Occurrence of False Positive Results for the Detection of Carbapenemases in Carbapenemase-Negative Escherichia coli and Klebsiella pneumoniae Isolates

Adequate detection of the production of carbapenemase in Enterobacteriaceae isolates is crucial for infection control measures and the appropriate choice of antimicrobial therapy. In this study, we investigated the frequency of false positive results for the detection of carbapenemases in carbapenemase-negative Escherichia coli and Klebsiella pneumoniae clinical isolates by the modified Hodge test (MHT). Three hundred and one E. coli and K. pneumoniae clinical isolates were investigated. All produced extended spectrum β-lactamases (ESBLs) but were susceptible to carbapenems. Antimicrobial susceptibility testing was performed by the disk diffusion and agar dilution methods. The MHT was performed using the standard inoculum of test organisms recommended by the CLSI. Genes that encoded ESBLs and carbapenemases were identified by PCR and DNA sequencing. Among the 301 clinical isolates, none of the isolates conformed to the criteria for carbapenemase screening recommended by the CLSI. The susceptibility rates for imipenem, meropenem, and ertapenem all were 100.0%, 100.0%, and 100.0%, respectively. Of the 301 E. coli and K. pneumoniae isolates, none produced carbapenemase. The MHT gave a positive result for 3.3% (10/301) of the isolates. False positive results can occur when the MHT is used to detect carbapenemase in ESBL-producing isolates and clinical laboratories must be aware of this fact.


Introduction
The production of extended-spectrum b-lactamases (ESBLs) is the most common mechanism of resistance to third-generation cephalosporins in Enterobacteriaceae bacteria, especially Escherichia coli and Klebsiella pneumoniae. Carbapenems are the drugs of last resort to treat severe infections caused by pathogens that produce ESBLs [1][2]. Although the rates of resistance to carbapenems are low, they have been increasing [3]. During the past decade the spread of antibiotic resistance in Enterobacteriaceae isolates, especially the increase in K. pneumoniae carbapenemase (KPC)-producing isolates, has become a major concern worldwide [4]. Infections due to KPCproducing isolates have become an important challenge in healthcare settings [5]. The rapid emergence of carbapenemresistant Enterobacteriaceae isolates in our hospital was revealed by bacterial surveillance data. The rate of resistance to imipenem in K. pneumoniae rose from 1.3% in 2006 to 11.1% in 2009, whereas the rate in E. coli rose from 0.3% 2006 to 1.0% in 2009 (data not shown).
The production of carbapenemases, especially KPC-type carbapenemases, is the most common mechanism for carbapenem resistance in Enterobacteriaceae isolates [6][7]. However, the detection of carbapenemases can be difficult because, for some carbapenemase-producing Enterobacteriaceae, the minimum inhibitory concentration (MIC) of carbapenem is high but still within the susceptible range as defined by the Clinical and Laboratory Standards Institute (CLSI) criteria [4,[8][9]. In 2009, the CLSI recommended the modified Hodge test (MHT) to screen for the production of carbapenemase in Enterobacteriaceae isolates with elevated MICs for carbapenems or reduced inhibition zones as measured by disc diffusion. The criteria were a diameter for the inhibition zones for meropenem and ertapenem of 19-21 mm and 16-21 mm, respectively, and MIC values of 2 mg/mL and 2-4 mg/mL, respectively (carbapenem breakpoints have been changed in M100-S20-U and M100-S21. According the new criteria, the initial screen test and the confirmatory test by MHT are no longer necessary for routine patient testing). Although the sensitivity and specificity of the MHT have been shown to exceed 90%, several studies have reported false positive or false negative results when this method was used to screen for carbapenemase in Enterobacteriaceae isolates [8,10]. In this study, we investigated the rate of false positives obtained when the MHT was used to test ESBL-producing and carbapenemase-nagetive E. coli and K. pneumoniae clinical isolates that did not conform to the CLSI criteria for carbapenemase screening.

Bacterial strains
Three hundred and one non-duplicate clinical isolates were analysed. The isolates included 153 E. coli isolates and 148 K. pneumoniae isolates and had been collected from January to December 2008 in Huashan Hospital, Fudan University, Shanghai, China. All the isolates produced ESBLs, as confirmed by the CLSI phenotypic confirmatory test initially, but were susceptible to carbapenems (zones of inhibition of $19 mm in diameter for ertapenem and $16 mm for meropenem, respectively) (9). As control, 18 carbapenem-resistant, KPC-2 type carbapenemaseproducing K. pneumoniae clinical isolates were used in this study (11).
Antimicrobial susceptibility testing and the MHT Antimicrobial susceptibility testing was performed using agar dilution methods recommended by the CLSI (9). Minimal inhibitory concentrations (MICs) of cefazolin, cefoxitin, cefotaxime, ceftriaxone, aztreonam, ceftazidime, cefepime, ertapenem, imipenem, meropenem, and piperacillin/tazobactam were determined in accordance with the CLSI criteria (9). The MHT was carried out on all isolates to detect carbapenemase using ertapenem and meropenem as described by the CLSI (9). A well characterized strain of C. freundii that produced IMP-9-type metallo-b-lactamase (MBL) and E. coli strain ATCC 25922 were used as positive and negative controls, respectively.

Carbapenem inactivation assay
In order to determine whether resistance to imipenem and meropenem was likely caused by production of carbapenemase, a disk diffusion bioassay was performed on Mueller-Hinton agar with imipenem, meropenem and ertapenem disks as described by Yigit et al [12].

