Characterization of Plasmid-Mediated AmpC and Carbapenemases among Iranain Nosocomial Isolates of Klebsiella pneumoniae Using Phenotyping and Genotyping Methods

Objectives Plasmid-mediated AmpC β-lactamases (PMABLs) and carbapenemases are emerging groups of antimicrobial-resistance determinants. The aims of the study were to evaluate the occurrence of PMABLs and carbapenemases in clinical isolates of Klebsiella pneumoniae and compare the test performance of various phenotypic methods for detection of these enzymes in Iran. Methods A total of 100 K. pneumoniae isolates were collected from clinical specimens obtained in Valiasr Hospital. AmpC production in all isolates was determined using the AmpC disk test, the cephamycin Hodge test, the AmpC Etest, and the boronic acid combined-disk test. In addition, carbapenemase production was determined using the modified Hodge test, the EDTA disk synergy test, and the boronic acid combined-disk test. The performances of various phenotypic methods were evaluated by the comparison of their results with polymerase chain reaction (PCR) method as the gold standard. Results Of the 100 isolates, 19 (19%) were demonstrated to harbor the PMABL-resistance gene by the multiplex PCR method. The PCR result indicated the presence of carbapenemase genes in 12 isolates. The performance of various phenotypic tests carried out for detection of carbapenemase-producing isolates varied widely, ranging in sensitivity from 30% to 100% and in specificity from 90.8% to 100%. Conclusion This is the first report of MOX-type AmpC β-lactamase and blaGES in K. pneumoniae in Iran. A comparison of the phenotypic methods showed that a combination of cefoxitin plus boronic acid is optimal for detecting plasmid-mediated AmpC enzymes in K. pneumoniae, whereas the implementation of molecular methods is often complex, requires specially trained personnel, and is associated with higher costs.


Introduction
AmpC b-lactamases are clinically important cephalosporinases encoded on the chromosome of many organisms such as Citrobacter spp., Enterobacter spp., Morganella spp., Hafnia spp., and Serratia spp. in which they mediate resistance to cephalothin, cefazolin, cefoxitin, most penicillins, and all b-lactamase inhibitor antibiotics except cefepime and imipenem [1]. AmpC b-lactamase hydrolyzes cephamycins and its activity is not inhibited by clavulanic acid. These features distinguish AmpC b-lactamase from extended-spectrum b-lactamases. Since 1989, plasmidmediated AmpC b-lactamases (PMABLs) have been detected worldwide in the strains of Escherichia coli, Klebsiella spp., Proteus mirabilis, and Salmonella spp. This is because transmissible plasmids have acquired genes for AmpC enzymes, which consequently can appear in bacteria lacking (e.g., Klebsiella pneumoniae) or poorly expressing a chromosomal ampC gene (e.g., E. coli).
Although many phenotypic tests are available for AmpC detection, none of these is validated by the Clinical and Laboratory Standards Institute (CLSI). Therefore, it is important to develop methods that can reliably detect PMABLs [2,3]. Significant spread of carbapenem-resistant K. pneumoniae was observed in the United States, Europe, and some Asian countries [4,5]. Spreading of different types of carbapenemases, such as K. pneumoniae carbapenemase and the New Delhi metallo-b-lactamase-1, was reported in Iran and elsewhere [6,7]. Therefore, we evaluated the prevalence of PMABLs and carbapenemases in Iranain clinical isolates of K. pneumoniae and compared the performance of phenotypic tests for detection of these enzymes.

Bacterial strains and susceptibility testing
In this cross-sectional study, 100 nonduplicate K. pneumoniae isolates were collected from clinical specimens obtained in Valiasr Hospital in the central province of Iran (MarcheSeptember 2011).
The identification of all isolates was confirmed by the API 20E system (bioMerieux, Marcy-l'Etoile, France). Susceptibilities to 21 different antibiotics (MAST, Bootle, Merseyside, UK) were defined by disk diffusion according to the CLSI guidelines ( Figure 1) [8].
The results of phenotypic methods for detection of AmpC-and carbapenemase-producing isolates are shown in Tables 1 and 2, respectively.

