Determining the Optimal Carbapenem MIC that Distinguishes Carbapenemase-Producing

AAC Accepted Manuscript Posted Online 8 August 2016 Antimicrob. Agents Chemother. doi:10.1128/aac.00838-16 Copyright 2016, American Society for Microbiology. All Rights Reserved. 1 1 2 Determining the Optimal Carbapenem MIC that Distinguishes Carbapenemase-Producing and Non-Carbapenemase-Producing Carbapenem-Resistant Enterobacteriaceae 3 4 5 Pranita D. Tamma, MD, MHS 1#, Yanjie Huang, ScM, BM 2,, Belita N.A. Opene, MS 3, & Patricia J. Simner, PhD 3 6 7 8 9 10 11 12 1 Division of Pediatric Infectious Disease, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA 2 Department of Health Policy and Management, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA 3 Division of Medical Microbiology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA 13 14 15 16 17 18 19 20 #Corresponding Author Pranita D. Tamma, M.D., M.H.S. The Johns Hopkins University School of Medicine 200 North Wolfe St., Suite 3149 Baltimore, MD 21287 Tel (443) 287-4099 ptamma1@jhmi.edu 21 22 23 24 25 Key words: CRE; KPCs; NDM; ROC curve; β-lactamases Abbreviated Title: Differentiating carbapenemase-producing and non-carbapenemase producing CRE Word count of body of text: 1043 26

2 27 28 29 30 31 32 Abstract Carbapenemase-producing (CP) Enterobacteriaceae are largely responsible for the rapid spread of carbapenem-resistant Enterobacteriaceae (CRE). Distinguishing CP-CRE and non- CP-CRE has important infection control implications. In a cohort of 198 CRE isolates, an ertapenem MIC of 0.5 µg/ml and meropenem, imipenem, or doripenem MICs of 2 µg/ml were best able to distinguish CP and non-cp CRE isolates. 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52

3 53 54 55 56 57 58 59 60 61 62 63 64 65 66 Carbapenem-resistant Enterobacteriaceae (CRE) encompass both carbapenemase-producers (CP) and non-carbapenemase-producers (non-cp). CP-CRE are particularly concerning from an infection control standpoint as the genes encoding carbapenemases are generally located on mobile genetic elements (i.e., plasmids, transposons, insertion sequences) and are easily transmissible to other gram-negative organisms [1]. In the United States, carbapenemase genes are most commonly bla KPCs [2]. However, in large part due to international migration and medical tourism, other carbapenemase genes (e.g., bla NDMs, bla VIMs, bla IMPs, and bla OXAs) have also been increasingly encountered [3]. Gram-negative organisms harboring these resistance genes can spread easily from patient to patient in healthcare settings. In fact, CP-CRE have been responsible for a number of outbreaks in the healthcare environment [1]. On the contrary, non-cp-cre generally emerge as the result of heterogeneous mechanisms, such as reduced outer membrane permeability, and have been associated with a loss of organism fitness and reduced transmissibility [1]. Distinguishing these resistance mechanisms has important infection control implications. 67 68 69 70 71 72 73 74 75 76 77 78 The Centers for Disease Control and Prevention (CDC) acknowledges that CP-CRE are currently believed to be primarily responsible for the increasing spread of CRE in the United States and have therefore been targeted for aggressive prevention and that a reliable way to differentiate CP-CRE from non-cp-cre might help guide such targeting by identifying the organisms of greatest epidemiological interest [4]. Determining whether a CRE is carbapenemase-producing involves several additional steps in the microbiology laboratory and can be resource intensive. As a result, many US clinical microbiology laboratories are not conducting additional phenotypic tests such as the Carba NP test or modified Hodge test to determine if CRE are indeed CP-CRE [2]. These tests have limitations in their ability to detect some CP genes (e.g., Carba NP are limited in detection of bla OXA-48-types and the modified Hodge test is limited in detecting metallo-β-lactamases) [5, 6].

