|Year : 2012 | Volume
| Issue : 3 | Page : 290-295
Detection of Amp C genes encoding for beta-lactamases in Escherichia coli and Klebsiella pneumoniae
M Shanthi1, U Sekar1, K Arunagiri2, B Sekar3
1 Department of Microbiology, Sri Ramachandra Medical College and Research Institute, Sri Ramachandra University, Porur, India
2 Department of Molecular Biology, Central Leprosy Teaching and Research Institute, Chengalpattu, India
3 Pasteur Institute of India, Coonoor, Nilgiris, Tamil Nadu, India
|Date of Submission||15-Jan-2012|
|Date of Acceptance||04-Apr-2012|
|Date of Web Publication||8-Aug-2012|
Department of Microbiology, Sri Ramachandra Medical College and Research Institute, Sri Ramachandra University, Porur
Source of Support: None, Conflict of Interest: None
Purpose : Amp C beta-lactamase are Ambler class C enzymes that confer resistance to extended spectrum cephalosporins and are not inhibited by beta-lactamase inhibitors. Their detection is crucial, since the phenotypic tests are not standardised leading to ambiguity in interpretation of results. This study was done to detect the types of Amp C prevalent in Escherichia coli and Klebsiella pneumoniae by multiplex polymerase chain reaction (PCR). Materials and Methods : Seventy-seven consecutive cefoxitin resistant clinical isolates of E. coli (n = 25) and K. pneumoniae (n = 52) were included in the study. Antibiotic susceptibility testing to various classes of antibiotics was performed by disc diffusion using Clinical Laboratory Standards Institute (CLSI) guidelines. Minimum inhibitory concentration (MIC) to cefoxitin, imipenem and meropenem were determined by broth microdilution method. Isolates were screened for production of Extended Spectrum Beta-Lactamase (ESBL). Multiplex PCR was performed for the detection of Amp C genes after phenotypic testing (Hodge test and inhibitor based test). Results : Cefoxitin Hodge test was positive in 40 isolates which included 20 E. coli and 20 K. pneumoniae. There was zone enhancement with boronic acid in 55 isolates, of which 36 were K. pneumoniae and 19 were E. coli. Multiplex PCR detected Amp C in 11/25 E. coli and 12/52 K. pneumoniae isolates. The Amp C genes detected were CIT (Amp C origin - Citrobacter freundii), DHA (Dhahran Hospital, Saudi Arabia), ACC (Ambler class C), EBC (Amp C origin - Enterobacter cloacae) groups. ESBL was co-produced in 54 isolates. Conclusions : Amp C was detected in 29.87% of the study isolates. Majority of them co-produced ESBL. The most common Amp C was the CIT family. Screen tests for cefoxitin resistance may be falsely positive due to production of carbapenamases.
Keywords: Amp C Beta-lactamases, Escherichia coli, Klebsiella pneumonia
|How to cite this article:|
Shanthi M, Sekar U, Arunagiri K, Sekar B. Detection of Amp C genes encoding for beta-lactamases in Escherichia coli and Klebsiella pneumoniae. Indian J Med Microbiol 2012;30:290-5
|How to cite this URL:|
Shanthi M, Sekar U, Arunagiri K, Sekar B. Detection of Amp C genes encoding for beta-lactamases in Escherichia coli and Klebsiella pneumoniae. Indian J Med Microbiol [serial online] 2012 [cited 2018 Aug 20];30:290-5. Available from: http://www.ijmm.org/text.asp?2012/30/3/290/99489
| ~ Introduction|| |
Beta-lactamase production is the most common mechanism of resistance in Gram negative bacteria. They are of significant concern because they restrict therapeutic options, cause treatment failures and are increasing in occurrence worldwide. Amp C beta-lactamase are Ambler class C or group I cephalosporinases that confer resistance to cephalosporins and cephamycins and are not affected by inhibitors (clavulanic acid, tazobactam and sulbactam). Of the two types of Amp C (plasmid-mediated and chromosomal), the former is encountered in Klebsiella sps., Proteus mirabilis and Salmonella More Details spp while the latter is seen in organisms such as Citrobacter freundii, Enterobacter cloacae, Morganella morganii, Hafnia alvei and Serratia marcescens. Escherichia More Details coli is unique in that as it also expresses chromosomal Amp C at low levels. , The plasmid determined enzymes are very closely related to chromosomal Amp C beta-lactamases. The Amp C beta-lactamases have been named based on their resistance to cephamycin (CMY), cefoxitin (FOX), moxalactam (MOX), latamoxef (LAT); site of discovery such as Miriam Hospital in Providence (MIR) or Dhahran Hospital in Saudi Arabia (DHA) or name of the source patient ,Bilal (BIL). Currently there are 43 CMY alleles, 7 varieties of FOX, 3 varieties to ACT and MOX, 2 varieties of DHA and 4 varieties of ACC, LAT and MIR each.These Amp C genes were grouped into six families based on the similarities in the gene sequence and/or origin as CIT (origin Citrobacter freundii), EBC (origin Enterobacter cloacae), DHA (origin Morganella morgannii), ACC( origin Hafnia alvei, FOX (origin unknown) and MOX( origin unknown). Plasmids carrying these genes often carry multiple other resistance genes. 
