Carbapenem-Resistant Enterobacteriaceae (CRE): Epidemiology, Duration of Carriage, and Progression to Infection in a Large Healthcare System in Miami, FL

Background: Carbapenem-resistant Enterobacteriaceae (CRE) is an urgent public health threat globally. Limited data are available regarding the epidemiology of CRE in South Florida. We describe the epidemiology of CRE within a large public healthcare system in Miami, FL, the experience with an internal registry, active surveillance testing, and the impact of infection prevention practices. Methods: Retrospective cohort study in four hospitals from a large healthcare system in Miami-Dade County, FL from 2012 to 2016. The internal registry included all CRE cases from active surveillance testing from rectal/tracheal screening occurring in the ICUs of two of the hospitals and clinical cultures across the healthcare system. All CRE cases were tagged in the electronic medical record and automatically entered into a platform for automatic infection control surveillance. The system alerted about new cases, readmissions, and transfers. Results: A total of 371 CRE cases were identified. The overall prevalence was 0.077

Control and Prevention (CDC) and the World Health Organization (WHO) classified CRE as an urgent threat that requires public health attention for treatment and prevention and a critical priority for research. 2,3 In 2019, the CDC estimated about 13,100 infections by CRE and about 1,100 deaths in the United States. 3 The most common species of CRE in the US are Klebsiella pneumoniae, Enterobacter aerogenes/cloacae, and Escherichia coli with reports of 5.7 %, 4.8 %, and 0.6 % of them being carbapenem-resistant respectively. 3,4 Most of the CRE cases in the US are reported from the East-North-Central, Mid-Atlantic, and South-Atlantic areas. 4 CRE infections are associated with increased morbidity, mortality, and extended hospital length of stay (LOS). 1,3,[5][6][7] CRE are not among the reportable diseases/conditions in Florida. 8 Therefore, there are limited data about the incidence of CRE in Miami-Dade County. CRE have been described in Florida since 2008 but mainly during various outbreaks. [9][10][11] Miami-Dade County is constantly receiving visitors and immigrants from different parts of the world known to be endemic with CRE; 6 [SSI]). The report excludes isolates from other HAIs, as well as communityacquired conditions, and isolates from active surveillance testing; therefore it does not reflect the total burden of CRE in the US. 4 Carbapenem-resistant Enterobacteriaceae (CRE) can be resistant to carbapenems by different mechanisms including augmented drug efflux, a mutation in or loss of outer membrane porins, and production of carbapenemases. 6,13,14 Carbapenemases are a type of β-lactamases that breakdown carbapenems, making the treatment with this class of antibiotics ineffective. Although CRE poses a challenge for the patient's antibiotic therapy, carbapenemase-producing Enterobacteriaceae (CPE) present a bigger problem for infection prevention, and treatment, since carbapenemase production is frequently plasmid-mediated; plasmids give the microorganism the capability to transfer genetic information for carbapenems resistance to other species of Gram-negative bacilli (i.e. other Enterobacteriaceae, Pseudomonas or Acinetobacter). 14 In 2017 Tamma et al., reported that the odds of dying among patients that developed bloodstream infection (BSI) by CPE are five times higher compared to those with BSI caused by non-CPE. 15 Since the first reports in the early 1990s, CPE has spread across the world. 6,7,14 There are four classes of β-lactamases associated with carbapenem resistance: A, B, C, and D. Classes A, C, and D are serine enzymes, and class B includes metalloenzymes.
This differentiation is important because metallo-β-lactamases are not inhibited by avibactam; combinations of avibactam are currently used to treat CPE infections. 16 In class A, Klebsiella pneumoniae carbapenemase (KPC) is the most important; KPC enzyme production is plasmid-mediated and considered endemic in the United States, Argentina, Brazil, Colombia, Italy, Greece, Israel, and China. 6,7,14,[17][18][19] Class A also includes the less common Guiana extended-spectrum β-lactamase (GES), and Serratia marcescens enzyme (SME). Class B carbapenemases are metallo-β-lactamases (MBLs) and include imipenemase (IMP), Verona integrin-encoded MBL (VIM), and New Delhi MBL (NDM); class B carbapenemases are more common in certain areas of Asia and Europe; nevertheless, there have been case reports and sporadic outbreaks across several states in the US caused by organisms carrying these carbapenemases. 6 1 Recent publications estimate that the CRE incidence in the US ranges between 0.46 and 4.17 cases per 10,000 patient-days, 3 and it is more prevalent in highly populated areas such as California, Chicago, and New York. [4][5][6] Risk factors for CRE acquisition have been extensively described and include the presence of medical devices, prolonged stay in healthcare facilities, surgery, antibiotic exposure, solid organ transplant, and travel to endemic areas. 1,7 Among the different mechanisms for carbapenem resistance, carbapenemase production is of major concern because it spreads easily, as it is frequently plasmid-mediated; genetic information can be shared among different bacterial species by this mechanism. [7][8][9] Despite recent efforts, little is known about the epidemiology of CRE in South Florida, in part because these organisms are not reportable in the state. 10

