Lab Matters Fall 2023 | Page 87

APHL 2023 POSTER ABSTRACTS

The Functional Role of Naturally Evolved Gene SNPs in Clinical Strains of Pseudomonas aeruginosa

K . Simanek 1 , L . Li 2 , J . Paczkowski 2 ; 1 SUNY Albany Biomedical Sciences , 2 New York State Department of Health , Wadsworth Center

Pseudomonas aeruginosa is an opportunistic pathogen of significant public health concern because it is highly antibiotic resistant , ubiquitous and persistent in healthcare environments , leading to tens of thousands of hospital-acquired infections each year . Healthcare settings drive evolution of clinical P . aeruginosa strains due to the use of antibiotics and antimicrobials . Clinical strains that cause chronic infections often evolve deleterious inactivating mutations in lasR , which encodes a master transcriptional regulator of virulence genes . LasR is part of a hierarchical network of quorum sensing signaling that coordinates virulent behaviors like biofilm formation , a major source of contamination of indwelling medical devices . Similarly , these clinical strains also evolve single nucleotide polymorphisms ( SNPs ) in a quorum sensing enzyme , rhlI . RhlI is the synthase of N-butyryl homoserine lactone ( C4HSL ), which is the ligand for a second major transcription factor , RhlR . However , the functional advantage of maintaining these evolved SNPs in rhlI is unclear . We chose three SNPs of interest to investigate the altered function of RhlI variants in clinical strains and their role in virulence . We found that these RhlI variants ( G62S , D83E , P159S ) partially compensate for deletion of lasR to restore virulence factor gene expression . Structural analysis of RhlI indicated that these variants could synthesize a homoserine lactone molecule with a longer acyl chain . To test this hypothesis , we used mass spectrometry to analyze the metabolites secreted in the supernatants of strains harboring RhlI variants . We found that these variants produce proportionally more C6HSL than C4HSL , implicating that a higher affinity interaction between RhlR and its ligand is advantageous in clinical strains of P . aeruginosa . Indeed , the structure of the RhlR ligand binding pocket revealed that ligand selection is driven by hydrophobic contacts with acyl tail groups , indicating that C6HSL is likely a higher-affinity ligand than C4HSL , the cognate ligand . Moving forward , we aim to understand the fitness advantage RhlI variants confer when these strains are challenged with antibiotics . It is important to understand the nuances of quorum sensing mediated virulence expression in clinical strains of P . aeruginosa to improve the treatment of lifethreatening infections , especially in the age of antibiotic resistance .

Presenter : Kayla Simanek , ksimanek @ albany . edu

The Toxic Crusaders Program

M . Koltunov , H . Gutierrez , Z . Xie , S . Ko ; California Department of Toxic Substances Control

The California Department of Toxic Substances Control ( DTSC ) seeks to promote scientific engagement , critical thinking skills and interest in STEM careers in high school students through the Toxic Crusaders Program , which provides a variety of handson experiments relating to environmental science and analytical chemistry . The Toxic Crusaders Program is coordinating with a high school in the Los Angeles Unified School District to work with 11th grade students on a STEM curriculum to simulate sampling , analysis and reporting of hazardous chemicals ( e . g ., poly- and per-fluoroalkyl substances ( PFAS ), hexavalent chromium , lead and cadmium ) found at a hypothetical contaminated site and communicating these results to the public . The framework of Toxic Crusaders consists of : 1 ) developing an interactive workbook with hands-on activities and relevant background information such as California laws and regulations on hazardous chemicals , sampling and lab procedures , worksheets and presentation templates ; 2 ) providing additional resources , such as links to videos , suggested readings and other relevant media for teachers to introduce concepts to the students at the beginning of the curriculum ; 3 ) hosting an initial lab visit to familiarize students with background information on the chemicals of interest , sampling and testing requirements , lab techniques and public engagement skills ; 4 ) leading a series of virtual workshops to reinforce concepts during class hours ; 5 ) hosting a second lab visit where students initiate their own investigation , sampling and testing of hazardous chemicals ; and 6 ) encouraging students to present their results to their community . To measure the effectiveness of Toxic Crusaders , preliminary evaluation surveys were administered after the initial lab visit , which indicated a self-reported increase in student knowledge of environmental chemistry topics and terminology , as well as increased interest in DTSC careers . Surveys will be administered before and after the second lab visit to further measure student engagement with scientific processes , critical thinking , and careers in STEM . After the completion of the program , students will have experienced working as researchers , environmental scientists , communication specialists and other professions within DTSC . They will have acquired practical field and laboratory techniques and used state-of-the-art analytical instruments such as Inductively Coupled Plasma / Optical Emission Spectroscopy ( ICP-OES ) and Liquid Chromatography / Tandem Mass Spectrometry ( LC-MS-MS ). Students will develop a better understanding of environmental issues within their community . Ultimately , students will discover possible career paths and professions in science .

Presenters : Hector Gutierrez ( hector . gutierrez @ dtsc . ca . gov ), Sarah Ko ( sarah . ko @ dtsc . ca . gov ), Zhenhui Xie ( zhenhui . xie @ dtsc . ca . gov )

Training the Next Generation of Laboratory Scientists during the COVID-19 Pandemic

R . G . Blackwell , S . Merid ; D . Edwards , J . Roberts , A . Murthi , J . Hauser ; District of Columbia Department of Forensic Sciences , Public Health Laboratory Division

Laboratory testing is a high-volume medical activity in the United States , with over 13 billion tests being performed each year . The pandemic exposed the severe shortage of trained laboratory personnel needed to run a variety of tests in clinical laboratories . Thus , there is an urgent need for training programs to encourage students to consider careers in clinical laboratories . To this end , the District of Columbia Public Laboratory ( DC PHL ) has been dedicated in their efforts to provide the highest levels of education and professional development . During the COVID-19 global pandemic , there was a severe shortage of CLIA laboratories available for training college and university students . Hospitals were limited in their ability to host clinical rotations due to the potential dangers of the pandemic . However , clinical rotations are an essential part of obtaining a degree in Clinical Laboratory Science and becoming testing personal in the laboratory . The DC PHL Microbiology Unit established a partnership with the Howard University Department of Clinical Laboratory Science to host the microbiology clinical practicum for their senior students . We collaborated and designed a program that exposed the students to modern instrumentation , cutting-edge scientific methods and high complexity testing .