Genotypic detection of b-Lactamase
The b-lactamase genes TEM, SHV, PER, SFO, VEB, CTX-M and GES, and carbapenemases including KPC, VIM, IPM, SPM, SME, GIM, NMC, IMI, IND and OXA, were investigated in all of clinical strains using previously described primers [13][14][15]. Conserved primers, forward primer KPC-F (59-AGGACTT-TGGCGGCTCCAT-39) and reverse primer KPC-R (59-TCC-CTCGAGCGCGAGTCTA-39), were designed against the KPC gene to detect the KPC-1 to KPC-11 sequences according to the published nucleotide sequences in Genbank, which encode different KPC-type carbapenemases and produced a 748-bp PCR product. PCR amplicons were sequenced, and the DNA sequences obtained were compared to the available sequences via National Center for Biotechnology Information BLAST search.    Analysis of outer membrane proteins The outer membrane proteins of all isolates with false-positive result on MHT were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, as described previously [14].

Ethics Statement
As this study was focused on bacterial isolates collected from routine samples, approval from ethics committee at Fudan University was not necessary. Neither was there any need for informed consent procedures as no extra sampling was performed and no personal data was stored in relation to the isolates.

Antimicrobial susceptibility testing
The results of the antimicrobial susceptibility testing indicated that, for the 301 isolates tested, the inhibition zones for meropenem and ertapenem all had a diameter greater than 22 mm, and the MIC values were all below 1 mg/mL. Therefore, none of the isolates conformed to the CLSI criteria for carbapenemase screening (namely, diameters for meropenem and ertapenem of 19-21 mm and 16-21 mm, respectively, and MIC values of 2 mg/mL and 2-4 mg/mL, respectively.). None of the isolates was found to be susceptible to cefazolin and 99.7%, 62.1%, 48.2%, and 19.9% were resistant to cefotaxime, ceftazidime, cefepime, and cefoxitin, respectively. The susceptibility rates for imipenem, meropenem, and ertapenem were 100.0%, 100.0%, and 100.0%, respectively (Table 1).  Table 2). Although all the isolates were sensitive to carbapenems and carbapenemase-negative, the MHT yielded a positive result (Figure 1) for 3.3% (10/301) of the isolates and all the MHTpositive isolates (except one isolate was SHV-12 type ESBL producer) were CTX-M type ESBL producing (Table 3). Among 18 carbapenem-resistant K. pneumoniae clinical isolates, all were produced KPC-2 type carbapenemase and all were producing a positive result of MHT.

Carbapenem inactivation assay
A carbapenem inactivation assay performed on all 301 carbapenems-susceptible isolates indicated that alterations in shape of the zones of inhibition around the test organism were not observed, suggesting no carbapenemase was involved in hydrolysis of carbapenems in these isolates. However The assay of all 18 KPC-2 type carbapenemase-producing K. pneumoniae isolates were positive.

Analysis of outer membrane proteins
Analysis of the outer membrane porin proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that all 10 isolates (except 08-438 E.coli) exhibited loss of one of porin protein compared with the sensitive strains ( Figure 2 and Figure 3). This suggests that the outer membrane porin proteins might play an important role in resulting to the false-positive result.

Discussion
Given the increasing prevalence of carbapenemase-producing Enterobacteriaceae isolates worldwide [4], simple and accurate tests are needed to detect isolates that produce carbapenemase. For this purpose, the CLSI have recommended the MHT for the detection of carbapenemase in Enterobacteriaceae isolates. Although the MHT is a simple method for detecting carbapenemase-positive isolates, false positive results are obtained occasionally [8,10]. In June 2010, CLSI changed the carbapenem breakpoints and reviewed the recommendation on carbapenemase detection by MHT among Enterobacteriacea (M100-S20-U) and informed that MHT would not be recommended, except for epidemiological or infection control purposes.. In this study, we used carbapenemsusceptible E. coli and K. pneumoniae isolates that did not conform to the CLSI criteria for screening for the presence of carbapenemase. False positive results for the MHT were obtained among 3.3% of the isolates. We presume that the false positive results are probably due to low-level hydrolysis of ertapenem by ESBLs, particularly those of the CTX-M type, because we found that 97.3% of the isolates in our study produced CTX-M-type ESBLs [10,16].
Although the false positive results were observed in our study, MHT is not recommended (and never was) to detect carbapenemase among isolates that yielded negative-result on screening test. Whether for epidemiological or infection control purposes, adequate detection of the production of carbapenemase in Enterobacteriaceae isolates is crucial for infection control measures and the appropriate choice of antimicrobial therapy [8]. To improve the detection of carbapenemase-producing Enterobacteriaceae in clinical microbiology laboratories, several phenotypic tests to detect KPCs have been developed [17][18][19][20]. In addition to the MHT, a second phenotypic method has been shown to be promising for the identification of KPCs. This method utilizes boronic acid and 3-aminophenyl boronic acid-based compounds, and has proved to be highly sensitive and specific for the detection of KPCs.
In summary, as reported, CTX-M-type ESBLs might be hydrolysing the ertapenem used in the assay [16]. Clinical laboratories must be aware that false positive results may occur, especially in geographical areas where the incidence of isolates that produce CTX-M-type ESBLs is relatively high, such as the Asia-Pacific region [21]. Hence, further work is necessary to determine the frequency of false positive results when the MHT is used to detect carbapenemase production in Enterobacteriaceae isolates and more accurate methods should be developed for use in clinical microbiology laboratories.