Carbapenem Disks on MacConkey agar as screening methods for the detection of. Carbapenem-Resistant Gram negative rods in stools.

Transcription

1 JCM Accepts, published online ahead of print on 7 November 2012 J. Clin. Microbiol. doi: /jcm Copyright 2012, American Society for Microbiology. All Rights Reserved. 1 2 Carbapenem Disks on MacConkey agar as screening methods for the detection of Carbapenem-Resistant Gram negative rods in stools Julie Blackburn 2, Catherine Tsimiklis 1-2, Valéry Lavergne 1-2, Josée Pilotte 3, Sophie Grenier 3, Andrée Gilbert 3, Brigitte Lefebvre 3, Marc-Christian Domingo# 3 Cécile Tremblay 2-3, Anne-Marie Bourgault Hôpital du Sacré-Coeur de Montréal, Canada 2 Département de microbiologie et immunologie, Faculté de médecine, Université de Montréal 3 Institut national de santé publique du Québec, Laboratoire de santé publique du Québec, Canada 4 McGill University Health Center, Montreal, Canada #Corresponding author : Institut national de santé publique du Québec, Laboratoire de santé publique du Québec, chemin Sainte-Marie, Sainte-Anne-de-Bellevue, H9X 3R5 Tel extension marc-christian.domingo@inspq.qc.ca Running title: Screening methods for carbapenem-resistant bacteria 1

2 24 ABSTRACT Direct plating of simulated stool specimens on MacConkey agar (MCA) with 10-µg of ertapenem, meropenem and imipenem disks, allowed to established the optimal zone diameters for screening of carbapenem-resistant gram negative rods (CRGNR) to: 24 mm (ertapenem), 34 mm (meropenem) and 32 mm (imipenem). Downloaded from on July 19, 2018 by guest 2

3 32 TEXT Screening of stool specimens is recommended by the Center for Disease Control and Prevention as well as the Institut national de santé publique du Québec to identify carriers of CRGNR and initiate appropriate infection control measures (1, 2). Lolans and colleagues reported that an ertapenem zone diameter of 27 mm on MCA was highly sensitive for detection of KPCproducing Enterobacteriaceae in rectal swab specimens (3). However, the zone diameter interpretive criteria for imipenem and meropenem directly put on MCA have not been established yet. This study compares the performance of the screening method using MCA + 10-µg carbapenem disks (ertapenem, meropenem and imipenem) and defines the optimal inhibition zone diameters for detecting CRGNR using simulated stool specimens. Thirty nine clinical isolates have been well characterized, phenotypically and genotypically as described in Table 1. Twenty carbapenemase-producing isolates (17 Enterobacteriaceae and 3 Non-Fermenters) were selected upon the presence of genes coding for different carbapenemases. Nineteen non-carbapenemase-producing Enterobacteriaceae [18 ESBL or plasmid-mediated AmpC (pampc)] and a susceptible wild-type Escherichia coli strain were also selected as negative control. The MICs of ceftazidime, cefotaxime, ertapenem, meropenem and imipenem were determined by the microdilution method according to the Clinical and Laboratory Standards Institute (4). A stool specimen obtained from a normal volunteer was used to prepare all the simulated clinical specimens. To ensure that the specimen did not harbour any beta-lactam resistant bacteria, screening tests were performed using ChromID ESBL, CHROMagar KPC and MCA with ertapenem, meropenem and imipenem disks. Each plate was inoculated with 100 μl of liquefied 3

