|Year : 2013 | Volume
| Issue : 1 | Page : 53-59
Detection of plasmid-mediated AmpC β-lactamase in Escherichia coli and Klebsiella pneumoniae
NO Yilmaz1, N Agus1, E Bozcal2, O Oner2, A Uzel2
1 Department of Microbiology Laboratory, Tepecik Educational and Research Hospital , Ege University, Izmir, Turkey
2 Department of Biology, Basic and Industrial Microbiology Section, Izmir, Turkey
|Date of Submission||13-Jul-2012|
|Date of Acceptance||18-Sep-2012|
|Date of Web Publication||15-Mar-2013|
N O Yilmaz
Department of Microbiology Laboratory, Tepecik Educational and Research Hospital , Ege University, Izmir
Source of Support: None, Conflict of Interest: None
Background: Detecting plasmid-mediated AmpC (pAmpC) β-lactamase-producing organism is important for optimal infection control and providing accurate and effective treatment option for physicians. Objectives: The aim of this study was to investigate the prevalence of pAmpC β-lactamase and compare the results of boronic acid (BA) disk test with other phenotypic tests detecting AmpC positive isolates. Materials and Methods: A total of 273 clinical isolates of Klebsiella pneumoniae (n: 82) and Escherichia coli (n: 191) were analysed. The presence of pAmpC β-lactamase was determined by BA disk test, cefoxitin (FOX) screening test, modified three dimensional test (M3DT), and multiplex polymerase chain reaction (PCR). Pulsed-field gel electrophoresis was performed to evaluate the genetic similarities between isolates. To detect extended spectrum β-lactamases (ESBL) in the presence of AmpC β-lactamase, ESBL confirmation test was carried out with and without BA solution. Results: Of the 273 strains tested, 127 strains were found FOX resistant, 114 were positive by M3DT, 108 were positive in BA disk test, and the multiplex PCR detected 24 pAmpC β-lactamase-positive isolate. The prevalence of AmpC-producing strains was 10.9% in E. coli and 3.6% in K. pneumoniae in the tested population by PCR. CIT and MOX group genes were predominant type in these strains. Conclusion: These results emphasize that clinical laboratories should consider testing the presence of pAmpC enzymes particularly in FOX-resistant isolates, and BA disk test will improve detection of this emerging resistance phenotype.
Keywords: AmpC β-lactamase, boronic acid, extended spectrum β-lactamases, pulsed-field gel electrophoresis
|How to cite this article:|
Yilmaz N O, Agus N, Bozcal E, Oner O, Uzel A. Detection of plasmid-mediated AmpC β-lactamase in Escherichia coli and Klebsiella pneumoniae. Indian J Med Microbiol 2013;31:53-9
|How to cite this URL:|
Yilmaz N O, Agus N, Bozcal E, Oner O, Uzel A. Detection of plasmid-mediated AmpC β-lactamase in Escherichia coli and Klebsiella pneumoniae. Indian J Med Microbiol [serial online] 2013 [cited 2019 May 26];31:53-9. Available from: http://www.ijmm.org/text.asp?2013/31/1/53/108723
| ~ Introduction|| |
AmpC β-lactamases, which belong to group 1 according to the classification of Bush et al.,  have gained importance since the late 1970s as one of the mediators of antimicrobial resistance in Gram-negative bacilli. AmpC β-lactamases are of two types: Plasmid-mediated and chromosomal. Chromosomal AmpC enzymes are seen in organisms such as Citrobacter freundii, Enterobacter cloacae, Morganella morganii, Hafnia alvei, and Serratia marcescens. These inducible chromosomal genes were also detected on plasmids in 1988.  The transfer of AmpC genes to plasmids has resulted in their dissemination among Enterobacteriaceae, with the consequence that AmpC-encoded β-lactamases are now present in strains of Klebsiella spp., Escherichia More Details coli, Proteus mirabilis, and Salmonella More Details spp.  Moreover, since the first report of transferable plasmid-mediated AmpC (pAmpC) β-lactamases in the late 1980s, their increasing presence worldwide is becoming of great concern. 