Detection of Amp C mediated resistance in clinical microbiology laboratory poses a problem because the phenotypic tests are not standardised. The Clinical Laboratory Standards Institute (CLSI) has not yet published guidelines for their detection. The Amp C beta-lactamases when co-produced with other beta-lactamases, can lead to indeterminate phenotypic test results, thus making genotypic characterisation mandatory. , Though there are few studies published from India on the prevalence and phenotypic detection of the Amp C beta- lactamases, there is a paucity of data on the distribution of the different types of plasmid Amp C genes in the Indian isolates. ,
This study was therefore undertaken to determine the prevalent plasmid Amp C beta-lactamases in E. coli and Klebsiella pneumoniae. An initial screening was performed using two phenotypic tests-Hodge test using cefoxitin and inhibitor based test using boronic acid. The study also aims to compare the performance of the phenotypic tests with polymerase chain reaction (PCR).
| ~ Materials and Methods|| |
The study was carried out in a 1600 bedded university teaching hospital for a period of 7 months (April 2010 to October 2010). It included a total of 77 cefoxitin resistant (by disc diffusion), consecutive, non-duplicate, clinically significant isolates of K. pneumoniae (n = 52) and E. coli (n = 25) from hospitalised patients. The study isolates were identified up to species level by standard biochemical tests and Microscan walkaway system using Gram negative panels.  They were obtained from various clinical specimens such as urine (n = 25), respiratory secretions (n = 22) which included sputum, endotracheal secretions and bronchoalveolar lavage, blood (n = 16) and exudative specimens (n = 14). E. coli (n = 25) isolates included in the study were from urine (n = 13), respiratory secretions (n = 7), exudates (n = 3) and blood (n = 2).The source of the K. pneumoniae (n = 52) were respiratory secretions (n = 15), blood (n = 14), urine (n = 12) and exudates (n = 11).
Antibiotic susceptibility testing
Antibiotic susceptibility test was done by disc diffusion method as per the CLSI guidelines.  The drugs tested were ceftazidime (30 μg), cefotaxime (30 μg), cefepime (30 μg), ciprofloxacin (5 μg), amikacin (30 μg), piperacillin-tazobactam (100/10 μg), polymyxin B (300units) [Himedia laboratories, Mumbai], cefoxitin (30 μg), ertapenem (10 μg), imipenem (10 μg) and meropenem (10 μg) [BD diagnostics]. E. coli ATCC 25922 and K. pneumoniae ATCC 700603 were used as control strains.
Minimum inhibitory concentration
Minimum Inhibitory Concentration (MIC) to cefoxitin, imipenem and meropenem were determined by broth microdilution method (Range: 0.008-128 μg/ml) according to CLSI guidelines. 
Extended Spectrum Beta-Lactamase (ESBL) Screening
Isolates were tested for ESBL production by the CLSI method. 
Phenotypic tests for amp C Production
Amp C production was tested by inhibitor-based method using boronic acid as inhibitor and Hodge test (HT) using cefoxitin. ,
MIC for cefoxitin in combination with 400 mg/L of boronic acid was determined by broth microdilution method. An eight fold or greater concentration decrease in MIC of cefoxitin tested in combination with boronic acid versus the MIC for cefoxitin when tested alone was considered indicative of Amp C production. ,
Multiplex PCR for Amp C Genes
Amp C genes were detected by multiplex PCR using the method described by Pénone rez-Pénone rez and Hanson with some modifications. DNA template was prepared by boiling the bacterial cell suspension for 10 min. , The primers used are listed in [Table 1].
The concentration of the primers in the PCR mixture was 0.6 μM primers MOXMF, MOXMR, CITMF, CITMR, DHAMF, DHAMR; 0.5 μM primers ACCMF, ACCMR, EBCMF, EBCMR; 0.4 μM primers FOXMF and FOXMR.
The PCR conditions consisted of an initial denaturation step at 94°C for 3 min, followed by 25 cycles of DNA denaturation at 94°C for 30s, primer annealing at 64°C for 30s and primer extension at 72°C for 1 min and a final extension step at 72°C for 7 min. Around 5 μl aliquots of PCR product were analysed by gel electrophoresis with 2% agarose. Gels were stained with ethidium bromide and visualized by UV transillumination.
