|Year : 2016 | Volume
| Issue : 1 | Page : 35-39
A study on detection of extended-spectrum beta-lactamases (ESBLs) and comparison of various phenotypic methods of AmpC detection in Pseudomonas aeruginosa from various clinical isolates in a tertiary care teaching hospital
Subbalakshmi Easwaran, Ranjani Chittur Yerat, Rajendran Ramaswamy
Department of Microbiology, Vydehi Institue of Medical Sciences and Research Centre, Bangalore, Karnataka, India
|Date of Web Publication||21-Jan-2016|
Flat no A-G, Alpine meadows, 2nd main, Kuvempu Road, Vignan Nagar, Bangalore-560075
Source of Support: None, Conflict of Interest: None
Introduction: Pseudomonas aeruginosa is one of the major etiological agents of nosocomial infection. They are difficult to treat as the majority of them exhibit varying degrees of innate resistance. Acquired resistance is mediated by the production of chromosomal and plasmid-mediated AmpC beta-lactamases, extended-spectrum beta-lactamases (ESBLs), and metallo-beta-lactamases (MBLs). Objectives: This study was conducted for the detection of ESBLs and the comparison of various phenotypic methods of AmpC beta-lactamases (both inducible and plasmid-mediated) from various clinical isolates of Pseudomonas aeruginosa. Materials and Methods: A prospective study was conducted for a period of 6 months from January 2014 to June 2014. Isolates of Pseudomonas aeruginosa were tested for ESBLs by combined disk diffusion method using ceftazidime/ceftazidime+clavulanic acid (CAZ+CAC). Inducible AmpC beta-lactamases were detected by both ceftazidime-imipenem antagonism test (CIAT) and cefotaxime/cefoxitin (CTX/CX) inducible disk diffusion test. Plasmid-mediated AmpC were detected using AmpC disk test and ethylenediaminetetraacetic acid (EDTA) disk test. All the isolates were tested for cefepime resistance. Results: Out of 116 Pseudomonas aeruginosa isolates, 70 were ceftazidime-resistant. While ESBLs were observed in 67 out of 70 isolates, 36 were positive for inducible AmpC by both the methods. Plasmid-mediated AmpC were detected in only 8 isolates. All the 70 strains showed resistance to cefepime. Conclusions: High prevalence of ESBL and chromosomal-mediated AmpC was observed in Pseudomonas aeruginosa. Both the inducible disk diffusion tests (CIAT, CTX/CX) were equally effective in the detection of inducible AmpC. As Pseudomonas is one of the major nosocomial pathogens, such high resistance poses a grave threat.
Keywords: AmpC beta-lactamases, extended-spectrum beta-lactamases (ESBLs), Pseudomonas aeruginosa
|How to cite this article:|
Easwaran S, Yerat RC, Ramaswamy R. A study on detection of extended-spectrum beta-lactamases (ESBLs) and comparison of various phenotypic methods of AmpC detection in Pseudomonas aeruginosa from various clinical isolates in a tertiary care teaching hospital. Muller J Med Sci Res 2016;7:35-9
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Easwaran S, Yerat RC, Ramaswamy R. A study on detection of extended-spectrum beta-lactamases (ESBLs) and comparison of various phenotypic methods of AmpC detection in Pseudomonas aeruginosa from various clinical isolates in a tertiary care teaching hospital. Muller J Med Sci Res [serial online] 2016 [cited 2019 Feb 24];7:35-9. Available from: http://www.mjmsr.net/text.asp?2016/7/1/35/174610
| Introduction|| |
Infections caused by Pseudomonas aeruginosa are difficult to treat as the majority of the isolates exhibit varying degrees of innate resistance. Acquired resistance is mediated by the production of plasmid-mediated AmpC beta-lactamase, extended-spectrum beta-lactamase (ESBL) and metallo-beta-lactamase (MBL) enzymes.  AmpC beta-lactamases play an important role in the resistance of Gram-negative bacilli. AmpC enzymes are grouped into Ambler class C classification.  These enzymes confer a high level of resistance to many beta-lactam antibiotics including the third-generation cephalosporins and cephamycins (cefoxitin and cefotetan). With the increase in occurrence and types of these multiple beta-lactamase enzymes, early detection is crucial for the initiation of proper antibiotic therapy and infection control policy. AmpC beta-lactamases are largely unknown due to difficulties in the phenotypic detection and are often misidentified as ESBLs. Detection of AmpC isolates is important not only because of their broader cephalosporin resistance but also because of carbapenem resistance by mutations resulting in reduced porin expression  (Manchanda). There are various phenotypic methods for AmpC detection and none of them are standardized as they are time-consuming when screening large number of isolates.  In addition to chromosomal AmpC, the production of plasmid-mediated AmpC presents a new threat in the treatment of infection caused by Pseudomonas aeruginosa. 