4 stool and incubated 24 hours aerobically at 35 C. These incubation conditions were further selected all over the study. There was no growth on the two selective chromogenic agar plates. For the MCA, inhibition diameters around the antibiotic disks were 29 mm, 39 mm and 35 mm respectively for ertapenem, meropenem, and imipenem. Dilutions of the 39 isolates were mixed with aliquots of stool. The simulated fecal material was inoculated onto the screening MCA media to obtain final challenge concentrations of 10 4 to 10 1 CFU/ml for each strain. The fecal inoculum was spread on MCA by rotation using a rake spreader and disks of the carbapenems were individually placed onto MCA. After incubation, the diameter of the inhibition zones around each carbapenem disk was measured. The results of all inhibition diameters obtained with the 4 dilutions tested for each strain were used to construct a Receiver Operating Characteristic (ROC) curve. A zone diameter breakpoint was identified in order to maximize the sensitivity (Se) and specificity (Sp) for the detection of CRGNR for each carbapenem disk. All ROC curve analysis followed the methodology of DeLong et al., and the binomial exact test for the determination of confidence interval for the area under the ROC curve was used (5). To evaluate the relative performance of the 3 different carbapenem disks, the ROC curves were compared using a pairwise comparison. Statistical analysis was conducted using the MedCalc software version (Mariakerke, Belgium). For all statistical tests, significance was set at an alpha-value = 0.05 and confidence interval at 95% (CI 95%). The optimal breakpoint for CRGNR screening on MCA was a zone diameter 24 mm for ertapenem, 34 mm for meropenem and 32 mm for imipenem. The area under the ROC curve was 0.94 with CI 95% ( ) for ertapenem (p-value <0.0001), 0.92 with CI 95% ( ) for meropenem (p-value <0.0001) and 0.90 with CI 95% ( ) for imipenem (p-value 4

5 <0.0001) (Figure 1). There was no statistical difference when comparing the ROC curves of the three carbapenems: ertapenem versus imipenem (p-value 0.14), ertapenem versus meropenem (pvalue 0.40), and meropenem versus imipenem (p-value 0.42). Using the optimal breakpoint identified for each carbapenem disk, the respective Se and Sp for each dilution tested were also calculated as shown in Table 2. The use of ROC curve analysis has allowed the establishment of an optimal zone diameter for each carbapenem disk that discriminates fairly well between CRGNR and non-crgnr. However, comparison of each curve did not demonstrate a significant statistical superiority of either one of carbapenem disk for the detection of CRGNR in stools. We might have been able to show a difference in performance amongst the three disks if we had tested a larger number of strains. A major strength of our study is the diversity of the carbapenemase enzymes produced by the strains tested. Furthermore, the CRGNR displayed a wide range of carbapenem MICs. Using standardized inocula, we were also able to identify breakpoint diameters for screening CRGNR in stools with either ertapenem, meropenem or imipenem disks (respectively 24 mm, 34 mm and 32 mm). Our study has some limitations. Firstly, the species selected for this study may not correspond to those that would be isolated from clinical specimens. Secondly, we used standardized dilutions of collection strains (10 1,10 2,10 3 and 10 4 CFU/mL) and correlation with real specimen inoculum concentration in patient stools or rectal swabs remains unknown. Thirdly, screening for CRGNR by direct carbapenem disk testing using the breakpoints established was not performed on rectal swab specimens owing to the very low prevalence of CRGNR in our hospital setting. Therefore, this might limit the external validity of our results. We should also underline the fact that our population of strains contained a very high proportion of CRGNR (51.3%). The overrepresentation of CRGNR may nevertheless underestimate the 5

6 negative predictive value, knowing that this parameter varies accordingly to prevalence, which is usually the most important value for a screening test In conclusion, screening stool for CRGNR using direct carbapenem disk method on MCA is reliable and easy to perform. Actually, the main limitation of this method is its poor sensitivity when a CRGNR is present at a low concentration (10 1 CFU/mL). To maximize the likelihood of finding CRGNR, two carbapenem disks per plate could be used. Our results suggest that the carbapenem disk method can be a useful tool for screening CRGNR in stool. Downloaded from on July 19, 2018 by guest 6

7 ACKNOWLEDGMENTS The plates of chromogenic media CHROMagar KPC (Alere Inc, Canada) and ChromID ESBL (Biomerieux, France) were kindly provided by the manufacturers

8 REFERENCES 1. Centers for Disease Control and Prevention Guidance for control of infections with carbapenem-resistant or carbapenem-producing Enterobacteriaceae in acute care facilities. MMWR. Morb. Mortal Wkly. 58: Comité sur les infections nosocomiales du Québec Prévention et contrôle de la transmission des entérobactéries productrices de carbapénèmases dans les milieux de soins aigus du Québec. Institut national de santé publique du Québec. enemases.pdf Lolans K, Calvert K, Won S, Clark J, Hayden MK Direct ertapenem disk screening method for identification of KPC-producing Klebsiella pneumoniae and Escherichia coli in surveillance swab specimens. J. Clin. Microbiol. 48: Clinical and Laboratory Standards Institute Methods For Dilution Antimicrobial Susceptibility Tests For Bacteria That Grow Aerobically Approved standard M7-A9. Clinical and Laboratory Standards Institute Wayne, PA. 5. DeLong ER, DeLong DM, Clarke-Pearson DL Comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach. Biometrics 44:

9 TABLE 1. Characteristics of the bacterial isolates tested at the Laboratoire de santé publique du Québec and National Microbiology Laboratory, Public Health Agency of Canada Type of β- Isolate Gene lactamase Citrobacter freundii Carb ase KPC Enterobacter cloacae Carb ase NMC Enterobacter cloacae Carb ase KPC Escherichia coli Carb ase KPC Escherichia coli Carb ase KPC Escherichia coli Carb ase KPC Escherichia coli Carb ase KPC Klebsiella pneumoniae Carb ase KPC Klebsiella pneumoniae Carb ase KPC Klebsiella pneumoniae Carb ase KPC Klebsiella pneumoniae Carb ase KPC Klebsiella pneumoniae Carb ase KPC Klebsiella pneumoniae Carb ase OXA-48 Klebsiella pneumoniae Carb ase NDM-1 Klebsiella pneumoniae Carb ase NDM-1 Klebsiella oxytoca Carb ase KPC Serratia marcescens Carb ase KPC Acinetobacter baumanii Carb ase IMP-4 OXA-23 Acinetobacter baumanii Carb ase OXA-51 Inhibition zone MIC (ug/ml) diameter (mm) at 10 2 dilution CAZ CTX ERTA MERO IMI ERTA MERO IMI > 64 > , > 64 > > 64 > > 64 > > 64 > > 64 > > 64 > 32 > 32 > > 64 > 32 > 32 > 32 > > 64 > 32 > > 64 > > 64 > 32 > 32 > 32 > > 64 > 32 > 32 > 32 > > 64 > 32 > > 64 > 32 > 32 > 32 > > 64 > 32 > 32 > 32 >

10 Pseudomonas aeruginosa Carb ase VIM-2 Escherichia coli ESBL TEM-26 TEM-1 Escherichia coli ESBL DHA Escherichia coli ESBL SHV-2a TEM-1 Escherichia coli ESBL CTX-M Escherichia coli ESBL CTX-M Escherichia coli ESBL TEM-19 SHV-11 Klebsiella pneumoniae ESBL CTX-M Klebsiella pneumoniae ESBL SHV-18 Klebsiella pneumoniae ESBL SHV-5 Citrobacter freundii pampc CMY-2 TEM-1 Escherichia coli pampc CMY-2 Escherichia coli pampc CMY-2 SHV-1 Klebsiella pneumoniae pampc CMY-2 SHV-1 Klebsiella pneumoniae pampc FOX Morganella morganii pampc DHA Proteus mirabilis pampc CMY-2 Proteus mirabilis pampc CMY-2 Escherichia coli None None > 64 > 32 > 32 > 32 > > < < 0.03 < < 0.03 < > 64 > > 64 > 32 < 0.03 < < 0.03 < > 64 > > ,06 0,06 0, < < 0.03 < < > > > < < 0.03 < ,

11 Carb ase : Carbapenemase ESBL: Extended spectrum ß-lactamase pampc: plasmid-mediated AmpC ß-lactamase 11

12 TABLE 2. Sensitivity and specificity of zone diameters around a 10-µg ertapenem ( 24 mm), meropenem ( 34 mm) and imipenem ( 32 mm) disk for detection of carbapenem- resistant gram negative rods Ertapenem Meropenem Imipenem Dilution (CFU/mL) Sen (%) Spe (%) Sen (%) Spe (%) Sen (%) Spe (%) , All dilutions Downloaded from on July 19, 2018 by guest 12

13 100 Ertapenem disk Sensitivity Sensitivity: 86.2 Specificity: 90.8 Criterion : <=24 A Sensitivity Sensitivity Sensitivity: 88.7 Specificity: 94.7 Criterion : <= Specificity Meropenem disk Sensitivity: 85.0 Specificity: 93.4 Criterion : <= Specificity Imipenem disk C B Specificity FIGURE 1. ROC curve for zones of inhibition around a 10-µg ertapenem disk (A), 10-µg meropenem disk (B), and 10-µg imipenem disk (C) for 39 challenge strains at different dilutions (10 1, 10 2, 10 3 and 10 4 CFU/mL). 13