Lauren A. Darling1#, Ann M. Evans1, Kathleen A. Stellrecht1,2, Seela M. Nattanmai1,

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1 JCM Accepted Manuscript Posted Online 20 September 2017 J. Clin. Microbiol. doi: /jcm Copyright 2017 American Society for Microbiology. All Rights Reserved. 1 JCM Letter to the Editor Submission 2 A Triple Disk Enrichment Method for CRE Screening Lauren A. Darling1#, Ann M. Evans1, Kathleen A. Stellrecht1,2, Seela M. Nattanmai1, Clemente I. Montero1,2 1Microbiology Laboratory, Department of Pathology and Laboratory Medicine, Albany Medical Center, Albany, NY 2Department of Pathology and Laboratory Medicine, Albany Medical College, Albany, New York, United States Running Title: Triple Disk Enrichment Method for CRE Screening Keywords: Carbapenem Resistant Enterobacteriaceae, CRE surveillance, KPC surveillance, Enrichment broth, CRE, CPE, KPC, NDM, Infection Control. Corresponding Author: Lauren Darling Microbiology Laboratory Department of Pathology and Laboratory Medicine Albany Medical Center Downloaded from on September 2, 2018 by guest New Scotland Ave. MC Albany, NY 12208

2 19 Phone: (518) Fax: (518) The results of this study were partially presented at the 114th American Society for Microbiology meeting held in Boston, MA. May 17-20, 2014 (poster 2527). Downloaded from on September 2, 2018 by guest

3 The global emergence of carbapenem resistant Enterobacteriaceae (CRE) is a major concern for public health. The CDC recommended procedure for surveillance is overnight incubation of a rectal/perianal swab in 5ml tryptic soy broth (TSB) supplemented with a 10µg ertapenem (ETP) or 5µg meropenem (MEM) disk followed by subculture to MacConkey agar (MAC). Any lactose fermenting Gram negative rods (LF) are identified and susceptibility and/or carbapenemase testing is performed (1). However, this method yields a high number of CRE-negative turbid broths. The development and validation of a tryptic soy broth enrichment method with ertapenem, fluconazole, and linezolid (TSB-EFL) is presented as an alternative to the CDC enrichment method. Rectal swabs from 487 ICU patients were collected and placed into 10ml TSB tubes (Hardy, Santa Maria, CA) and homogenized for 10 seconds. A 5ml aliquot was immediately taken and placed into a separate tube with three antimicrobial disks TSB-EFL. In the remaining 5ml aliquot (including original patient swab), a single ETP disk was added. Both tubes were incubated aerobically for 24 hours at 35 C and visually inspected (after inversion) for turbidity. Non-turbid tubes were considered negative, whereas, all turbid tubes were further evaluated using both culture and molecular methods of detection. All turbid screening broths were tested using a laboratory developed, New York State Department of Health Clinical Laboratory Evaluation Program (CLEP) approved (2) PCR targeting the bla KPC gene. In addition, a small subset of nonturbid broths were tested by PCR and culture to demonstrate that non-turbid broths are true negatives. PCR positive broths were subcultured to chromogenic KPC agar (ChromeAgar, France) from which ID and sensitivity were performed for epidemiological purposes. Identification was performed using the MALDI-TOF (Biotyper LT, Bruker Billerica MA) and sensitivity was performed using the Sensititre System (ThermoFisher, Waltham, MA). Downloaded from on September 2, 2018 by guest

4 For LOD experiments, 90 CRE isolates with varying ETP/ MEM MICs (table 1) were serially diluted 100-fold in saline solution starting with 0.5 McFarland. Then 100µl was inoculated into TSB-EFL, a small amount of CRE negative stool was added and incubated for 24 hours. Estimation of colony forming units for LOD experiments was performed in triplicate. During the clinical parallel testing, 290 out of 483 CRE negative turbid broths were observed using TSB-ETP. In contrast, only 47 CRE negative TSB-EFL broths were turbid (table 2). The cause of the false turbid TSB-EFL broths are as follows:19 broths growing Pseudomonas aeruginosa, 10 broths with Gram positive flora, 7 were turbid due to excess specimen, 5 grew other non-enterobacteriaceae species, and 6 grew Enterobacteriaceae of varying species (All Enterobacteriaceae were confirmed CRE negative by Check-Direct CPE). Our laboratory uses the TSB-EFL enrichment in combination with molecular methods for the screening of any patient admitted to our ICU. Since its implementation in 2012 we have identified at least 93 new CRE carriers. And incidentally, the first Citrobacter freundii NDM reported in the United States was recovered using the TSB-EFL method (confirmation by NYSDOH Wadsworth Center, Albany NY, May 2013). We found that by adding a FCA and LZD disk to the CDC TSB-ETP enrichment method the TSB-EFL decreased the number of turbid CRE negative broths by 6-fold saving a substantial amount of time and money. A study by Cheng, reported the average level of colonization in a CRE carrier was as low as 500 CFU/g of stool (3). LOD testing reveled over 75% of positives were recovered with 200 CFU/mL or less and our low LOD was 2 CFU/ ml. The implementation of the TSB-EFL enrichment broth is an inexpensive, readily available, and Downloaded from on September 2, 2018 by guest

5 68 69 simple screening method that can be used to reduce the burden associated with screening of CREs Acknowledgements The authors thank Dr. Markus Stein and Dr. Mary George for reviewing the manuscript and Ms. Cynthia Vanner from the Rhode Island Department of Health for sharing their NDM-1 strains. Downloaded from on September 2, 2018 by guest

6 75 REFERENCES Centers for Disease Control and Prevention Laboratory Protocol for Detection of Carbapenem-Resistant or Carbapenemase-Producing, Klebsiella spp. and E. coli from Rectal Swabs, on Center for Disease Control and Prevention New York State Department of Health at Wadsworth. Clinical Laboratory Evaluation Program Cheng VC, Chan JF, Wong SC, Chen JH, Tai JW, Yan MK, Kwan GS, Tse H, To KK, Ho PL, Yuen KY Proactive infection control measures to prevent nosocomial transmission of carbapenem-resistant Enterobacteriaceae in a non-endemic area. Chin Med J (Engl) 126: Downloaded from on September 2, 2018 by guest

7 Table 1: MIC of reference and previous positive strains used for performance evaluation of broth enrichment methods Ertapenem MIC 1 (µg/ml) Meropenem MIC 1 Organism N 0.5 (S) 1.0 (I) (R) >4.0 (R) 1.0 (S) 2.0 (I) 4.0 (R) >4.0 (R) Aeromonas hydrophilia Citrobacter freundii Citrobacter amalonaticus Enterobacter aerogenes Escherichia coli Enterobacter cloacae Klebsiella oxytoca Klebsiella pneumoniae Providencia rettgeri Pantoea species TOTAL Clinical interpretation of MIC (µg/ml) following CLSI document M100-S25 (S) = Susceptible (I) = Intermediate (R)= Resistant Downloaded from on September 2, 2018 by guest

8 Table 2 Method Comparison: Patient Performance of Enrichment Methods Using Surveillance Specimens TSB-ETP KPC Positive KPC Negative Total Turbidity Positive Turbidity Negative Total TSB-EFL KPC Positive KPC Negative Total Turbidity Positive Turbidity Negative Total Downloaded from on September 2, 2018 by guest