|Year : 2014 | Volume
| Issue : 1 | Page : 23-28
Necessity of detection of extended spectrum beta-lactamase, AmpC and metallo-beta-lactamases in Gram-negative bacteria isolated from clinical specimens
Mehvash Haider, Meher Rizvi, Nazish Fatima, Indu Shukla, Abida Malik
Department of Microbiology, JNMCH, AMU, Aligarh, Uttar Pradesh, India
|Date of Web Publication||15-Mar-2014|
Department of Microbiology, JNMC, AMU, Aligarh, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Background: With increasing incidence of resistance to antibiotics carbapenems are used as the last resort because they are stable even in response to extended spectrum and AmpC beta-lactamases. However, Gram-negative bacilli producing the acquired metallo-beta-lactamases (MBL) are on the rise. Aim: The aim of the following study is to detect phenotypically the presence of extended spectrum beta-lactamase (ESBL), AmpC and MBL in Gram-negative bacteria isolated from clinical specimen. Materials and Methods: Gram-negative isolates from clinical samples were screened and confirmed for the presence of ESBLs by double disk synergy test (DDST), for AmpC by disk approximation assay and for MBL by Modified Hodge Test and imipenem-ethylenediaminetetraacetic acid DDST. Results: Among 251 isolates studied, 138 (54.98%) were ESBL producers, 49 (19.52%) were AmpC producers and 45 (17.93%) were MBL producers. Highest rates of ESBL detection was by Cefoperazone sulbactam (109/138) 78.98%. Out of 92 of the AmpC producing strains 20 (21.73%) were inducible and 72 were stably derepressed (78.26%). Out of 251 strains studied 45 (17.93%) were phenotypically identified as MBL producers, the highest no. being of Pseudomonas aeruginosa. Among the methods employed for detection of MBL production, Hodge test (62.22%) proved better than DDST (40%). Conclusions: High level of antibiotic resistance pattern exists in various clinical isolates. ESBL production should be looked for routinely in Gram-negative bacteria other than Escherichia coli and Klebsiella. A high percentage of derepressed AmpC mutants are noteworthy and alarming. We recommend phenotypic identification methods as routine practice in laboratories as genotypic methods are not cost-effective.
Keywords: Drug resistance, extended spectrum beta-lactamase, Gram-negative bacteria
|How to cite this article:|
Haider M, Rizvi M, Fatima N, Shukla I, Malik A. Necessity of detection of extended spectrum beta-lactamase, AmpC and metallo-beta-lactamases in Gram-negative bacteria isolated from clinical specimens. Muller J Med Sci Res 2014;5:23-8
|How to cite this URL:|
Haider M, Rizvi M, Fatima N, Shukla I, Malik A. Necessity of detection of extended spectrum beta-lactamase, AmpC and metallo-beta-lactamases in Gram-negative bacteria isolated from clinical specimens. Muller J Med Sci Res [serial online] 2014 [cited 2020 May 31];5:23-8. Available from: http://www.mjmsr.net/text.asp?2014/5/1/23/128939
| Introduction|| |
Extended spectrum beta-lactamases (ESBL's) were first reported in 1983  and plasmid mediated AmpC beta-lactamases were reported in 1988.  In general ESBL's are mutant, plasmid mediated beta-lactamases derived from older broad-spectrum beta-lactamases which have an extended profile that permits hydrolysis of all cephalosporins, penicillins and aztreonam. These enzymes are most commonly produced by Klebsiella pneumoniae and Escherichia More Details coli but may also occur in other Gram-negative bacteria. Plasmids responsible for ESBL production may carry resistance to many antibiotics such as aminoglycosides, fluoroquinolones, tetracyclines, chloramphenicol and cotrimoxazole.
AmpC class beta-lactamases are cephalosporinases that are poorly inhibited by clavulanic acid. They are differentiated from other ESBLs by their ability to hydrolyze cephamycins as well as other extended spectrum cephalosporins. Plasmid mediated AmpC beta-lactamases have originated through the transfer of chromosomal genes onto plasmids.  Until date all plasmid mediated AmpC beta-lactamases have similar substrate profiles to the parental enzymes from which they appear to be derived. Plasmid mediated AmpCs differ from chromosomal AmpCs in being constitutive. Both ESBLs and plasmid mediated AmpC beta-lactamases are typically associated with multi-drug-resistance, usually a consequence of genes for other antibiotic resistance mechanisms residing on the same plasmids as the ESBL and AmpC genes.