For clinical microbiologist, detection of AmpC-mediated resistance in Gram-negative organisms poses a problem because phenotypic tests may be misleading resulting in misreporting and treatment failures. Although there are some recommendations of Clinical and Laboratory Standards Institute (CLSI) for detecting extended spectrum β-lactamases (ESBLs)-producing isolates of E. coli, Klebsiella spp., and P. mirabilis, there is no recommendation for detecting pAmpC-producing organisms or organisms harboring both ESBL and pAmpC.  They typically have a negative confirmatory test for ESBLs and therefore laboratories may report AmpC producers as susceptible to broad-spectrum cephalosporins. This may have disastrous consequences if physicians use broad-spectrum cephalosporins to treat serious infections such as bacteremia. 
Several methods have been developed for detection the AmpC β-lactamases. Screening with cefoxitin (FOX) disk is recommended for initial detection. However, it does not reliably indicate AmpC production.  The phenotypic methods like the Kirby-Bauer disk potentiation method with some β-lactamase inhibitors or the three-dimensional method, modified double disk test and the cefoxitin-Hodge test have been performed. But these methods are labour-intensive, technically intricate, and not suitable for routine clinical use in clinical microbiology laboratories and may not detect all AmpC β-lactamases. , Also, phenotypic tests are not able to differentiate between chromosomal AmpC genes and AmpC genes that are carried on plasmids.  Polymerase chain reaction (PCR) analysis may be used to detect the presence of externally acquired AmpC genes. But it is expensive and requires time-consuming techniques, and not yet available for routine use in clinical laboratories.  There is need for practical and simple method to detect the resistance mediated by pAmpC β-lactamase. The inhibitor-based methods similar to the CLSI ESBL confirmatory test are simple to perform and easy to interpret. ,, Inhibitors of the AmpC enzyme are well described and include boronic acid (BA) compounds, cloxacillin, and novel inhibitors such as Syn2190.  In 1982, BAs were reported as reversible inhibitors of AmpC β-lactamases.  There are several reports for the detection of AmpC β-lactamases with BA. ,,,
The aims of this study were to investigate the presence of the AmpC β-lactamases with different methods, compare the results of BA disk test with other phenotypic tests and to evaluate the genetic similarities using pulsed-field gel electrophoresis (PFGE) analysis in E. coli and Klebsiella pneumoniae isolates. To detect ESBL in the presence of AmpC β-lactamase, ESBL confirmation test was carried out with and without BA solution.
Antimicrobial susceptibility tests were performed to evaluate the results of the isolates with pAmpC β-lactamases.
To the best of our knowledge, and as supported by the absence of reports in PubMed, this is the first study to research AmpC β-lactamases by BA inhibition test method in Turkey.
| ~ Materials and Methods|| |
Clinical isolates were collected and identified by the conventional methods  or VITEK 2 automatic system (bioMerieux, Marcy I'Etoile, France). A total of 273 non-repeat K. pneumoniae (n:82) and E. coli (n:191) isolates showing resistance to one or several extended-spectrum cephalosporins were collected between January and December 2009. Isolates were screened for FOX susceptibility by the standard disk diffusion method using 30-μg disks (Becton Dickinson Microbiology System, Cockeysville, MD, USA).  Isolates that yielded a zone diameter less than 18 mm were suspected to be AmpC producers, described as screen positive, and further subjected confirmatory testing.
ESBL and AmpC enzyme detection
All the screen positive isolates were tested by modified three-dimensional test (M3DT) as described by Coudran et al.  and BA disk tests. M3DT was briefly as follows: Crude enzyme extract of the test organism was prepared by repeated freeze thawing in -80°C for seven times. A lawn culture of E. coli ATCC 25922 was made on MHA and a FOX (30 μg) disk was placed at the centre. Linear slit was cut, 3 mm away from the disk and 30 μl of the enzyme extract was added to a well-made at the outer edge of the slit. The plate was incubated overnight at 37°C. Clear distortion of zone of inhibition of FOX is considered positive test.