Setting
This was a retrospective cohort study of patients admitted to hospitals of a large health care system in Miami-Dade County between January 2012 and December 2016.
The health system has over 2,000 licensed beds; it is composed of four hospitals. Facility A is a 1,100-bed major-teaching hospital with 134 adult intensive care unit (ICU) beds; this facility is associated to the University of Miami, and it is also a national and international referral center providing services in several specialties including one of the

Infection prevention interventions
In addition to the NHSN requirements for HAI surveillance, 20  In early 2015, Facility A experienced an outbreak of Klebsiella pneumoniae carbapenemase (KPC)-producing Citrobacter freundii in one of its ICUs. As previously reported, in response to the outbreak, the staff of the ICU received infection prevention education; at the same time, the unit went into plumbing repairs. 13 In addition to the above-mentioned interventions, the system participated in the FL-DOH CRE initiatives, which provided education and expert support.

Microbiology testing
Surveillance cultures for CRE were performed by perirectal swab or tracheal aspirate sample cultured on a MacConkey plate with meropenem and ertapenem disks followed by full identification and susceptibility testing as previously described. 12  test was done using CarbaNP test. 22 No further testing was routinely performed. In addition, the isolates associated with the KPC-Citrobacter outbreak in 2015 were sent to the University of Pittsburgh for molecular testing, 13 and the frozen isolates underwent further molecular testing later as part of this study.

Molecular testing
Crude DNA was prepared by the boiling method and used as the template for polymerase chain reaction (PCR). Multiplex PCR was performed to detect blaKPC, blaNDM, blaOXA-48, as described previously. 23 For S. marcescens, a separate PCR was performed to detect blaSME, also as previously described. 24 Pulsed-field gel electrophoresis (PFGE) analysis was performed using restriction enzyme XbaI (New England Biolabs, Ipswich, MA) and a CHEF III DR electrophoresis system (Bio-Rad, Hercules, CA). The relatedness of PFGE patterns was determined by the unweighted-pair group method using average linkages and cluster analysis with the Dice setting on Bionumerics software (Applied Maths, Sint-Martens-Latem, Belgium).
During the study period, 47,963 Enterobacteriaceae isolates were tested against any of the carbapenems from all hospitals; 2.0% were reported as resistant to at least one carbapenem. (Table 3). Facilities A and B had more than twice the incidence density than facilities C and D. The incidence density for CR-KP was 7.69 per 10,000 patient days (95% CI, 6.5 -9.0); this result was significantly higher than the one found for the state during the 2014 FL-DOH collaborative of 0.47 per 10,000 patient-days (P = 0.003).

Rates of CRE
The overall prevalence and incidence density increased from 2012 to 2014 and then declined from 2015 to 2016; the maximum peaks were between March 2015 and June 2015 (Figure 1). These peaks were driven by a polyclonal cluster of KPC-producing Klebsiella pneumoniae and a KPC-producing Citrobacter outbreak in Facility A. 13 The introduction of active surveillance testing in Facility B in 2014 was also responsible for the apparent increase of the incidence density in 2014 (Figures 1 and 2 After performing molecular testing on the saved isolates, we found five cases associated with CP-CRE harboring blaNDM.. Three of these cases were unnoticed during the patients' admissions, and they were "assumed" to be KPC producers as we did not have molecular diagnostics available. Not knowing the specific mechanism of carbapenemase production has serious implications for antimicrobial stewardship as the treatment might be inappropriate based on the spectrum of activity of each agent and the need for combination therapy for metallo-β-lactamases. 28 The study found an incidence density of CR-KP notably higher than the one found among participating facilities in the 2014 FL-DOH CRE collaborative ( indicate that the use of regional registries is effective in controlling the spread of CRE. 18,30,31 In our case, the internal registry played an important role in controlling the initially increasing CRE rates. Having a county-wide or state-wide registry would have a greater impact in the regional efforts to control the spread of CRE. 31 We urge state and local public health authorities to implement a regional registry to facilitate timely, accurate communication between healthcare facilities to ensure that when a patient with a history of highly drug-resistant organisms is admitted, prompt, appropriate infection prevention practices and targeted antimicrobial stewardship management can be implemented. In addition, it is important to differentiate CP-CREs vs non-CP-CREs as the mechanism of resistance and transmission are different, and hospitals need to cohort patients appropriately. Facilities with a similar prevalence of CRE as our institutions should consider routine testing of carbapenemase types to determine the specific mechanism of carbapenemase production taking advantage of available technologies.