Screening for carbapenamases production
In isolates that were resistant to both cefoxitin and carbapenems by disc diffusion, co-production of carbapenamase was screened by Modified Hodge test (MHT) using meropenem (10 μg) disc in accordance with CLSI guidelines. 
| ~ Results|| |
All the study isolates were resistant to cefepime, piperacillin/tazobactam and ciprofloxacin. Among the E. coli (n = 25) resistance to amikacin was observed in 16 isolates, ertapenem resistance in 12 isolates and 7 were resistant to imipenem and meropenem. Of the 52 K. pneumoniae isolates included in the study, 48 were resistant to amikacin, 29 to ertapenem, 27 isolates to imipenem and meropenem. The study isolates were uniformly susceptible to polymyxin B.
Phenotypic screening tests for Amp C beta lactamases
The results of cefoxitin screen test both by disc diffusion and MIC estimation were comparable. The Boronic acid Inhibition (BAI) test was positive in 55 isolates which included 19 E. coli and 36 K. pneumoniae. Hodge test using cefoxitin was positive in 40 isolates which included 20 of each E. coli and K. pneumoniae.
PCR for Amp C Beta-Lactamases
Multiplex PCR detected plasmid Amp C in 23 isolates, of which 12 were K. pneumoniae and 11 were E. coli. The amplicon size were 462 bp (CIT), 405 bp (DHA), 346 bp (ACC), 302 bp (EBC). The different Amp C genes detected were CIT, DHA, ACC and EBC types [Figure 1]. Notably, MOX and FOX types were not detected in the study isolates. The types of Amp C detected and their distribution among the study isolates is shown in [Table 2]. Comparison of the phenotypic tests and the PCR results is shown in [Table 3] and [Table 4].
|Table 2: Distribution of the plasmid mediated Amp C genes within the study isolates|
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|Table 4: Sensitivity and specifi city of the plasmid mediated Amp C beta-lactamase detection methods|
when compared with PCR
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|Figure 1: Multiplex PCR for detection of Amp C beta lactamases, Lane1— Molecular mass marker (100 bp DNA ladder);lane 2, 3— EBC; lane 4— DHA; lane 5— Molecular mass marker (100 bp DNA ladder); lanes 6, 7— CIT; Lane 8— CIT and EBC; lane 9— CIT; lane 10— DHA; lane 11— Molecular mass marker (100 bp DNA ladder)|
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ESBL was co-produced in 54 isolates. Ten isolates, which were ESBL non producers, harboured Amp C.
Screening for carbapenamases: Resistance to carbapenems was detected in 41 isolates among which 34 were resistant to imipenem, meropenem and ertapenem and 7 isolates were resistant to ertapenem alone. The MIC 90 values for imipenem and meropenem were 4 mg/L and 2 mg/L, respectively. Among the 41 carbapenem resistant isolates, MHT using meropenem was positive in 40 isolates. [Table 5] shows the results of Amp C detection by phenotypic methods and multiplex PCR in carbapenem resistant isolates.
|Table 5: Results of the Amp C screen test and plasmid multiplex PCR in carbapenem resistant isolates (n = 41)|
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| ~ Discussion|| |
The Amp C resistance phenotypes occur due to over expression of the chromosomal Amp C gene, acquisition of a plasmid Amp C, alteration in the permeability of the cell to cefoxitin, or a combination of the above factors. Lack of a standardised phenotypic method for screening and detection of this type of resistance, makes the surveillance and characterisation of such strains problematic. ,
We screened for Amp C producers by disc diffusion and MIC determination using cefoxitin and results of both were comparable. Hence it may be assumed that cefoxitin MIC determination is not mandatory for Amp C screening. These isolates were then subjected to phenotypic tests namely cefoxitin Hodge test and boronic acid inhibitor method. The presence of Amp C genes was confirmed by multiplex PCR.
Considering PCR as the gold standard, we compared the sensitivity and specificity of the phenotypic tests employed. The Hodge test fared better in terms of sensitivity and specificity when compared with the inhibitor-based test (78.2% and 59.2% vs 65.2% and 25.9%). Both the tests had better negative predictive values.