The BA disk test was performed by using commercially available antibiotic and BA-containing disks as described previously with some modification.  Briefly, 120 mg of phenylboronic acid (benzeneboronic acid, Schuchardt CGH, Germany) was dissolved in 3 ml of dimethylsulphoxide (Carlo Erba, Italy) and 3 ml of sterile distilled water was added to this solution. Twenty microliter of the stock solution was dispensed onto disks containing 30 μg FOX, ceftazidime (CAZ), cefotaxime (CTX), ceftazidime-clavulanic acid (CAZ-CLA), cefotaxime-clavulanic acid (CTX-CLA). Disks were allowed to dry for 30-60 min and used immediately. The BA disk test was performed by inoculating Mueller-Hinton agar by the standard disk diffusion method and placing the disks containing antibiotics with and without BA onto the agar. Inoculated plates were incubated overnight at 35 o C. An organism demonstrating a zone diameter around the FOX, and/or CAZ, and/or CTX disk containing with BA ≥5 mm than containing alone these disks were considered an AmpC producer.
Similar to the CLSI confirmatory test, an organism exhibiting an increase of ≥5 mm zone size around the BA containing CAZ-CLA or CTX-CLA disk (CAZ-CLA-BA and/or CTX-CLA-BA) compared with CAZ or CTX disk alone was considered positive for ESBL production. E. coli J53 Tc (CMY-2) and K. pneumoniae FOR (DHA-2) were used as positive control. As negative controls, ATCC E. coli 25922, was used.
Detecting of AmpC 0β-lactamase genes by PCR
Multiplex PCR was performed on the M3DT-positive isolates (E. coli: 99, K. pneumoniae: 15). DNA isolation was performed with PureLink Genomic DNA Mini Kit (Invitrogen, Carlsbad, USA). Organisms were tested for the presence of six families of pAmpC β-lactamase genes (FOX, CIT, DHA, EBC, MOX, and ACC) using the multiplex PCR assay described by Perez-Perez and Hanson. 
ESBL detection by CLSI confirmation test
The CLSI confirmatory method using CAZ and CTX disks with and without clavulanic acid (10 μg) was also carried out.  E. coli ATCC 25922 and K. pneumoniae ATCC 700603 were used as negative and positive controls.
Pulsed-field gel electrophoresis
The clonal relatedness of M3DT-positive E. coli and K. pneumoniae isolates were investigated with PFGE method. PFGE was carried out according to procedure described by Turabelidze et al. with minor modifications.  After digestion of the cells and washing of the plugs, genomic DNA was digested with XbaI (Invitrogen, Carlsbad, CA, USA). The DNA restriction fragments in plugs were separated by electrophoresis through 2% pulsed-field certified agarose (Bio-Rad Laboratories, CA, USA) in CHEF-Mapper System (Bio-Rad Laboratories, Nazareth, Belgium). DNA bands were visualized by staining of gel with ethidium bromide and viewed by Versa-Doc Imaging System (Bio-Rad Laboratories, Nazareth, Belgium). The DNA band profiles were analysed with Bio-1D ++ (UPGMA) software (Bio-Rad Laboratories, Nazareth, Belgium) program coefficient with Nei and Li Dice. The results of band patterns were evaluated according to criteria of Tenover et al.,  and the strains were classified as indistinguishable, closely related, possible related, or different.
Antimicrobial susceptibility test
Antibiotic susceptibility testing was performed by the Kirby-Bauer disk diffusion method according to CLSI protocols.  Six β-lactams (CAZ [30 μg], CTX [30 μg], cefepime [FEP] [30 μg], piperacillin-tazobactam [TZP] [100/10 μg], imipenem [IMP] [10 μg], and meropenem [MEM] [10 μg]) and three non-β-lactam antibiotics (gentamicin [CN] [10 μg], amikacin [AK] [30 μg], trimethoprim/sulfametaxazol [SXT] [1.25/23.75 μg]) susceptibilities were evaluated. Intermediate and resistant strains were grouped together and classified as resistant. E. coli ATCC 25922 was used as a quality control strain.
