OCORRÊNCIA DE blaKPC E blaVIM EM ISOLADOS DE Pseudomonas aeruginosa MULTIDROGA RESISTENTES PROVENIENTES DE INFECÇÃO EM PACIENTES DE UM HOSPITAL PÚBLICO EM RECIFEPE.

Introdução: Pseudomona aeruginosa é uma bactéria gram negativa oportunista considerada uma séria ameaça à saúde pública e são responsáveis por uma variedade de Infecções Relacionadas à Assistência à Saúde (IRAS), como pneumonias, infecções em tecidos moles, no trato urinário, e bacteremias(1).A emergência de P. aeruginosa resistentes aos carbapenêmicos, produtora de carbapenemases, dificulta ainda mais o tratamento e são associadas a altas taxas de mortalidade(2). Objetivos: O objetivo desse estudo foi analisar o perfil de susceptibilidade bem como detectar a presença dos genes de resistência para carbapenemases (blaKPC, blaNDM, blaVIM, blaIMP e blaGES) em dois isolados de P. aeruginosa (Ps1-A2 e Ps2-A2) provenientes de secreção traqueal e ponta de cateter respectivamente, de dois pacientes de um hospital público de Recife-PE no ano de 2021. Métodos: A identificação bioquímica e o perfil de susceptibilidade dos isolados foi realizada através do sistema automatizado BD PhoenixTM. O DNA total foi extraído utilizando o kit comercial Wizard genomic DNA Purification e a pesquisa dos genes de resistência foram realizadas por Reação em Cadeia da Polimerase (PCR). Resultados: O isolado Ps1-A2 foi resistente a todos os antibióticos testados, com exceção da polimixina B. Já o isolado Ps1-A2 apresentou sensibilidade a ampicilina, gentamicina e polimixina B. Os genes blaNDM, blaIMP e blaGES não foram detectados. O gene blaKPC foi detectado no isolado Ps2-A2, e o isolado Ps1-A2 apresentou simultaneamente os genes blaKPC e blaVIM. A concomitância desses genes merece destaque pelo acúmulo desses mecanismos genéticos de resistência em uma mesma espécie bacteriana. Conclusão: Os genes para carbapenemases detectados nos isolados de P. aeruginosa, revelam o grande potencial dessa espécie em adquirir e consequentemente disseminar esses mecanismos de resistência no ambiente hospitalar. Precisamos considerar isso como um grande problema de saúde pública, levando em conta as limitações terapêuticas para tratar as infecções causadas por essa espécie. Sendo assim, devemos ressaltar a importância da detecção dessas enzimas nos laboratórios de microbiologia para que sejam adotadas medidas de controle e prevenção da disseminação desses microrganismos multirresistentes(3).

Utilization of Non-Invasive Sample Sources for Identification of Somatic and Germline Mutations in Hematologic Malignancies and Clonal Hematopoiesis

Introduction A matched "tumor" and germline sample is required to unambiguously identify somatically acquired mutations in the blood or bone marrow of patients with hematologic malignancies or clonal hematopoiesis, particularly when variants of unknown significance (VUS) with a variant allele frequency (VAF) approximating 50% are encountered. Our goal was to identify sample sources for matched blood and germline samples to fit the following criteria: 1) non-invasive, 2) amenable to home collection by participants, and 3) stable at room temperature for an extended period. We hypothesized that saliva, comprised of mainly white blood cells as the DNA source, is a viable alternative to blood for identifying somatic hematopoietic mutations and that nail clippings provide adequate DNA devoid of contamination from blood cells to serve as a germline sample. To test this, a targeted NGS myeloid panel was performed on concurrently collected blood, saliva, and nail samples from patients with myeloproliferative neoplasms (MPN) or other clonal disorders. Methods Peripheral blood was collected in EDTA tubes, aliquoted into microcentrifuge tubes, and stored at -80°C until use. Saliva was collected using a DNA/RNA shield saliva collection kit (Zymo) and stored at -80°C until use. Fingernail or toenails were clipped into a plastic bag and stored at room temperature until use. For isolation of DNA from blood the DSP DNA Blood mini kit (QIAGEN) was used. For saliva and nails, the QIAmp DNA Investigator kit (QIAGEN) was used. After DNA quantification targeted NGS libraries were prepared from 100ng of DNA per sample using the ArcherDX VariantPlex Myeloid (SK0123) workflow. The resulting libraries were sequenced on an Illumina NextSeq500 instrument using v2 chemistry (Illumina, San Diego, CA), obtaining at least 4 millionreads per sample. The FASTQ data files were analyzed on ArcherDX Suite Analysis software (v.5.1.7) to identify SNPs, Indels, structural rearrangements, and Copy Number Variations. Results First, to test if nail clippings are a feasible source of germline DNA, we performed a targeted NGS myeloid panel on paired blood and nail from a patient with Polycythemia Vera (Patient #1). Clippings from approximately five nails was sufficient to yield 100ng of DNA. JAK2 V617F was detected at a low VAF in his nail sample (4% in nail vs 23% in blood), suggesting the presence of blood contamination. On subsequent samples, nail clippings were rinsed with water prior to DNA purification, and no MPN driver mutations were detected in washed nails (Patients #2-5). However, since processing of nails for DNA purification is time consuming and laborious, and the DNA yield from nails is quite limited, we are investigating alternative non-invasive sources for germline samples including nasal swabs. Sequencing of matched blood and saliva was performed from patients with all three MPN driver mutations, and in a patient with Clonal Cytopenia of Undetermined Significance (CCUS). All somatic mutations identified in the blood were also seen in the saliva, almost all had identical VAFs from both sample sources, even with VAFs as low as 3%. Conclusion Nail clippings are a feasible source for germline DNA in patients with hematologic malignancies and clonal hematopoiesis. Saliva is equivalent to peripheral blood for identifying somatic mutations in hematopoietic cells. These methods of sample collection are non-invasive, amenable to in home collection, and are temperature stable. Collection of saliva and nails could be easily utilized for remotely administered studies by mailing collection kits to participants, thus avoiding the cost and labor of a blood draw. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Ultra Low-cost DNA Miniprep Spin-Columns for Rapid Filter-Trapped Proteomics Sample Preparation

