1. Materials and Methods (Figure 1)
Discordance in CLSI standards among the Carbapenem Producing Enterobacteriaceae
David R. Woodard, MSc1, Fidelis Uzoma Enyinnaya, MBBS, MPH1,2, Patricia Cruz, Ph.D.1, Mark P. Buttner, Ph.D.1, and Chad Cross, Ph.D.3
Contact Information
4505 S. Maryland Pkwy.
Box
Las Vegas, NV
(702)
1 School of Community Health Sciences, University of Nevada, Las Vegas, Las Vegas, NV; 2 Le Moyne College, Syracuse, NY; 3 PaLS-Mathematics, Nevada State College, Henderson, NV
Abstract
BACKGROUND: This study was conducted to compare antimicrobial susceptibility testing (AST) results using the CLSI 2009 and CLSI 2012 guidelines. In 2012, the National Healthcare Safety Network reported a carbapenem resistance rate of 10.4% among Klebsiella pneumoniae infections, and indicated that the mortality rate associated with carbapenem resistant Enterobacteriaceae (CRE) infections was approximately 50%.
METHODS: A total of 56 CRE isolates obtained from various clinical settings were evaluated using real time polymerase chain reaction (PCR) to detect the Klebsiella pneumoniae carbapenemase (KPC) gene. The MIC results for these isolates were compared using the S19 (2009) and the S22 (2012) Clinical Laboratory Improvement Amendments (CLIA) standards with the Vitek 2 Compact instrument.
RESULTS: When isolates were compared with previous and current CLSI criteria in defining carbapenem resistance, five isolates that were “susceptible” changed to non-susceptible, and four of these were blaKPC gene negative. The overall difference in susceptibility was 8%. This is not statistically significant. In addition to the blaKPC gene, other mechanisms of resistance observed included ESBL, AmpC, and impermeability. The prevalence of the blaKPC gene in these CRE isolates was 83.3%.
CONCLUSIONS: The results from this study demonstrated the presence of the KPC gene and determined antimicrobial susceptibility profiles among CRE isolates in a discrete population. To ensure proper detection of emerging resistance, species- related zones of inhibition or MIC breakpoints should be published as a means to target control of CREs. The mis-characterization of antimicrobial susceptibility has significant consequences in the management of infections caused by these pathogens; which could cause under reporting of susceptibility and potential treatment failures or delay in implementation of effective therapy. 
Materials and Methods (Figure 1)
• Pure cultures from clinical isolates suspected of being carbapenem-resistant Enterobacteriaceae were obtained from a local core microbiology laboratory (Figure 2)
• Bacterial DNA was extracted using the MoBio PowerSoil DNA® extraction kit
• The 7900 HT Fast PCR System® (Life Technologies, Foster City, CA) was used for detection and amplification of the blaKPC gene
• Primers and a fluorescent probe specific for the blaKPC gene were employed 4, 5
• Primers were obtained from Eurofins MWG Operon (Huntsville, AL) and the probe was obtained from Life Technologies
• The master mix was prepared using TaqMan 1X Universal Master Mix, sterile nuclease-free water, 0.3 µM of forward and reverse primers, and 0.2 µM of probe
• A commercially-available TaqMan Exogenous Internal Positive Control® (Life Technologies) was used to detect PCR inhibition and rule out false negatives
• Antibiotic Susceptibility Testing (AST) using Gram-negative (GN) AST and identification cards (GN69 and XN06) for the Vitek 2 Compact System® (bioMerieux, Durham, NC) following the manufacturer’s protocol was conducted on all isolates (Figure 3)
• Data for the CRE isolates were subjected to a test of marginal homogeneity using counts of susceptibility (S) and resistance (R) as the outcome variable
• The ‘irr’ package in R (v )® was used and reported exact p-values for two sets of tests: (1) determination of statistical differences between S and R isolates, and (2) determination of statistical differences between the carbapenem profiles among blaKPC gene-positive isolates when using the vs breakpoint standards
• Data for doripenem were not analyzed owing to the absence of an established cut-point for 2009
Results
• A total of 54 of 56 isolates were subjected to analysis
• The prevalence rate of the KPC gene among the 54 suspected CRE isolates was 83.3% (45 out of 54)
• Klebsiella pneumoniae comprised the majority of CRE isolates (Table 1), and 93.6% of these had the blaKPC gene