Data were analysed using SPSS software (Version 10.0; SPSS Inc., Chicago). Fisher's exact test was performed to determine statistically significant differences among the antibiotic susceptibility rate of AmpC and non-AmpC-producing E. coli and K. pneumoniae isolates. The standard significance level, P < 0.05, was used, and all tests of statistical significance were two-tailed.
| ~ Results|| |
Bacterial strain distribution
A total of 273 clinical isolates of K. pneumoniae (n:82) and E. coli (n:191) were analysed. Distribution of the different wards and specimens among isolates are shown in [Table 1]. Specimens were isolated from the urinary tract (n:237, 86.8%), blood cultures (n:21, 7.7%) and other body sites (n:15, 5.5%). Of the 273 test isolates 163 (60%) were from outpatients, 78 (28%) were from hospitalized patients and 32 (12%) were from intensive care unit.
The occurrence rates of plasmid-mediated AmpC producers in E. coli and K. pneumoniae
Of the 273 strains tested, 127 (46.5%) strains were found FOX resistant (E. coli: 109, K. pneumoniae: 18), 114 (41.7%) were positive by M3DT (E. coli: 99, K. pneumoniae: 15), 108 (39.5%) were positive by BA disk test (E. coli: 93, K. pneumoniae: 15). The multiplex PCR detected 24 (8.7%) pAmpC β-lactamase-positive isolates (E. coli: 21, K. pneumoniae: 3) [Table 2]. To determine AmpC β-lactamases among the three disks (FOX, CAZ, CTX) by BA disk test CAZ showed the best performance in combination with BA.
|Table 2: Detection of AmpC β-lactamase by different methods and screening results from the Clinical and Laboratory Standards Institute1 confi rmatory and boronic acid2 disk tests for extended spectrum β-lactamases3|
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The prevalence of AmpC-producing strains was 10.9% (21/191) in E. coli and 3.6% (3/82) K. pneumoniae in the tested population by PCR. The occurrence rate of pAmpC-producing strains in E. coli was higher than that of the K. pneumoniae strains.
In this test population, the detection of AmpC activity using resistance to FOX by disk testing as a screening criterion showed a sensitivity of 70%, a specificity of 55%. The BA disk tests showed sensitivity of 100%, a specificity of 66%.
The distribution of genotypes in plasmid-mediated AmpC-producing isolates
MOX group genes (including MOX-1, MOX-2, CMY-1, and CMY-8 to CMY-11) were the predominant type in all isolates. Among the pAmpC-producing E. coli strains, the MOX and CIT group genes (CMY like genes originated from Citrobacter freundii, including LAT-1 to LAT-4, CMY-2 to CMY-7, and BIL-1) were harboured by 18 (18/21, 85%) of the isolates. The distributions of the AmpC β-lactamase genes for E coli were: 10 CIT, 8 MOX, 3 EBC, and 1 FOX group genes (one of the isolate harboured both FOX and CIT genes). All K. pneumoniae isolates carried MOX group genes [Figure 1]. Meanwhile DHA and ACC group genes were not detected.
|Figure 1: Detection of Amp-C genes by multiplex PCR. M: Marker, A: Control strain|
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ESBL detection by BA disk test and the CLSI confirmatory test
One hundred and twenty-seven strains (46%) harboured only ESBLs, 105 strains (38%) harboured both ESBLs and pAmpC, and nine strains (3%) harboured only pAmpC β-lactamases.
Presence of ESBLs can be masked by the expression of AmpC β-lactamase. In our study, AmpC production also covered and masked underlying ESBL production in five additional isolates (all of them were E. coli) which were initially labelled as ESBL negative by the CAZ-CLA versus CAZ and CTX-CLA versus CTX tests. One of the major findings of this study was CTX-CLA-BA versus CTX disk tests determined all masked ESBL producing isolates.
