <i>Candida glabrata</i>; GUI: <i>Candida guilliermondii</i>; LUS: <i>Candida lusitaniae</i>; PAR: <i>Candida parapsilosis</i>; ROB: <i>Candida robusta</i>; TRO: <i>Candida tropicalis</i>; ACHXYL: <i>Achromobacter xylosoxidans</i>; >: The left species is more abundant than the right species; m: minor discrepancy; M: Major discrepancy; N: No species detected, below detection level; S: Sample; D: Direct method; I: Indirect method; +: Indication of the quantity of a species, on the basis of number of colonies cultured;?: Unknown melting peak pattern.</p><p>^a: Number of samples for which the direct method established the presence of more species than the indirect method (total number: 44).</p><p>^b: Pattern of low intensity with unknown peak. Sequencing yielded an unreliable identification (closest match <i>Cladosporium cladosporioides</i>).</p><p>^c: Pattern of low intensity, similar to the pattern observed with a mixture of <i>C</i>. <i>glabrata</i> and <i>C</i>. <i>robusta</i>. Sequencing yielded an unreliable identification (closest match <i>Physarum loratum</i>).</p><p>^d: Unknown peak at a Tm of 88.78°C</p><p>Detailed overview of the discrepancies, divided into following categories: Category A: direct method positive, but indirect method negative (n = 38); Category B: direct method more species than indirect method (n = 6); Category C: indirect method positive, but direct method negative (n = 2); Category D: indirect method more species than direct method (n = 9); and Category E: aspecific detection of nonfungal species by indirect method (n = 3).</p