• PCR inhibitors were observed in several KPC gene negative isolates
• Serial dilution (1:10 and 1:100) of the inhibited samples resulted in an additional positive KPC isolate by PCR
• Statistical differences in antimicrobial susceptibility between the 2 standards were not demonstrated for ertapenem (χ2 = 0.90, p = 0.342), imipenem (χ2 = 2.90, p = ), or meropenem (χ2 = 1.90, p = 0.168)
• AST results with the Vitek 2 Cards GN 69 and GN XN 06 (bioMerieux) (Table 2), showed that there was a demonstrable, but statistically insignificant change in the susceptibility observed when the organisms are carbapenemase (metallo- or KPC) positive (83.3%-90.7%) (p = 0.150)
• In addition to the blaKPC gene, other mechanisms of resistance observed included ESBL, AmpC, and impermeability (Table 3)
Discussion
• In this study, the prevalence of the blaKPC gene was 83.3% among suspected CRE isolates from a local core microbiology laboratory. Isolates identified as Klebsiella pneumoniae comprised the majority of our CRE isolates, and 93.6% of these had the blaKPC gene. These findings are similar to those of other studies in the U.S. reporting that the blaKPC gene is the most commonly implicated gene in carbapenem resistance among the Enterobacteriaceae. 6,7
• Our results imply that the current CLSI criteria may not offer additional benefits in the fight against CREs. These results are similar to others reported in the literature that showed either no change between the two breakpoints or unnecessary increase in the estimation of carbapenem resistance. 6,7
• Because the quantity of antibiotics used on patients and the development of resistance are directly proportional, increased reports of Enterobacteriaceae resistant to carbapenems and reduced MIC breakpoints will increase the number of Enterobacteriaceae determined to be resistant to at least one agent in any antimicrobial category. Therefore, it is anticipated that clinicians will most likely prescribe increased doses of carbapenems or other antimicrobial classes which may lead to the development of more resistance. 8
• Molecular detection of the blaKPC gene was only positive in isolates that possess the KPC gene and thus may have underestimated the presence of other resistance mechanisms implicated in CREs, such as the MBL, the OXA, and the AmpC enzymes in blaKPC gene negative isolates. The PCR methodology also provides an advantage to the clinician in terms of time required to produce a more complete picture of the susceptibility pattern of an organism.
• Research questions that have yet to be fully addressed are the prevalence of KPC occurring in the Long-Term Acute Care hospital compared to the General Acute Care Hospital, and the epidemiological pattern of resistance across Nevada as well as nationally.
Table 1.  Vitek 2 identification results for individual organisms among our CRE isolates.  
Organism Identified  N 
Acinetobacter baumannii  1 
Citrobacter  freundii  2 
Klebsiella pneumoniae 
46 
Proteus mirabilis 
Enterobacter aerogenes 
Escherichia coli 
References
1. Jacob, JT, Klein, E, Laxminarayan, R, Beldavs, Z, Lynfield, R, Kallen, AJ, ... Fridkin, S.  2013.  Vital signs: carbapenem-resistant Enterobacteriaceae.  MMWR.  62(9):  
Clinical and Laboratory Standards Institute.  2009.  Performance Standards for Antimicrobial Susceptibility Testing. Nineteenth Informational Supplement (M100-S19). Wayne, PA: Clinical and Laboratory Standards Institute. 
Clinical and Laboratory Standards Institute.  2012.  Performance Standards for Antimicrobial Susceptibility Testing; Twenty Second Informational Supplement (M100-S22). Wayne, Pennsylvania: Clinical and Laboratory Standards Institute 
Tenover, FC, Kalsi, RK, Williams, PP, Carey, RB, Stocker, S, Lonsway, D, ... Hanna, B.  2006.  Carbapenem resistance in Klebsiella pneumoniae not detected by automated susceptibility testing.  Emerg. Infect. Dis.  12(8):1209. 
Hindiyeh, M, Smollen, G, Grossman, Z, Ram, D, Davidson, Y, Mileguir, F, ... Rahav, G.  2008.  Rapid detection of blaKPC carbapenemase genes by real-time PCR.  J. Clin. Microbiol.  46(9):  
Hombach M, Bloemberg GV., and Böttger EC.  2012.  Effects of clinical breakpoint changes in CLSI guidelines 2010/2011 and EUCAST guidelines 2011 on antibiotic susceptibility test reporting of Gram-negative bacilli.  J. Antimicrob. Chemother.  67(3):   doi: /jac/dkr524.  
