Lab Matters Fall 2024 | Page 59

APHL 2024 POSTER ABSTRACTS the Aptima Combo 2 ( AC2 ) assay ( Hologic ) on a new pharyngeal specimen which tested negative . Attempts were made to isolate a gonococcal culture from the patient using ESwab ( Copan ) and plating onto modified Thayer-Martin agar and using the InTray GC ( Biomed Diagnostics ) culture detection device . While both culture methods did not isolate NG , a commensal bacterial strain was isolated . To detect antimicrobial resistance markers from the suspected gonococcal infection , we PCR amplified and sequenced NG-STAR targets ( penA , parC and 23S rRNA ) from the remnant Cobas NAAT specimen , which resulted in partial sequences with sequence identity to several commensal and pathogenic Neisseria spp . The negative result from the AC2 test and the inability to obtain a gonococcal culture or to PCR amplify NG sequences from the remnant clinical specimen resulted in no further testing or antibiotic treatment for the patient . By using MALDI-TOF ( Bruker ) we identified the commensal bacterial isolate as belonging to the genus Neisseria but could not identify the species . Similarly , whole genome sequencing and BLAST analyses of the strain could not identify a single Neisseria species , as 74 % of the genome had sequence identity to the unclassified Neisseria sp . Marseille-Q6792 , 5 % to N . cinerea , 3 % to NG , 2 % to N . meningitidis and 1 % to N . lactamica , suggesting the commensal is an unidentified Neisseria species or a subspecies variation within an established species . Interestingly , we found three short regions of recombination within the genome of this commensal isolate encoding the gonococcal diagnostic signature DR-9 ( with 96 , 92 and 82 % sequence identity to NG DR-9 ), which is the target of the Cobas CT / NG test used to initially diagnose the patient . PCR amplification using the Cobas assay primers NG-519 and NG-514 produced the DR-9 band . Additional investigations of the cross-reactivity of this new commensal isolate with different versions of the Cobas CT / NG and other diagnostic NAAT tests as well as the phylogenetic origin of the strain are underway . To our knowledge this is the second commensal Neisseria sp . strain , isolated from the oropharynx , that has been described to contain the DR-9 gonococcal marker .

Presenter : Julio Ayala Figueredo , pyd0 @ cdc . gov

MALDI-TOF-MS Library Enrichment of Medically Important Rare Yeasts in a Clinical Laboratory

A . Marathe 1 , S . Chaturvedi 2 , Wadsworth Center , New York State Department of Health 1 , University of Albany 2

Background : Pathogenic yeasts are a leading cause of hospitalacquired infections ( HAIs ) and have high mortality rates exceeding 50 %. Identification of these pathogenic yeasts to species-level is essential for rapid diagnosis and timely implementation of appropriate antifungal therapy . The molecular identification of yeasts by ribosomal genes ( ITS & D1 / D2 ) PCR and Sanger sequencing followed by BLAST search is an excellent technique , but they are both labor and time intensive . Matrix-assisted laser desorption ionization-time of flight mass spectrometry ( MALDI- TOF MS ) has revolutionized the diagnostic mycology laboratory by efficiently identifying yeasts to species level . One limitation of MALDI-TOF MS could be inadequate main spectra profiles ( MSPs ) in the reference library which results in low scores leading to no organism identification . Hence library enrichment becomes crucial to obtain adequate MSPs in the reference library which ensures good scores ( indicating high-confidence identification ). In this study , we performed addition of 141 rare yeast isolates representing 68 unique species to the MALDI In-house mycology library .

Methods : Rare yeast isolates were grown on Sabaraud Dextrose Agar ( SDA ) plates at 30 ° C overnight . For MALDI-TOF MS , all samples were prepared with the ethanol / formic acid protein extraction protocol and 1 µ l of each sample was spotted twelve times on a 96-spot target plate ( Bruker Daltonics , Germany ). Bacterial Test Standard ( BTS , Bruker Daltonics , Germany ) was used as a positive control on one spot . The air-dried samples were overlaid with 1 µ l of HCCA matrix , measured three times using MALDI Biotyper FlexControl . Additional step of bead-beating with 70 % formic acid was used for six rare yeast isolates that had no peaks upon standard protein extraction . Upon analysis , a minimum of 20 spectra were selected to create a reference MSP stored in the In-house Mycology library .

Results : In this study , we added 141 rare yeast isolates comprising of 68 unique species and 25 genera to the In-house Mycology library . Library addition was accomplished with accurate identification to species level and a score value > 2.0 . Of a total of 141 rare yeast isolates tested , 135 isolates ( 95.7 %) were identified to species level with a score value > 2.0 using full protein extraction , whereas 6 isolates ( 4.3 %) required an additional bead-beating step for identification to species level with a score value > 2.0 . Out of the 68 species added , 33 species of 13 genera had no reference spectra present , while the rest were poorly represented in the Bruker library .

Conclusion : Enrichment of the in-house MALDI-TOF MS library will improve laboratory work efficiency by reducing the turnaround time for identification of rare yeasts . This will further lead to timely and accurate patient treatment .

Presenter : Anuradha Marathe , anuradha . marathe @ health . ny . gov

Mycobacterial Conjugation Reveals a Role for Calcium in Cell Contact Signaling

T . Farrington , T . Gray , K . Derbyshire , New York State Department of Health , Wadsworth Center

A novel form of horizontal gene transfer ( HGT ) has been described in mycobacteria that is called distributive conjugal transfer ( DCT ). DCT serves as an experimental system to study mycobacterial cell-cell interactions and the signal transduction networks that respond to cell contact . Previous studies have shown that activation of the ESX- 4 secretion system is required for DCT in the recipient strain . SigM , an extra cytoplasmic sigma factor , is triggered by cell-to-cell contact and activates genes that encode the ESX-4 secretion apparatus as well as other genes that putatively support ESX-4 function . Here we show that a solo gene of the SigM-ESX-4 regulon is also required for DCT . Annotation of the predicted encoded protein product of MSMEG _ 6925 does not suggest functional association with ESX secretion systems . Rather , repeated motifs consistent with calcium binding run the length of the protein , strongly suggesting that binding calcium is its function . We call this MSMEG _ 6925 encoded protein product CasM for its calcium and SigM associations . Identifying CasM led us to investigate whether calcium has a role in DCT . We find that free environmental calcium is required for DCT and for the contact-dependent signal transduction that supports DCT . This work reveals a novel role for calcium in mycobacterial cell