PFGE and analysis of genetic similarities
According to PFGE band patterns, the studied 99 E. coli isolates were highly heterogeneous indicating there was no predominant epidemic clone [Figure 2] and [Figure 3]. The strains carrying resistance genes were not found clonally related with each other. The genetic relatedness of pAmpC encoding-K. pneumoniae isolates showed about 50% homology according to their band pattern. Regarding the criteria of Tenover et al., 15 K. pneumoniae strains were found in seven different genetic clones. This similarity might be a result of the fact that the same K. pneumoniae carried variable ESBL and/or AmpC genes, resulting in various antimicrobial susceptibility patterns. It was interesting that these clinical isolates were collected from the patients who were hospitalized in the recent past.
|Figure 2: A representative pulsed-field gel electrophoresis (PFGE) typing results of 29 E. coli strains|
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|Figure 3: Dendrogram to illustrate the genetic relatedness of 15 AmpC β-lactamase producing K. p neumoniae isolates examined by pulsed-fi eld gel electrophoresis (PFGE)|
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Antibiotic susceptibility of plasmid-mediated AmpC-producing isolates
A comparison of antimicrobial susceptibilities of the AmpC and non-AmpC producers E. coli and K. pneumoniae isolates is shown in [Table 3]. When isolates produced AmpC β-lactamase they were more resistant than others. Non-AmpC-producing isolates were more resistant to third-generation cephalosporins than AmpC-producing isolates (P < 0.05). All isolates were susceptible to IMP and MEM.
|Table 3: Antibiotics susceptibility rate of AmpC and Non-AmpC producers E. coli and K. pneumoniae isolates (%)|
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| ~ Discussion|| |
The increasing prevalence of AmpC β-lactamase resistance among E. coli and K. pneumoniae, which are the most commonly isolated species of Enterobacteriaceae family in the clinical laboratory, is becoming a serious worldwide problem. High-level AmpC production is typically associated with in-vitro resistance to third-generation cephalosporins and cephamycins. In connection with this, high clinical treatment failures with broad-spectrum cephalosporins have been documented. , However, in this study, one-third of bacterial isolates possessing pAmpC have been documented as susceptible to third-generation cephalosporins. So, detection of AmpC β-lactamase is very important for the treatment of patients and infection control purposes.
Due to the lack of simple and reliable detection methods for AmpC β-lactamases those can be performed in clinical laboratories, their exact prevalence is unknown. The prevalence of pAmpC-producing strains were 10.9% (21/191) in E. coli and 3.6% (3/82) K. pneumoniae in the tested population (overall 8.7%) by PCR. This rate of prevalence was much higher than that reported from other countries (China: 2.79%, Japan: 0.13%); on the other hand, it is lower than in some countries such as Korea (39.3% K. pneumoniae, 3.1% E. coli). ,, Differences between these results may be related to the features of the selected isolates (different geographic regions, antimicrobial resistance phenotype of isolates, etc.) or detection methods (phenotypic or genotypic tests). Results from a single centre study should be interpreted with care, further population-based prevalence studies are required to observe the true spread of pAmpC β-lactamases.
Although there is no CLSI guidelines for phenotypic methods to screen and detect AmpC activity in E. coli and Klebsiella spp.,  several methods have been developed for detection the pAmpC β-lactamases. Reduced susceptibility to FOX in the Enterobacteriaceae may be an indicator of AmpC activity. Unfortunately, FOX resistance is not only due to AmpC β-lactamase production, but may also due to alterations to outer membrane permeability.  Coudron showed that 55 of the 271 FOX resistant isolates were AmpC-PCR positive and the BA disk test detected 54 of the isolates.  The present study demonstrated that 114 and 108 of the 127 FOX-resistant isolates were AmpC β-lactamase positive by M3DT and BA disk test, respectively. The cause of FOX resistance in the remaining isolates probably due to non-enzymatic-resistance mechanisms such as altered permeability. Screening for AmpC-producing organisms using resistance to FOX was less sensitive and specific than BA disk test. The results of this study underline the need for reliable laboratory tests that confirm the presence of AmpC β-lactamases in clinical isolates.