Metwally, L, Gomaa, N, Attallah, M, and Kamel, N.  2013.  High prevalence of Klebsiella pneumoniae carbapenemase-mediated resistance in K. pneumoniae isolates from Egypt.  Eastern Mediterr. Health J.  19(11):  
Magiorakos, AP, Srinivasan, A, Carey, R, Carmeli, Y, Falagas, M, Giske, C, ... Olsson‐Liljequist, B.  2012.  Multidrug‐resistant, extensively drug‐resistant and pandrug‐resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance.  Clin. Microbiol. Infect.  18(3):
Image sources:
Introduction
In the U.S., the most common mechanism of resistance to carbapenem antibiotics is Klebsiella pneumoniae carbapenemase (KPC). In 2012, the National Healthcare Safety Network reported a carbapenem resistance rate of 10.4% among K. pneumoniae infections, and indicated that the mortality rate associated with carbapenem resistant Enterobacteriaceae (CRE) infections was approximately 50%1. We conducted a study to compare the minimum inhibitory concentration (MIC) results in a population of Klebsiella pneumoniae obtained from a regional microbiology laboratory using the S19 (2009) 2 and the S22 (2012) 3 Clinical Laboratory Improvement Amendments (CLIA) standards. A significant problem in the laboratory detection of CRE is the fact that some bacterial isolates carry the KPC gene, but demonstrate susceptibility according to MICs. In this study, the carbapenem antimicrobial susceptibility testing (AST) profiles were determined and the resistance rates were compared between the previous and current Clinical Laboratory Science Institute (CLSI) criteria. While the standardized susceptibility results are published by the CLSI, the adoption of these by the Food and Drug Administration (FDA) and subsequent implementation by the manufacturers of automated AST methods lags behind. We evaluated the impact of this disconnect using Klebsiella isolates that were reported as resistant to carbapenem.
Table 2. Individual carbapenem susceptibility among all CRE isolates analyzed.
Individual carbapenem
Previous Breakpoints  
(M100-S19) 
Current Breakpoints  
(M100-S22) 
Susceptible 
Non-susceptible  N  % 
Ertapenema 6 
11.3 
47 
88.7  5 
9.4 
48 
90.6 
Imipenem  8 
14.8 
46 
85.2 
9.3 
49 
90.7 
Doripenemb
nd 
9.6 
90.4 
Meropenem 7 
13.0 
87.0 
a Ertapenem use was not indicated for 1 isolate. 
b Doripenem use was not indicated for 2 isolates. 
nd = Doripenem interpretation was not defined in the previous breakpoints.
Figure 1. Flow chart of the experimental design.
Table 3.  Mechanisms of antibiotic resistance as determined with the Vitek 2 instrument.
Organism ID  N 
Resistance Phenotype (GN 69/XN 06 AST Card) 
Acinetobacter baumannii  1 
Impermeability 
Citrobacter freundii 
Carbapenemase (Metallo- or KPC) 
Klebsiella pneumoniae  3 
Penicillinase, Inhibitor Resistant PASE (IRT or OXA), ESBL, Impermeability 
Proteus mirabilis 
ESBL/Carbapenemase (Metallo- or KPC) 
Enterobacter aerogenes  2 
ESBL/HL-Case (AmpC)a, Impermeability, Carbapenemase (Metallo- or KPC) 
Escherichia coli 
Penicillinase/ Inhibitor Resistant PASE (IRT or OXA)
Objectives
• To determine the carbapenem antimicrobial susceptibility testing (AST) profiles and the resistance rates for carbapenem resistant Enterobacteriaceae (CRE) isolates obtained from various clinical settings
• To compare AST results between the previous and current Clinical Laboratory Science Institute (CLSI) criteria (i.e., CLSI 2009 and CLSI 2012 guidelines)
• To evaluate CRE isolates using real time polymerase chain reaction (PCR) to detect the Klebsiella pneumoniae carbapenemase (KPC) gene
Figure 2. Microscopic image of K. pneumoniae cells.
a HL-Case (AmpC) – High Level AmpC
Figure 3. Procedures used with the Vitek 2 Compact instrument.