4 79 80 81 82 83 84 85 Currently, most healthcare facilities institute contact precautions when patients with CRE are identified and subsequently perform unit-wide point prevalence studies to identify other unrecognized carriers on the unit when CRE clinical isolates are identified, according to CDC recommendations [7]. We sought to determine whether an optimal carbapenem MIC can reliably distinguish CP-CRE and non-cp-cre, potentially lessening the burden on microbiology laboratories. 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 We included well-characterized CRE isolates from the Antimicrobial Resistance Isolate Bank between 2012 and 2015 [8] (n=145) and all CRE bloodstream isolates from unique patients at The Johns Hopkins Hospital between 2014 and 2015 (n=53) (Table 1). The Antimicrobial Resistance Isolate Bank is a repository of bacteria with genotypic and susceptibility data that have been assembled by the CDC and Food and Drug Administration. For The Johns Hopkins Hospital isolates, ertapenem, meropenem, imipenem, and doripenem MICs were determined using the Etest (biomérieux). Carbapenem resistance was defined according to the Clinical Laboratory and Standards Institute criteria (CLSI) with ertapenem resistance defined as 2mcg/ml and meropenem, imipenem, and doripenem resistance defined as MICs 4 mcg/ml [9]. β-lactamase gene identification was performed using the Check-MDR CT103XL assay (Check-Points, Wageningen, The Netherlands). The Check-MDR CT103XL assay combines PCR amplification and micro-array technologies for the detection of an extended panel of carbapenemase genes (bla KPC, bla NDM, bla VIM, bla IMP, bla OXA-48-like, bla GES, bla GIM, bla SPM, bla OXA-23- like, bla OXA-24/40-like, bla OXA-58-like ). [10]. 101 102 103 104 We created histograms to distinguish the MIC range of CP and non-cp-cre individually for ertapenem, meropenem, imipenem, and doripenem. The Wilcoxan rank-sum test was used to compare medians and interquartile ranges (IQR) between carbapenem MICs for CP-CRE and

5 105 106 107 108 109 110 111 112 non-cp-cre for each of the antibiotics, with a two-tailed P value of <0.05 considered statistically significant. A receiver operating characteristic (ROC) curve was generated to determine the optimal MIC for the detection of CP-CRE using various carbapenem MICs. The discriminatory power was evaluated using the area under the ROC curve (AUC), with an AUC value of 0.5 indicating no discriminative ability and an AUC exceeding 0.8 indicating good to excellent prediction. The sensitivity and specificity of the prediction rule were calculated at various carbapenem MIC values. Statistical analyses were performed using the R statistical package. 113 114 115 116 117 118 119 120 121 122 There were 135 (68%) CP-CRE and 63 (32%) non-cp-cre isolates. The genus and species of the full cohort are displayed in Table 1. For the 53 Johns Hopkins Hospital isolates, the most commonly recovered organisms were Klebsiella pneumoniae (53%), Enterobacter spp. (42%), and E. coli (5%). Evaluating the full cohort, the CP-CRE isolates contained a variety of serine and metallo-β-lactamase carbapenemase genes; encoding Class A carbapenemases [bla KPC (n=58), bla SME (n=7), bla IMI (n=2)]; Class B carbapenemases [bla NDM (n=38), bla IMP (n=4), bla VIM (n=9)]; and Class D carbapenemases [bla OXA-48-type (n=17)], Table 1. Twenty-three (43%) of the Johns Hopkins Hospital isolates were CP-CRE, including 21 (92%) bla KPCs, 1 (4%) bla NDM, and 1 (4%) bla OXA-48-type. 123 124 125 126 127 128 129 130 The distribution of MICs for each of the carbapenems is displayed in Figure 1. The median and IQR for the carbapenems tested against CP-CRE were as follows: ertapenem [16 µg/ml, 8-16 µg/ml], meropenem [16 µg/ml, 4-16 µg/ml], imipenem [16 µg/ml, 4-64 µg/ml], doripenem [16 µg/ml, 4-16 µg/ml]. The median and IQR amongst non-cp CRE were as follows: ertapenem [2 µg/ml, 0.125-16 µg/ml], meropenem [1 µg/ml, 1-2 µg/ml], imipenem [1 µg/ml, 1-2 µg/ml], doripenem [1 µg/ml, 0.5-2 µg/ml]. For each of these antibiotics, the distributions of carbapenem MICs for CP and non-cp-cre were significantly different (P <0.01). Comparing isolates