There are several reports of studies which used BA compounds for the identification of AmpC β-lactamase-producing bacteria. ,, Although the BA disk test is simple enough to be used in clinical microbiology laboratories, the evaluation of the results of susceptibility tests is subjective. The difference of the disks inhibition zone diameters with and without BA solution affects the results. Differences of 4 or 5 mm cause critic results. So, evaluation must be done carefully. In this study, pAmpC genes were detected in 24 of the 108 (22%) BA disk test positive isolates. Based on these findings, the BA disk test exhibited 100% sensitivity, 66% specificity. Tenover et al.  found that only 18 of 31 (58%) BA disk test positive isolates give positive result in the multiplex PCR. Authors suggest that the diversity of pAmpC β-lactamase genes continues to expand beyond those contained in the six families of genes covered by multiplex assay. In this study, only six families of pAmpC β-lactamases genes were used. It should be noted that BA disk tests are unable to distinguish E. coli isolates that produce AmpC-β-lactamase due to imported genes with isolates carrying altered AmpC chromosomal genes.  A chromosomal AmpC gene is present in E. coli, but it is not usually expressed because of the lack of a strong promotor region.  Differentiation between M3DT or BA disk test and PCR tests results can be explained by the increased activities of chromosomal AmpCs or porin mutations or an unknown AmpC gene.
It has been stated that the AmpC β-lactamases when present along with ESBL can mask the phenotype of the latter.  Thus, the coexistence of pAmpC and ESBL in the same strain may give false-negative tests results for the detection of ESBL. Although, in new CLSI interpretive criteria, routine ESBL testing is no longer necessary while reporting results may still be useful for epidemiological or infection control purposes.  When ESBL production is suspected but the confirmatory test is negative, the strain should be screened for the presence of Amp-C β-lactamases. In the present study, five isolates which harboured pAmpC β-lactamases showed ESBL-negative result by the CLSI ESBL confirmatory test (CAZ-CLA vs. CAZ and CTX-CLA vs. CTX), but positive with BA disk test (CAZ vs. CAZ-CLA-BA and/or CTX vs. CTX-CLA-BA). If CLSI ESBL confirmatory test was used alone, five ESBL-producing organisms were missed.
Plasmids containing genes that encode for AmpC and/or ESBLs often contain resistant determinants for other classes of antimicrobial agents and are readily transmissible from strain to strain and among different species in Enterobacteriaceae. According to the result of susceptibility test, AmpC-producing isolates showed high resistance rates to cephalosporins than non-AmpC-producing isolates [Table 3]. Therefore, it is important to identify AmpC-producing isolates, and physicians can consider treatment with cephalosporins. Although the detection of only AmpC enzymes do not change the antimicrobial therapy in individual patients; moreover, if they are accompanied by other resistance mechanisms (such as porin channel block and ESBL), they affect susceptibility to other antimicrobial agents like quinolones, aminoglycosides, or carbapenems and limitations in therapeutic option.
| ~ Conclusion|| |
The exact detection of pAmpC and ESBLs in isolates is important for epidemiology and infection control purposes. pAmpC β-lactamase prevalence was 10.9% in E. coli and 3.6% K. pneumoniae isolates in our centre. Inhibitor-based method using BA is a practical and efficient method to detect plasmid-mediated AmpC-β-lactamases in E. coli and K. pneumoniae showing FOX resistance. Although phenotypic tests are unable to differentiate between pAmpC activity and chromosomally encoded AmpC activity, they are suitable for routine clinical microbiology laboratories. Further genotyping tests such as sequencing and typing the isolates may be required for better understanding of the genetic relatedness and molecular epidemiology of this resistance mechanisms.
| ~ Acknowledgments|| |
We are grateful to Professor Dr. Zeynep Gulay and Professor Dr. Deniz Gur for their valuable advice and suggestions during the development of this study. We would like to thank Professor Dr. Deniz Gur and Dr. G. Vardar Unlu for supplying the control strains. We thank to Izmir Institute of Technology, Biotechnology and Bioengineering Research and Application Center and Professor Dr. Baris Otlu for their help during PFGE.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]
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