With the extensive use of third and fourth generation cephalosporins as an important component of empirical therapy, resistance to these drugs has become a major problem all over the world.
Carbapenems are often used as antibiotics of last resort for treating infections due to multi-drug resistant Gram-negative bacilli because they are stable even in response to extended spectrum and AmpC beta-lactamases. However, Gram-negative bacilli producing the acquired metallo-beta-lactamases (MBLs) are on the rise. ,,,, Given that MBLs hydrolyse virtually all classes of beta-lactams and that we are several years away from the development of an alternative and effective antimicrobial; the continued spread of these enzymes would be a clinical disaster.
The aims of our study included:
- To find out the prevalence of ESBL, AmpC and MBL in Gram-negative bacteria isolated from clinical specimens.
- To evaluate various phenotypic methods for identification of ESBL, AmpC and MBL production. We concentrated on phenotypic methods as molecular methods are not cost effective, especially in developing countries.
- To devise a standard operating procedure for antibiotic susceptibility reporting to aid in prompt and precise patient management.
| Materials and Methods|| |
The study was carried out in the Department of Microbiology, Jawaharlal Nehru Medical College, Aligarh, India over a 9 month period (2007-2008).
Samples received in the clinical bacteriology laboratory were processed for microbiological confirmation of clinically suspected infection. Patients in whom Gram-negative bacteria were isolated during routine diagnostic testing were included in the study.
Clinical information including age, sex, occupation, previous hospitalization, history of antibiotic use, current hospital stay, type of sample, invasive procedures undergone (if any) were recorded for each patient.
Samples were collected from patients as per standard procedures. 
Pus samples were collected after cleaning the lesions with sterile normal saline. Aspirated pus specimens were preferred over swabs. Special care was taken to avoid contamination by normal flora of skin or mucous membrane. Specimen was collected in sterile leak proof containers.
Urine samples (early morning clean catch mid-stream specimen) were collected in Universal containers. If the patient was catheterized, the tube was clamped above the port so as to let fresh urine collect. The tubing was then cleaned with 70% alcohol vigorously and with the help of a sterile syringe, urine was aspirated from the tubing.
Respiratory tract specimens (tracheal aspirates, bronchial aspirates, bronchoalveolar lavage fluids) were collected through bronchoscopes as per the standard method of injecting 30-50 ml of physiological saline into peripheral bronchiolar ramifications. The saline is then aspirated and submitted for smear preparation and culture. Early morning expectorated sputum samples were collected in sterile, disposable, wide mouthed, screw capped plastic containers.
Cerebrospinal fluid was collected by lumber spinal puncture in sterile leek proof containers under strict aseptic conditions after adequate skin disinfection.
Pleural or peritoneal fluid specimens were collected by thoracentesis and paracentesis, respectively under aseptic conditions after adequate skin disinfection.
Identification of Beta-lactamases
Initial screening according to Clinical and Laboratory Standards Institute (CLSI) (2012)  on day 1 [Figure 1].
|Figure 1: Double disc approximation test for extended spectrum beta-lactamase detection showing increase in zone diameter with addition of inhibitor by ≤5 mm with special reference to ceftazidime and ceftazidime sulbactam combination|
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It is noteworthy that we attempted screening and confirmation on the same day, also an isolate resistant to cefoxitin or showing no increase in zone diameter with the addition of inhibitor was suspected to be an AmpC producer; tests for which were put up on day 2.
Criteria followed for inducible AmpC on day 2.
- Blunting of zone toward inducer namely imipenem by 2 mm.
- No increase in zone size with the addition of inhibitor specifically sulbactam, clavulanic acid and tazobactam.
- Susceptible to cefepime.
Criteria followed for derepressed AmpC mutant:
- Resistant to all cephalosporins.
- No increase in zone size with the addition of an inhibitor.
MBL was suspected when,
- Resistant to imipenem (zone size <16 mm)
- Heaping and zone size >16 mm and <20 mm.