Overall, plasmid mediated Amp C was detected in 29.8% (n = 23) of the study isolates, which included 11/25 E. coli and 12/52 K. pneumoniae. The prevalence of plasmid mediated Amp C varied widely in different parts of the world from 2% to 46%. ,, In Indian studies, the prevalence of Amp C ranged from 8% to 47%. ,,,
We looked for the following plasmid Amp C types-CIT, DHA, ACC, EBC, MOX and FOX of which the latter two were not detected. Coexistence of multiple types was observed. The most common were the CIT family (LAT-1 to LAT-4, CMY-2 to CMY-7 and BIL-1), followed by the DHA which was found alone in three isolates and together with EBC in four. ACC was detected in one isolate only. Worldwide, blaCMY-2 is the most prevalent plasmid mediated Amp C. The other commonly reported Amp C is the DHA. ,, Outbreaks of infections have been traced to strains harbouring CMY-2, MIR-1, BIL-1, ACT-1, ACC-1, etc. FOX enzymes have been described in Spain, Argentina and Italy, while MOX has been reported mainly from Japan and France. ,, There are very few reports on the prevalence of different plasmid Amp C types in Indian isolates. In one study, out of 455 E. coli isolates, multiplex PCR detected 103 (22.6%) isolates harbouring different families of Amp C gene with bla CIT being predominant. 
In K. pneumoniae which do not possess chromosomal Amp C, detection of Amp C is presumed as plasmid borne. However in E. coli isolates that are known to hyper produce chromosomal Amp C, the distinction between plasmid and chromosomal Amp C is possible only by performing isoelectric focusing or by transfer experiments.  This study did not include isoelectric focusing or transfer experiments.
The cefoxitin Hodge test was positive in all the CIT producing isolates (n = 14), whereas the BAI test was positive only in 7 isolates. Previous studies have reported that the CMY-2 could be screened effectively by cefoxitin Hodge test. It is a simpler test because it does not require a beta-lactamase releasing procedure. However it is unreliable in the detection of DHA-1 producing isolates.  In our study also, the DHA alone producing isolates (n = 3) were all cefoxitin Hodge test negative. In those which co-produced EBC, this test was positive. The BAI test was positive in all the DHA producing isolates thus providing a good correlation between the two. The ACC enzyme appears to be inhibited by cefoxitin and isolates may appear susceptible to cefoxitin in the screen test. , In our study the lone ACC producing K. pneumoniae isolate was in-fact resistant to cefoxitin, but Hodge test and BAI tests were negative. This isolate was from the wound swab of a patient in the multidisciplinary intensive care unit (ICU) of the hospital. The MIC to cefoxitin was >128 μg/Ml. Resistance to cefoxitin in this isolate can be attributed to the presence of other beta-lactamase and/or porin defects.  Since cefoxitin resistance by disc diffusion was the inclusion criteria in this study, the ACC producing cefoxitin susceptible Klebsiella pneumoniae could have been missed.
Often Amp C enzymes are co-produced with other beta- lactamases such as TEM or ESBLs. In our study ESBL screening tests was positive in 54 isolates, indicating co-production. Of the 23 ESBL screen test negative isolates, 10 harboured Amp C. ESBL and Amp C masking each other when co-produced is a common occurrence.
In a small proportion of cefoxitin resistant study isolates (n = 6), Amp C was not detected by the Hodge test, inhibitor-based test and multiplex PCR. In these isolates the resistance is probably due to alteration in permeability of the cell to cefoxitin due to loss of porin or presence of other beta-lactamases. ,
Among the study isolates, 41 were resistant to carbapenems. Screening for carbapenamases by MHT was positive in 40. In one MHT negative, ertapenem resistant isolate, the attributable resistance mechanism may be ESBL production together with loss of porin. 
Amp C genes were detected in 12 of the carbapenem resistant isolates. In 10 isolates at least one of the Amp C screen test was positive. In two isolates where both the screen tests were negative, the attributable cause may be masking of the Amp C phenotype by carbapenamases such as KPC/metallobeta-lactamases (MBL). In the remaining carbapenem resistant isolates with negative PCR for plasmid Amp C (n = 29), at least one of the Amp C screen test were positive in 26 isolates. In these isolates presence of Amp C genes other than those in the multiplex assay is one possibility or these phenotypic tests could have reflected false positivity due to the co-production of carbapenamases such as KPC/MBLs. , In three carbapenem and cefoxitin resistant isolates, the causes could be presence of other beta- lactamases and/or porin loss.
To conclude, PCR and isoelectric focussing remain the gold standard for detection of amp C. Co-production of ESBLs and carbapenamases is not uncommon. The CIT family is the most common type of Amp C in E. coli and K. pneumoniae. DHA, EBC and ACC are the other families. Multiple Amp C types coexist in many isolates. Presence of beta-lactamases and/or porin defects render the phenotypic tests unreliable since many of these mechanisms mask each other. The phenotypic tests for Amp C detection have low sensitivity and specificity. However they seem to have reasonable negative predictive value.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]