Lab Matters Fall 2024 | Page 61

APHL 2024 POSTER ABSTRACTS a phenazine biosynthesis protein , expression respectively . These data indicate that the RhlI variants convey not just altered AI levels , but also varying antibiotic susceptibility and QS gene expression profiles .

Presenter : Megan Schumacher , megan . schumacher @ health . ny . gov

Pseudomonas aeruginosa Strains with Quorum Sensing Mutations Form Small Colony Variants in Co-culture

K . Simanek , A . Kurtz , J . Paczkowski , New York State Public Health Lab , Wadsworth Center

Pseudomonas aeruginosa causes thousands of hospital-acquired infections every year , which are difficult to treat because they employ a wide range of antibiotic resistance mechanisms . It is a notorious pathogen of immune compromised individuals , like cystic fibrosis patients , where it establishes chronic infections that persist throughout the life of the patient . How P . aeruginosa establishes chronic infections in the lungs is still a largely unknown phenomenon . One hallmark of chronic P . aeruginosa infections is the evolution of quorum sensing mutants . Quorum sensing is a mechanism of interbacterial communication that relies on the production , secretion and detection of small molecules called autoinducers . Chronic infection strains evolve deleterious mutations in lasR , an autoinducer receptor and transcription factor that upregulates numerous virulence genes . We recently found that secondary mutations in rhlI , an autoinducer synthase , restore virulence phenotypes in strains lacking a functional LasR . We hypothesized that these secondary mutations mark a reversion of the chronic phenotype to an acute , infectious phenotype . To test this hypothesis , we competed lasR deletion strains harboring rhlI mutations against the parent lasR deletion strain and found that in co-culture , both strains produced small colony variants ( SCVs ) and large colonies . Strikingly , strains with a rhlI mutation produced more SCVs and at a faster rate , than the parent lasR deletion strain . RNAsequencing identified the genes that were differentially regulated in the SCVs compared to the large colonies . Altogether , we purport that SCVs are a mechanism of P . aeruginosa to establish chronic infections in the lung niche . Moreover , strains that evolve mutations in lasR and rhlI form heterogenous populations in the lung , which serve to either establish chronic infections or disseminate to seed new infections . This research can help us understand the progression of P . aeruginosa pathogenesis in the lung and lead to the identification of new targets for drug treatments against infections .

Presenter : Kayla Simanek , ksimanek @ albany . edu

Rapid and Quantitative Detection of Intact Legionella Using a Novel Viability Real-time PCR Assay

N . Feirer 1 , N . Kuchi 2 , S . Paladugu 3 , T . Kirkland 1 , M . Scurria 3 , J . Cali 1 , A . Rehman 2 , S . Mondal 1 , Promega Corporation 1 , Research and Productivity Council ( RPC ) 2 , Promega Biosciences LLC 3

Legionellosis has been an increasing public health concern in New Brunswick and throughout the world . Culture-based detection of Legionella is commonly used as part of routine microbial surveillance protocols of water supplies including cooling towers , car washes and showers . Despite this widespread acceptance , culture-based approaches are severely limited regarding time-toanswer ( 7-10 days ). These approaches also lack the ability to detect “ viable but nonculturable ” ( VBNC ) Legionella bacteria that are often residually present after common disinfection treatments ( e . g ., chlorination or heat ). PCR-based detection directly addresses these issues and is quickly becoming a widely adopted method for rapid detection of Legionella .

The main drawback of existing PCR approaches is the lack of livedead discrimination in the analysis of test results . In this study we show that by using a cell-impermeable intercalating molecule ( Viability PCR Reagent ) that exclusively binds to and covalently modifies DNA from membrane-compromised dead , dying , or lysed cells , we can discriminate between viable and non-viable Legionella cells .

We developed and validated a qPCR method to detect Legionella spp ., L . pneumophila , L . pneumophila Serogroup 1 and report the efficiency , linearity and specificity of this assay . In addition to Legionella targets , the qPCR assay design includes an internal positive control ( IPC ) to detect PCR inhibitors . Next , we used the Viability PCR Reagent in conjunction with the qPCR assay to determine viable genomic units per milliliter ( vGU / mL ) in water samples spiked with a range of concentrations of viable and heat-killed L . pneumophila . Samples were filter concentrated and treated with Viability PCR Reagent , followed by DNA purification and quantitation by qPCR . In conjunction , the spiked water samples were also enumerated using two commonly used culture-based Legionella detection techniques . The vGU / mL values resulting from the Viability qPCR assay were comparable to both culture-based methods , demonstrating that our molecular viability assay can be used as a same-day surrogate test for traditional Legionella monitoring methods . This rapid detection method ( 3-6 hours ) can be used to assist public / health professionals in performing risk assessment and allows for rapid response to mitigate outbreaks .

Presenter : Nathan Feirer , nathan . feirer @ promega . com

Self-collect Chlamydia trachomatis and Neisseria gonorrhea Prevalence Rates in 2022-2023 and the Benefit of Testing Multiple Sites Beyond Just Genital

O . Knobbe , B . Reiser , M . Nakano , K . Koach , C . Sailey , Molecular Testing Labs

The 2021 STI surveillance report from the Center for Disease Control and Prevention ( CDC ) shows that Chlamydia trachomatis ( CT ) and Neisseria gonorrhoeae ( NG ) are the most common sexually transmitted infections ( STI ) in the United States , other than human papillomavirus . The latest report shows a significant disruption in reporting due to the COVID-19 pandemic but still displays a consistent increase in cases nationwide . Here , we report the prevalence of CT and NG positivity of 1,667,195 samples from 357,468 individuals in 2022 and 2023 . The CT / NG positivity of four collection sites was examined .

This retrospective study was conducted using de-identified CT and NG test results in 2022 and 2023 . The specimens were selfcollected at home or in a non-clinical setting and shipped to the laboratory for analysis . These samples were analyzed at a CAP / CLIA clinical laboratory with the validated lab-developed assay according to manufacturer specifications with additional studies such as