Conclusion
In conclusion, our study demonstrated that the burden of CRE in our population of patients from Miami-Dade is similar to that of other highly populated cities in the US.
Rising rates of CRE can be controlled by infection prevention strategies supported by tools such as registries and testing that allow rapid detection of carries and early interventions.    most studies are limited to rectal colonization while colonization of other body sites and associated infection has not been often studied. 4 We aimed to identify patient characteristics associated with CPE infections among patients previously colonized by the same organism.

Methods
This was a retrospective cohort study of CPE-colonized patients admitted to any of four hospitals in a large healthcare system (three adult and one pediatric, 2,500-beds)

Results
In total, 152 patients with CPE were identified during the study period; 98 were excluded, resulting in 54 (35%) included in the analysis (Supplementary Figure 1) Figure 2) showed that the probability of development CPE-related infections among colonized patients decreased over time, with a higher probability in the first three months after the identification of colonization.
After adjusting for significant variables in the multivariable Cox regression  Table 2). Evaluation of those exposed to colistin showed that patients who developed CPE infection were exposed via intravenous route while those exposed to inhaled colistin did not; colistin was administered for treatment of extensively-drug-resistant (XDR) Pseudomonas, Acinetobacter, or Stenotrophomonas.

Discussion
We Antimicrobial stewardship programs should tailor interventions to align with the recent recommendations for the use of colistin and recently available antimicrobials.
IUC are among the most commonly utilized devices in hospitals. 10 We found that exposure of these devices was associated with an increased probability of developing

Supplementary materials (Available only online)
Carbapenemase-producing Enterobacteriaceae cultures process description.
The surveillance samples were cultured on MacConkey agar plates with 10 µg meropenem and ertapenem disks. Organism identification and full susceptibility testing from surveillance and clinical isolates were performed using the Vitek2® system.
Carbapenemase production was tested phenotypically with the Modified Hodge Test until 2014 and using CarbaNP Test starting in 2015. Isolates were frozen for epidemiological reasons. For the purpose of this study, viable isolates were tested by PCR in 2019 to detect blaKPC, blaNDM, and blaOXA-48; Serratia marcescens isolates were also tested for blaSME.

Supplementary Figure 2. Kaplan-Meier curve for CPE-colonized patients hazard of developing infections by CPE over time.
CPE = Carbapenemase-producing Enterobacteriaceae.

© Copyright 2020
Duration of Carbapenemase-producing Enterobacteriaceae Carriage among Hospitalized Patients in Miami, FL: a Retrospective Cohort Study.

Current recommendations by the Centers for Disease Control and Prevention (CDC)
suggest placing patients with carbapenem-producing Enterobacteriaceae (CPE) in contact precautions (CP), but there is no consensus on the appropriate duration of precautions.
We aimed to evaluate predictors for prolonged CPE carriage and median clearance time.

Methods
Hospitalized patients with first isolated CPE identified from 2012 to 2016 were followed for clearance of CPE using at least two rectal or tracheal aspirate surveillance cultures and clinical cultures. Predictors associated with prolonged CPE carriage were assessed using Cox proportional hazards.

Results
Out of 75 eligible patients, 25 (33%) cleared their CPE-carrier status; the median time to clearance was 80 days (Range, . Patients who were immunocompromised, had mechanical ventilation exposure, or exposure to carbapenems had 66%, 66%, and 86 % (HR, 0.34, 0.34, and 0.14, respectively [P-value <0.05]) lower probability of clearing their CPE carrier status compared to those immunocompetent or without such exposures.
Patients with CPE isolated from more than one anatomical body site had a 5.3 times higher probability of clearing their CPE-carrier status (P-value <0.001).