The suspected isolate was then subjected to Modified Hodge Test  [Figure 2] and imipenem- ethylenediaminetetraacetic acid (EDTA) double disk synergy test (DDST) [Figure 3].
|Figure 2: Modified Hodge Test showing the presence of a distorted inhibition zone around imipenem disc was interpreted as a positive result for carbapenem hydrolysis screening|
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|Figure 3: Imipenem ethylenediaminetetraacetic acid (EDTA) test for metallo-beta-lactamase (MBL) detection. EDTA acts as chelating agent. Even a small zone of synergistic inhibition of MBL around imipenem EDTA disc was interpreted as positive|
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Modified Hodge Test
E. coli ATCC 25922 at turbidity 0.5 Mc Farland standard was used to swab inoculate on the surface of a Mueller Hinton Agar plate and test strain was heavily streaked from center to plate periphery. After the plate was allowed to stand for 10 min at room temperature a 10 μg IPM disk was placed in the center and the plate was incubated overnight. The presence of a distorted inhibition zone was interpreted as a positive result for carbapenem hydrolysis screening.
EDTA acts as a chelating agent.
Suspected isolate was swabbed onto a plate of Mueller Hinton agar (turbidity 0.5 Mc Farland units). 10 μg IPM disk and a disk onto which 10 μl 0.5 M EDTA solutions was added were placed at a distance of 10 mm edge to edge.
After overnight incubation the presence of even a small zone of synergistic inhibition was interpreted as positive.
Criteria followed were:
- Resistant to cefoxitin (AmpC).
- Blunting of zone toward inducer (inducible AmpC).
- Increase in zone size with the addition of an inhibitor by >5 mm (ESBL).
Decrease in zone diameter around imipenem, confirmed by Modified Hodge Test or DDST (MBL).
| Results|| |
Among 251 isolates studied 138 (54.98%) were ESBL producers, 49 (19.52%) were AmpC producers and 45 (17.93%) were MBL producers [Table 1]. Range of susceptibility to other antimicrobials were as follows: Aminoglycosides - 10-50%, fluroquinolones - 10-30%, cephalosporins - 5-30%. [Table 1] shows the number of ESBL producers in each of the microorganisms isolated. Our study analysis of 138 phenotypically identified ESBL isolates revealed that ESBL were predominantly produced by E. coli (35.5%) followed by Pseudomonas (31.88%), Citrobacter (11.59%), Klebsiella (9.42%), Serratia (5.07%), Proteus mirabilis (4.34%) and Acinetobacter (2.17%). Highest producers of ESBLs in their individual categories were among Acinetobacter (100%), Klebsiella (86.66%), Proteus (85.71%) followed by Pseudomonas (59.45%) [Table 1]. Phenotypic detection of ESBL production was attempted by beta-lactam and beta-lactamase inhibitor combination. The combinations used were cefoperazone sulbactam, piperacillin tazobactam and ceftazidime clavulanic acid. Highest rates of detection was by cefoperazone sulbactam (109/138) 78.98% followed by piperacillin tazobactam (58/138) 42.02% and ceftazidime clavulanic acid (20/138) 14.5%. In the isolates studied cefoperazone sulbactam combination proved to be most sensitive. In E. coli CFS detected 77.55% isolates, in Pseudomonas 88.63%, in Klebsiella 76.92%, in Proteus 83.33%, in Serratia 57.14%, in Acinetobacter 100% and in Citrobacter 62.5% [Table 2].
|Table 1: Distribution of ESBLs, AmpC and MBL (percentages are present in brackets)|
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|Table 2: Distribution of detection methods for ESBLs (percentages are present in brackets)|
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Out of 92 of the AmpC producing strains 20 (21.73%) were inducible and 72 were stably derepressed (78.26%). Such a high percentage of derepressed mutants is noteworthy and alarming [Table 3].
|Table 3: Percentages of inducible and derepressed AmpC β -lactamases (percentages are present in brackets)|
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Out of 251 strains studied 45 (17.93%) were phenotypically identified as MBL producers, the highest no. being of Pseudomonas aeruginosa. In their individual categories 5 of 15 Klebsiella and 1 of 3 Acinetobacter (33.3% each) followed by 4 of 17 Serratia (23.52%), 2 of 7 Proteus (28.57%), 19 of 74 Pseudomonas (25.67%), 5 of 25 Citrobacter (20%) and 9 of 109 E. coli (8.25%) were identified to be MBL producers [Table 1].
| Discussion|| |
This study aimed to study the prevalence rates of beta-lactamases among various Gram-negative bacteria isolated in our clinical labs and also to evaluate various phenotypic detection methods.
ESBL and AmpC detection rates were in the range reported in other studies from India. But 17.93% isolates detected as MBL producers on phenotypic identification is very alarming. Thus, high level of antibiotic resistance pattern exists in various clinical isolates. This fact is also confirmed by the presence of low susceptibility of our isolates to other classes of antimicrobials such as aminoglycosides and fluoroquinolones.