Discussion
The prevalence of PMABLs evaluated in this study (19%) is much higher than described in several other parts of the world. In an earlier study from Iran, the prevalence of PMABLs was 8.4% in K. pneumoniae. In Japan, the prevalence of PMABLs was 13% in K. pneumoniae [18]. In China, the rate was 10.1% in K. pneumoniae and the lowest rates of AmpC genes were reported from Switzerland (0.4%) [19]. High prevalence of AmpC genes was reported from Korea (77%) and Singapore (26%) [19]. In this study, similar to other reports, CIT-(CMY-2) and MOX-type genes were predominantly present in K. pneumoniae, followed by the EBC and DHA types, whereas the ACC and FOX genes were not detected. The CMY-2 is one of the most prevalent and most widely distributed PMABLs and has been previously found in several countries [1].
The rate of resistance to cefoxitin in our isolates was 46% (n Z 46); only 12 isolates were AmpC producers and 34 isolates were negative. Cefoxitin resistance in these nonproducers could be due to some other resistance mechanism such as alterations in outer membrane permeability [2,20]. In this study, 63% (12/19 isolates) and 68.4% (13/19) of AmpC producers were found to be resistant to cefoxitin and amoxicillineclavulanic acid, respectively.
Cefotetan has a lower sensitivity than cefoxitin concerning the detection of AmpC production (52.6% vs. 63%). These data indicate that although screening methods using cefoxitin and amoxicillineclavulanic acid for detection of AmpC-harboring isolates are useful, they are not perfect [20,21]. Therefore, in contrast to Ingram et al [22], our study cannot recommend cefoxitin, cefotetan, and amoxicillineclavulanic-acid-susceptibility testing for initial AmpC screening. Resistance to cefepime among 11/19 (58%) AmpCpositive K. pneumoniae isolates is another important finding. No synergy was found between expandedspectrum cephalosporins and clavulanic acid using the double-disk synergy test (data not shown). Only 1/11 cefepime-resistant isolates in this study had carbapenemase genes (bla VIM ) and thus, resistance to cefepime may be mediated by structurally modified AmpC blactamases [23].
We found that the cefoxitin AE boronic acid disk test detected 19/19 AmpC-positive K. pneumoniae isolates and gave one false-positive result. The AmpC disk test and CHT detected 7/19 and 5/19 AmpC-positive strains (12 and 14 false negatives), respectively. Possibly, inhibitory compounds exhibit different levels of inhibition among heterogeneous groups of AmpCs [24].
The AmpC disk test and CHT gave five false-positive results. Of these five isolates, three were MBL (bla VIM ) producers and the two others were bla GES positive.
The AmpC Etest was easy to use, but was unsuitable for AmpC detection. Overall, 17/19 AmpC-producing isolates were not detected by this method. Test performance could be improved by developing an Etest strip with a broader minimum inhibitory concentration range. Inconclusive results using this test have been previously reported [21,22]. However, we indicate that combination-disk testing with boronic acid is potentially useful for clinical laboratories for the detection of PMABLs [13].
The prevalence of carbapenemases among our isolates was found to be 12%. The sensitivity and specificity of disk diffusion for their identification using imipenem disk were 66.6% and 100%, respectively, whereas meropenem disk demonstrated 100% sensitivity and specificity. Apparently, application of meropenem disk is superior for screening, which is in contrast to the study carried out by Benenson et al [25]. This is the first report of bla GES and bla MOX in Iran. Guiana extended-spectrum b-lactamase (GES b-lactamase) has been reported in Europe [26]. In our region, carbapenem resistance has emerged in K. pneumoniae, mostly as VIM-type MBLs. VIM-MBL producers have been reported in Greece, Kuwait, and Korea [27].
Four VIM-producing isolates were not detected by MHT (Table 2). It has been suggested that addition of

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A. Japoni-Nejad, et al zinc sulfate or the use of MacConkey agar may improve the sensitivity of the MHT for MBL producers [28]. In addition, the MHT gave five false positives. The overall sensitivity and specificity of the MHT were 66% and 94.3%, respectively. The sensitivity and specificity of the imipenem AE boronic acid disk test were 100% and 90.8%, respectively. Although two GES-producing isolates were detected using the MHT, there were also nine false-positive results. Of these nine isolates, six were MBL (bla VIM ) producers and three were AmpC producers. It is possible that other AmpC families, not detected by the multiplex PCR, were present in the six VIM-producer isolates in our study.
The MBL Etest was the most specific test (100%) for MBL detection in this study. In the EDS test, the EDTAeceftazidime combination detected additional MBL producers, which were not identified by the EDTAeimipenem combination (90% sensitivity vs. 30% sensitivity). Therefore, ceftazidime appears to be Weakly positive. CAZ Z ceftazidime; IMI Z imipenem; MBL Z metallo-b-lactamases; MEM Z meropenem; MHT Z modified Hodge test.
Results were rated inconclusive if the minimum inhibitory concentrations exceeded the scale of the Etest for cefotetan alone and/or cefotetan in combination with cloxacillin (Etest AmpC). CAZ Z ceftazidime; CT Z cefotetan; CTX Z cefotaxime; FEP Z cefepime; FOX Z cefoxitin; IMI Z imipenem; IR Z inconclusive result. the better substrate for the EDS test. Similar results were observed by Noyal et al [29].
In conclusion, this study confirmed the prevalence of AmpC-producing K. pneumoniae and carbapenemase (bla VIM and bla GES ) in the central hospital of Arak (Iran) to be very high. By understanding the resistance pattern and prevalence of the b-lactamase-producing organisms, especially K. pneumoniae, we would be able to manage infection-control policy as well as proper and rational antibiotics prescription. The results indicate that a cefoxitin plus boronic acid is potential method for clinical laboratories to detect emerging AmpC-plasmid-mediated enzymes in K. pneumoniae, whereas the implementation of molecular methods is often complex, requires specially trained personnel, and is associated with higher costs.

Conflicts of interest
The authors declare no conflicts of interest.