6 131 132 133 134 producing the most frequent carbapenemase genes identified in the United States, bla KPC, bla NDM, and bla OXA-48-like, no differences in the carbapenem MIC distributions were noted; however, there were small numbers of carbapenemases other than bla KPCs and bla NDMs in our cohort, limiting a robust comparison of the MIC distributions for the different carbapenemases. 135 136 137 138 139 140 141 142 143 144 145 146 We developed an ROC curve for each of the carbapenem antibiotics to identify the carbapenem MIC that best distinguishes CP and non-cp-cre (Figure 2). For ertapenem, a group 1 carbapenem, the AUC of the ROC curve was 0.79. An MIC cutoff of 0.5 µg/ml had the greatest overall sensitivity and specificity for distinguishing CP-CRE from non-cp CRE, which were 98.2% and 61.1%, respectively. In contrast, the AUC was over 0.90 for meropenem, imipenem, and doripenem, group 2 carbapenems, with sensitivities ranging from 89.5% to 94.5% and specificities ranging from 77.8% to 83.3% - indicating these agents had excellent discriminatory ability to distinguish CP and non-cp-cre at an MIC of 2 µg/ml. Of note, as has been previously described in the literature (11), the 3 included Proteus mirabilis isolates had imipenem MICs well above 2 µg/ml (i.e., 16-32 µg/ml) and significantly higher than the respective meropenem MICs (0.5-4 µg/ml). 147 148 149 150 151 152 153 154 155 156 The use of an MIC cutoff to distinguish CP-CRE and non-cp-cre, if a useful cutoff exists, could reduce the burden of clinical microbiology laboratories when deciding to conduct point prevalence surveillance studies if a patient with a CRE is identified. Additionally, a reliable MIC cutoff could avoid the additional steps involved in implementing phenotypic methods to distinguish CP and non-cp-cre. Our data suggest that based on a carbapenem ROC curve, group 2 carbapenem MICs of 2 µg/ml represent the carbapenem antibiotics and MIC cutoff that are best able to discriminate between CP and non-cp CRE. However, using group 2 carbapenem MICs to distinguish CP-CRE and non-cp-cre would translate to about 5-10% of CP-CRE remaining undetected. Although an ertapenem MIC of 0.5 µg/ml is highly sensitive for

7 157 detecting CP-CRE, it is associated with a specificity of only approximately 60%. 158 159 160 161 162 163 164 165 166 There are pros and cons to using ertapenem versus group 2 carbapenems to make the distinction between CP-CRE and non-cp-cre. Using nonsusceptibility of ertapenem to distinguish CP and non-cp-cre could result in a large portion of non-cp-cre erroneously designated as CP-CRE, however, it would ensure that almost all patients with a CP-CRE could be rapidly identified and placed on contact precautions, potentially limiting their spread in healthcare institutions. Because of the significant morbidity and mortality associated with CP- CRE infections, we favor approaches to optimize the sensitivity of CP-CRE detection, even if it means some compromise in the specificity. 167 168 169 170 171 172 173 174 175 There are limitations with our study. We realize that susceptibility patterns may change over time and that these data need to be periodically reviewed to remain accurate. Additionally, we realize that the assortment of carbapenemases and proportion of CP-CRE and non-cp-cre may not be generalizable to what is found in clinical practice and that these data will vary based on local epidemiology. In our cohort, the majority of carbapenemases were bla KPCs, followed by bla NDMs. While this is consistent with what others have observed in the United States, it differs from carbapenemase distributions observed globally and it is not known if our findings would be replicated in settings outside of the United States. 176 177 178 179 180 181 Nonetheless, until rapid, accurate, cost-effective, and standardized methods of carbapenemase detection are available in clinical microbiology laboratories across the country, we believe that other approaches might be useful to differentiate CP and non-cp-cre. We encourage other research groups to repeat these analyses with a larger cohort to increase the accuracy of carbapenem MICs to distinguish CP and non-cp-cre. 182

8 183 184 185 186 187 Acknowledgements P.D.T. received support from the inhealth Pilot Project Discovery Program and P.J.S. received support from the Fisher Center Discovery Program award supported by Sherrilyn and Ken Fisher Center for Environmental Infectious Diseases. All authors report no conflicts of interest relevant to this article. 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208

9 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 References 1. Goodman KE, Simner PJ, Tamma PD, & Milstone AM. Infection control implications of heterogeneous resistant mechanisms in carbapenem-resistant Enterobacteriaceae (CRE). Expert Rev Infect Ther 2016;1:95-108. 2. Chea N, Bulens SN, Knogphet-Tran T, et al. Improved phenotype-based definition for identifying carbapenemase-producers among carbapenem-resistant Enterobacteriaceae. Emerg Infect Dis 2015;21(9):1611-16. 3. Nordmann P, Naas T, Poirel L. Global spread of carbapenemase-producing Enterobacteriaceae. Emerg Infect Dis 2011; 10:1791-8. 4. Centers for Disease Control and Prevention. FAQs about choosing and implementing a CRE definition. http://www.cdc.gov/hai/organisms/cre/definition.html. Accessed June 27 th, 2016. 5. Tijet N, Boyd D, Patel SN, Mulvey MR, and Melano RG. Evaluation of the Carba NP test for rapid detection of carbapenemase-producing Enterobacteriaceae and Pseudomonas aeruginosa. Antimicrob Agents Chemother 2103; 57(9):4578-4580. 6. Girlich D, Poirel L, and Nordmann P. Value of the modified Hodge test for detection of emerging carbapenemases in Enterobacteriaceae. J Clin Microbiol 2012; 50(2):477-479. 7. Facility Guidance for Control of Carbapenem-resistant Enterobacteriaceae (CRE) November 2015 Update CRE Toolkit. http://www.cdc.gov/hai/organisms/cre/cre-toolkit/. Accessed June 27 th, 2016. 8. FDA-CDC Antimicrobial Resistance Isolate Bank. http://www.cdc.gov/drugresistance/resistance-bank/. Accessed June 27 th, 2016. 9. CLSI. Performance standards for antimicrobial susceptibility testing. 26th edition. CLSI supplement M100S. Wayne, PA: Clinical and Laboratory Standards Institute; 2016. 10. Suwantarat N, Logan LK, Carroll KC, et al. The prevalence and molecular epidemiology of multidrug-resistant Enterobacteriaceae colonization in a pediatric intensive care unit. Infect Cont Hosp Epidemiol 2016 doi:10.1017/ice.2016.16.