Our study analysis of 138 phenotypically identified ESBL isolates revealed that ESBL were predominantly produced by E. coli (35.5%) followed by Pseudomonas (31.88%), Citrobacter (11.59%), Klebsiella (9.42%), Serratia (5.07%), P. mirabilis (4.34%) and Acinetobacter (2.17%). Our finding was similar to that of Ananthakrishna et al. who reported a high prevalence of ESBL among E. coli. This high incidence of ESBLs in E. coli may be peculiar to the Indian subcontinent.
These figures reinforce the fact that ESBL production should be routinely screened in Gram-negative bacteria other than E. coli and Klebsiella. Although CLSI recommendations exist, but they are limited to E. coli and Klebsiella spp. No guidelines exist for detection of AmpC beta-lactamases. 
The increased efficacy of cefoperazone-sulbactam in phenotypic detection of ESBL production may be attributed to its increased stability when compared to a penicillin inhibitor combination. This may be the case especially in those isolates co-producing an ESBL and penicillinase at high levels since the concentration of inhibitor in their periplasmic space may be insufficient to protect penicillin. Although on weight basis clavulanic acid is more potent than sulbactam, its ability to induce AmpC production may interfere with ESBL production. 
The rise in resistance mechanisms poses a serious threat to effective antimicrobial therapy in the near future. E. coli was the most common organism producing AmpC both inducible and derepressed (65% and 12.5% respectively). In the individual categories highest number of AmpC producers were present in Serratia 7/17 (41.17%). If a strain is susceptible to cefepime and resistant to cefotaxime and cefoxitin then AmpC is a likely mechanism. If it is cefotaxime resistant and cefoxitin sensitive then the strain is a probable ESBL producer which needs further confirmation. High Level AmpC expression may prevent ESBL recognition but this high level of AmpC has minimal effect on activity of cefepime making this drug more reliable for ESBL detection in the presence of AmpC.  The coexistence of different classes of beta-lactamases in a single bacterial isolate may pose diagnostic and treatment challenges. The AmpC producing organisms can act as a hidden reservoir for the ESBLs. 
Gram-negative bacilli producing acquired MBLs have been increasingly reported in Asia and Europe. ,,,, Infection with carbapenem-resistant Enterobacteriaceae (CRE) or carbapenemase-producing Enterobacteriaceae is emerging as an important challenge in health-care settings.  Currently, carbapenem-resistant K. pneumoniae is the species of CRE most commonly encountered in the United States. Among detection methods employed for MBL detection, Hodge Test and DDST combined was highly efficacious [Table 4]. Heaping may be used as a screening tool. Microbiological excellence is needed more than ever and it is critical that ESBLs, AmpC beta-lactamases and carbapenemases be promptly and accurately detected. 
|Table 4: Distribution of detection methods for MBL (percentages are present in brackets)|
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MBLs efficiently hydrolyze all beta-lactams except for aztreonam in vitro.  Therefore detection of MBL producing Gram-negative bacilli is crucial for optimal treatment of patients and to control the spread of infection. The MHT is a phenotypic test used to detect carbapenemases in isolates demonstrating elevated but susceptible carbapenem MICs and has demonstrated sensitivity and specificity exceeding 90% in identifying carbapenemase-producing Enterobacteriacea (CLSI 2012). Among the methods employed for detection of MBL production, Hodge test (62.22%) proved better than DDST (40%) [Table 4] in contrast with other studies. 
Our study was limited by the fact that final confirmation of resistance is by detection of molecular markers by gene demonstration, which could not be performed due to constraint of resources. Correlation of results of drug therapy with the presence of resistance mechanisms may also provide insight. Similar multi centric studies on a larger scale need to be done to validate these results.
| Conclusion|| |
ESBL and AmpC detection rates were in the range reported in other studies from India. However 17.93% isolates detected as MBL producers on phenotypic identification is very alarming. Thus, high level of antibiotic resistance pattern exists in various clinical isolates.
One of our aims was to provide a holistic antibiotic susceptibility report and this is how we plan to do it.
Day 1: Screen and confirm ESBL, also screen for AmpC
Day 2: Confirm presence of AmpC (inducible/derepressed), also screen for MBL
Day 3: Confirm presence of MBL.
We recommend these methods of phenotypic identification as genotypic methods are not feasible in at least developing countries like India.
Due to high levels of prevalence of drug resistance, there should be routine identification of ESBLs, AmpC and MBL of clinical isolates especially if the patient's condition merits so.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]