Complete proteolytic digestion in the preparation of proteins for bottom-up proteomic analysis is substantially improved by the use of detergents for complete denaturation. This however is incompatible with many proteases, and highly detrimental to LC-MS/MS data collection. Recently, filter-based methods such as FASP (Filter-Aided Sample Prep) have seen wide use due to their ability to remove detergents and other harmful reagents prior to digestion and mass spectrometric analysis. Unfortunately, these techniques can be variable and time consuming. Suspension trapping (S-Trapping) is a newer method that utilizes a depth-filter to trap flocculated proteins, and has proven to be a faster approach for proteomic analysis. Sample preparation by these methods requires careful control of protein concentrations in order to flocculate the sample for collection, and the cost of commercial solutions can be high. We hypothesized that protein suspensions also retain on silica-based filters due to ionic interactions mediated by the presence of sodium (Na+), SO42- and PO43-. As such, we sought to investigate if very low-cost DNA purification spin-filters, so called ‘minipreps’ could efficiently and reproducibly trap proteins for digest and LC-MS/MS analysis. Using model proteins and whole-cell lysates we compared digestion efficiencies, capacities, recovery and identification rates from samples prepared using DNA-minipreps and FASP-based protocols. Samples were analyzed using nano uHPLC MS-MS/MS and Label-Free-Quantitative (LFQ) proteomics. DNA-filters show low variability, excellent recovery, sensitivity, and proteome depth from a commercially obtainable device which costs < $0.25 (US) per sample.

A Simple, Cost-Effective, and Automation-Friendly Direct PCR Approach for Bacterial Community Analysis

Understanding bacterial interactions and assembly in complex microbial communities using 16S rRNA sequencing normally requires a large experimental load. However, the current DNA extraction methods, including cell disruption and genomic DNA purification, are normally biased, costly, time-consuming, labor-intensive, and not amenable to miniaturization by droplets or 1,536-well plates due to the significant DNA loss during the purification step for tiny-volume and low-cell-density samples.

eDNA extraction: phenol-chloroform-isoamyl alcohol DNA purification from filters stored in Longmire buffer v1

This is an organic DNA extraction method for filters preserved in 2 ml of Longmire buffer that uses a phase lock to allow easy decanting of the aqueous layer instead of pipetting.

eDNA extraction: phenol-chloroform-isoamyl alcohol DNA purification from filters stored in Longmire buffer v1

This is an organic DNA extraction method for filters preserved in 2 ml of Longmire buffer that uses a phase lock to allow easy decanting of the aqueous layer instead of pipetting.

Recombinant expression of Barnase in Escherichia coli and its application in plasmid purification

Background The use of bovine-origin ribonucleases has been part of the standard protocol for plasmid DNA purification. As the field of gene therapy now enters the clinical stage, such enzymes need to be phased out or alternative purification protocols need to be developed to ensure product safety and regulatory compliance. The recombinant expression of bacterial RNase is fraught with toxicity problems making it a challenging enzyme to express. The current study describes a plasmid construct that allowed expression of barnase in Escherichia coli under co-expression of its native inhibitor barstar. Results The pure enzyme without the inhibitor barstar was exported to the extracellular space through the periplasm and then purified from the cell-free supernatant. Cation exchange chromatography was employed as a primary purification step. This was followed by hydrophobic interaction chromatography which resulted in a concentrated fraction of active enzyme. Although current levels of volumetric activity achieved are quite meagre (4 Kunitz units mL− 1), in principle its application to plasmid DNA purification could be proved. Currently, this is capable of processing small amounts (13 g) of bacterial biomass for plasmid production. Conclusions The current work focusses on the downstream purification strategies for a recombinant RNase and sets a framework for higher scale production if specific productivity is increased by optimal hosts and/or re-engineered plasmids. Also important is to curtail the massive enzyme loss during purification by cation exchange chromatography. Application of even a relatively small amount of recombinant RNase would contribute to greatly reducing the initial RNA levels in alkaline lysates thereby augmenting further downstream plasmid purification steps.