KPC-3–Producing Serratia marcescens Outbreak between Acute and Long-Term Care Facilities, Florida, USA

We describe an outbreak caused by Serratia marcescens carrying blaKPC-3 that was sourced to a long-term care facility in Florida, USA. Whole-genome sequencing and plasmid profiling showed involvement of 3 clonal lineages of S. marcescens and 2 blaKPC-3-carrying plasmids. Determining the resistance mechanism is critical for timely implementation of infection control measures.


The Study
In June 2018, a 382-bed hospital that is part of a large hospital health system network in Miami, Florida, identified an increase of CP-S. marcescens. A retrospective search for more cases included all patients admitted to any facility in the 4-hospital network during October 2017-June 2018 using the automatic surveillance system (VigiLanz; VigiLanz Corporation, https://vigilanzcorp.com) with interface to the electronic medical record (EMR).
We defined cases as patients with carbapenemresistant S. marcescens by Clinical and Laboratory Standards Institute (CLSI) breakpoints (5) isolated from any source, including clinical or surveillance cultures, during October 2017-December 2018. Based on Centers for Disease Control and Prevention guidelines, community-onset events (CO) were those cases identified <3 days after hospital admission; hospitalonset (HO) were those for which the specimens were collected >4 days after hospital admission (6).
In response to the outbreak, and in addition to interventions in place to prevent hospital-acquired infections (Appendix Table 1, https://wwwnc.cdc. gov/EID/article/26/11/20-2203-App1.pdf), all possible cases were prospectively identified upon admission to any of the network facilities via automatic surveillance system. Transfer forms and regular communication with the local Department of Health (DOH) notified hospitals when a known case-patient was transferred from the LTCF. All patients admitted from the source LTCF were placed in contact precautions at admission and screened for CPE. If positive, patients were placed in enhanced contact precautions (Appendix Table 1) for the duration of their stay. Miami-Dade DOH provided infection prevention and control education and support to the LTCF.
We performed matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometry (bioMérieux, https://www.biomerieux-diagnostics.com) and Biofire BCID panel (bioMérieux) for bacterial identification. We conducted susceptibility testing using Vitek2 (bioMérieux) following CLSI guidelines. We tested carbapenemase production with CarbaNP test (Hardy Diagnostics, We describe an outbreak caused by Serratia marcescens carrying bla KPC-3 that was sourced to a long-term care facility in Florida, USA. Whole-genome sequencing and plasmid profiling showed involvement of 3 clonal lineages of S. marcescens and 2 bla KPC-3 -carrying plasmids. Determining the resistance mechanism is critical for timely implementation of infection control measures. https://hardydiagnostics.com). We processed active surveillance testing (AST) using a 10 μg meropenem disk on MacConkey plate after broth enrichment; possible CPE colonies were identified and tested for carbapenemase production.
We purified plasmids by alkaline-lysis method and used them to transform Escherichia coli TOP10 by electroporation (8). We selected transformants harboring bla KPC-3 on lysogenic agar with ampicillin, and confirmed acquisition of plasmids by PCR. The plasmids were extracted from the E. coli transformants, digested (EcoRI or HindIII), and run on 0.7% gel to obtain restriction patterns.
During October 2017-December 2018, a total of 14 patients with CP-S. marcescens were identified in our hospitals ( Figure 1); all patients resided at a neighboring LTCF (Table 1). Five cases (36%) were HO, but 4 were detected <15 days after admission and did not coincide in location or time with the other cases. Transmission within the hospital was not suspected; those patients were possibly colonized at admission but undetected due to low sensitivity of AST protocols. The fifth patient had long length-of-stay and previous bloodstream infection (BSI) with KPCproducing Klebsiella pneumoniae.
Ten patients had >1 rectal AST; all were negative. Twelve patients had >1 tracheal aspirate AST; 2 were positive for CP-S. marcescens (suceptibilities in Table  2; Appendix Table 2). Ten cases had clinical infections by CP-S. marcescens including pneumonia (n = 9) and bloodstream infection (n = 4). Most cases were treated empirically with piperacillin/tazobactam, cefepime, and vancomycin. Targeted treatments included ceftazidime/avibactam. Four cases were colonized without signs or symptoms of CP-S. marcescens infection during hospital admission. Three patients died (21% in-hospital mortality); these deaths were not associated with infection by CP-S.marcescens.
During June 2018-January 2019, the 67 notifications of admissions from the source LTCF were related to 30 individual patients. In 7 cases (23%), CP-S. marcescens was present at admission.
We performed molecular testing on 12 isolates, 1 per patient. Core genome analysis demonstrated the presence of 3 clonal lineages of bla KPC-3 -carrying  (9); confirmation was by an identical plasmid restriction profile. Lineage 1 isolates shared a 19-kb ColRNAI-type bla K-PC-3 -harboring plasmid, 99% identical to previously reported pNJST258C2 from K. pneumoniae (GenBank accession no. CP006919.1), except for the absence of the transposable element containing aminoglycoside resistance genes aacA1 and aacA4 (10). The outlying isolate, 520, only contained a pNJST258C2-like bla K-PC-3 -harboring plasmid identical to that found in lineage 1. The bla KPC-3 sequence was identical between the 2 plasmids and was located on Tn4401b-like elements, which were identical except for a 70-bp deletion in tnpA leading to frameshift and premature stop in the pNJST258C2-like plasmid. This deletion did not seem to affect KPC expression; isolate 520, carrying only the pNJST258C2-like plasmid, was still resistant to carbapenems. In addition to bla KPC-3 , the pKPC_Kp46-like plasmid contained bla TEM-1 , Δbla OXA-9 , and qnrB19. The pNJST258C2-like plasmid did not contain additional antimicrobial resistance genes; however, it contained an operon encoding production of colicin, an antimicrobial substance that is lethal against related strains that lack it (11). Isolate 520 was from the patient with a history of KPC-producing K. pneumoniae, suggesting that the bla KPC-3 -harboring plasmid was transferred to S. marcescens in the patient in a separate event from the infection of the other 11 cases.

Conclusions
Use of an automatic surveillance system enabled retrospective and prospective detection of cases and identification of their common exposure in an LTCF on the basis of shared address. Prospective identification of residents of the source LTCF enabled screening at point of entry and implementation of interventions to prevent hospital transmission. Direct communication between the infection control department and the LTCF was difficult and relied upon the local DOH to share information about known cases. Unfortunately, a regional registry of patients with CPE is not available in Florida (12,13). The pKPC-KP4-like plasmid shared among the