10 235 236 237 11. Villar HE, Danel F, Livermore DM: Permeability to carbapenems of Proteus mirabilis mutants selected for resistance to imipenem or other β-lactams. J Antimicrob Chemother. 1997, 40: 365-370. 10.1093/jac/40.3.365. 238 239 240 241 242 243 244 Figure Legend Figure 1. Distributions of carbapenem-resistant Enterobacteriaceae carbapenem MICs comparing carbapenemase producers and non-carbapenemase producers; (A) Ertapenem, (B) Meropenem, (C) Imipenem, (D) Doripenem; Gray represents non-carbapenemase producers and black represents carbapenemase producers 245 246 247 248 Figure 2. Receiver operating characteristic curve using carbapenem minimum inhibitory concentrations for the detection of carbapenemase-producing carbapenem-resistant Enterobacteriaceae; (A) Ertapenem, (B) Meropenem, (C) Imipenem, and (D) Doripenem 249 250 251 252 253 254 255 256

Type of β-lactamases Citrobacter amalonaticus (n=1) Citrobacter freundii (n=2) Enterobacter aerogenes (n=7) Enterobacter asburiae (n=1) Enterobacter cloacae (n=40) Escherichia coli (n=30) Klebsiella oxytoca (n=4) Klebsiella ozanae (n=2) Klebsiella pneumoniae (n=96) Morganella morgganii (n=2) Proteus mirabilis (n=3) Providencia rettgeri (n=1) Raoultella spp. (n=1) Salmonella spp. (n=1) Serratia marcescens (n=7) Class A TEM (n=8) ESBL 1 4 3 TEM, ACT/MIR (n=3) ESBL + AmpC 3 SHV (n=4) ESBL 2 2 SHV, ACT/MIR (n=3) ESBL + AmpC 3 TEM, CTX-M, SHV (n=6) ESBL 6 CTX-M, SHV (n=4) ESBL 4 CTX-M (n=5) ESBL 1 4 CTX-M, DHA (n=3) ESBL + AmpC 3 TEM, SHV (n=4) ESBL 4 TEM, CTX-M, SHV (n=1) ESBL 1 KPC (n=29) Carbapenemase 1 6 3 2 1 12 1 2 1 KPC, TEM, CTX-M, SHV (n=6) ESBL + Carbapenemase 6 KPC, CTX-M, SHV (n=3) ESBL + Carbapenemase 3 KPC, SHV, TEM (n=5) ESBL + Carbapenemase 2 3 KPC, ACT/MIR (n=1) AmpC + Carbapenemase 1 KPC, CTX-M, ACT/MIR (n=1) ESBL + AmpC + Carbapenemase 1 KPC, ACT/MIR, TEM (n=1) ESBL + AmpC+ Carbapenemase 1 KPC, TEM (n=4) ESBL + Carbapenemase 2 2 KPC, SHV (n=8) ESBL + Carbapenemase 8 IMI (n=2) Carbapenemase 2 SME (n=7) Carbapenemase 7 Class B NDM (n=28) Carbapenemase 1 1 10 12 1 1 1 1

NDM, CTX-M (n=4) ESBL + Carbapenemase 4 VIM (n=8) Carbapenemase 2 6 VIM, CMY (n=1) ESBL + AmpC + Carbapenemase 1 IMP (n=4) Carbapenemase 2 2 NDM, TEM, CTX-M, SHV (n=3) ESBL + Carbapenemase 3 NDM, DHA, CTX-M, SHV (n=3) ESBL + AmpC + Carbapenemase 3 Class C campc (n=8) AmpC 2 6 CMY (n=7) AmpC 7 ACT/MIR (n=7) AmpC 4 3 Class D OXA-48 (n=7) Carbapenemase 3 4 OXA-232 (n=2) Carbapenemase 2 OXA-181 (n=7) Carbapenemase 1 6 OXA-48, CTX-M, SHV, TEM (n=1) ESBL + Carbapenemase 1