Conclusion
Patients immunocompromised, with mechanical ventilation exposure, or exposure to carbapenems had higher risk for prolonged CPE carriage. Infection prevention programs should consider these predictors as part of their assessment of discontinuing contact precautions among CPE carriers to prevent horizontal transmission and outbreaks within healthcare facilities.

Background
Since the first reports in the early 1990s, Carbapenem-resistant Enterobacteriaceae (CRE) have spread across the world 1-3 . CRE are a public health threat globally due to their rapid spread, and limited treatment options. CRE can be resistant to carbapenems by different mechanisms including production of carbapenemases 1,4-6 .
The In 2018, the Society for Healthcare Epidemiology of America (SHEA) published expert guidance regarding the duration of CP for acute-care facilities in the case of CPE 13 . In their systematic review, the authors found wide variability in the median time for CPE clearance. Most of the studies included in the review were from Israel, China, or Europe.
They also reported that only 32% of the facilities in the US had policies allowing the discontinuation of CP for CRE. Of those with policies, 28% reported using screening tests for discontinuing CP, and 38% reported using a 'greater than one year since the last positive test' rule to discontinue CP. The expert guidance recommends maintaining CP indefinitely for extensively drug-resistant Enterobacteriaceae such as CPE 13 . We therefore aimed to evaluate the duration of CPE carriage and factors associated with its prolonged carriage among patients admitted to the hospitals of the largest healthcare system in Miami, FL.

Methods
This was a retrospective cohort study among patients admitted between January 2012 and December 2016 to any of the four hospitals of the health system. The system comprises two community hospitals and two major referral tertiary-care teaching centers, one adult and one pediatric. The system has over 2,000-licensed hospital beds and includes several medical specialties including trauma, burns, one of the largest solid organ transplant centers in the US, an 80-bed inpatient rehabilitation facility, two longterm care facilities, and numerous outpatient clinics. As previously described 16  Baseline characteristics of groups based on clearance are presented in Table 1. There was no statistically significant difference in mortality rates between the two groups.
Bivariable Cox proportional regression showed crude hazard ratios with P-value < 0.1 for females, immunocompromised patients, exposure to mechanical ventilator, exposure to carbapenems, and CPE isolated from more than 1 body site ( Table 2).
Analysis of the effect of interactions between different classes of antibiotics and time to clearance did not show any statistically significant associations. After adjusting for all variables in the Cox proportional multivariable analysis model, immunocompromised patients had a 66% (P-value, 0.014) lower probability of clearing CPE compared to immunocompetent patients. Patients who experienced mechanical ventilator also had a 66% lower probability of clearance compared to those without such exposure (P-value, 0.016 ). Patients with exposure to carbapenems had a 86% lower probability of clearance compared to those without exposure to carbapenems(P-value, 0.010). Interestingly, patients with CPE isolated from more than one anatomical site had a 5.3 times higher probability of CPE clearance than those who had CPE isolated from only 1 body site (Pvalue <0.001) ( Table 3). A deeper look into the cleared patients who had CPE isolated from more than one anatomical site revealed that 12 (86%) had CPE-related infections and only 2 (14%) were deemed as colonized. Figure 1 shows the probabilities over time of CPE clearance based on exposure to mechanical ventilator, exposure to carbapenems, and being immunocompromised, or having CPE isolate from more than one body site. was not very sensitive, introducing potential measurement bias. Using more than one surveillance culture and requiring agreement with the initial anatomical source of CPE isolation likely compensated for the lower sensitivity of the procedure.

Conclusion
In conclusion, infection prevention and antimicrobial stewardship practices should consider risk factors for prolonged CPE carriage to limit transmission and select suitable treatment options to optimize success at eradication, limit transmission, and safely discontinue isolation precautions. Further studies are needed to identify risk factors for long-term CPE carriage in different populations and to clarify possible differences for duration of CPE carriage related to microorganism specific species and type of carbapenemase.   or Clostridioides difficile. These multidrug resistant organisms are also considered urgent threats by the CDC and are of increasing concern because of their rapid spread around the world.