The present study was designed to investigate the presence of different classes of beta-lactamase enzymes in clinical isolates of Pseudomonas aeruginosa with special reference to inducible and plasmid-mediated AmpC beta-lactamases in a tertiary care teaching hospital.
| Materials and Methods|| |
This prospective study was conducted over a period of 6 months from January 2014 to June 2014. This work was approved by the Ethical Committee of the institute. A total of 116 nonconsecutive clinical samples of Pseudomonas aeruginosa were isolated from among various clinical isolates from both inpatients and outpatients.  As per Clinical and Laboratory Standards Institute (CLSI) 2011  guidelines, routine ESBL testing is no longer necessary before reporting results. However, ESBL testing may still be useful for epidemiological or infection control purposes.  All the isolates that showed resistance to ceftazidime were evaluated for ESBL production by using the phenotypic confirmatory test. A difference of ≥5 mm between zone diameter of either the cephalosporin disks or their respective cephalosporin-clavulanate disk was taken to be phenotypic confirmation of ESBL production.
Chromosomal AmpC detection
A test isolate with a turbidity equivalent to that of 0.5 McFarland standard was spread over a Mueller-Hinton agar (HiMedia) plate. Cefotaxime 30 mcg and cefoxitin 30 mcg disks were placed 20 mm apart from center to center. Isolates showing blunting of the cefotaxime zone of inhibition adjacent to the cefoxitin disk were screened as positive for AmpC beta-lactamase.
Ceftazidime-imipenem antagonism test (CIAT)
In this test, evidence of AmpC beta-lactamase production was judged by the CIAT, which consisted of imipenem (10 ug) disk placed 20 mm apart from a ceftazidime disk on a Mueller-Hinton agar plate previously inoculated with a 0.5 McFarland bacterial suspension and then incubated for 24 h at 35° . Antagonism indicated by the reduction in inhibition of the zone around the ceftazidime adjacent to the imipenem disk was interpreted as being positive for AmpC producers.
Plasmid-mediated AmpC detection
The surface of a Mueller-Hinton agar plate was inoculated with a lawn of cefoxitin-susceptible Escherichia More Details coli ATCC 25922 according to the standard disk diffusion method. Immediately prior to use, AmpC disks were rehydrated with 20 μL of saline and several colonies of each test organism were applied to a disk. A 30-mcg cefoxitin disk was placed on the inoculated surface of the Mueller-Hinton agar. The inoculated AmpC disk was then placed almost touching the antibiotic disk, the inoculated disk face in contact with the agar surface. The plate was then inverted and incubated overnight at 35°C in ambient air. After incubation, plates were examined for either an indentation or a flattening of the zone of inhibition, indicating enzymatic inactivation of cefoxitin (positive result), or the absence of distortion, indicating no significant inactivation of cefoxitin (negative result). Enhancement of inhibition was observed with ethylenediaminetetraacetic acid (EDTA).
| Results|| |
Out of 116 Pseudomonas aeruginosa isolates, 70 were ceftazidime-resistant (60.34%). Out of 70 ceftazidime-resistant isolates, 67 isolates exhibited a zone of enhancement with clavulanic acid, confirming their ESBL production. All the 70 ceftazidime-resistant isolates showed resistance to cefoxitin. Phenotypic detection of chromosomal AmpC detection was done by both cefotaxime/ cefoxitin (CTX/CX) and CIAT [Figure 1]. Out of 70 ceftazidime-resistant isolates, 36 isolates were positive for inducible AmpC by both CTX/CX and CAZ/IMP (51.42%) methods separately, which showed that they were inducible AmpC producers, while 11.42% were plasmid AmpC producers [Table 1]. Chromosomal AmpC was detected by both the methods in 22 out of 70 (31.42%) isolates. Inducible AmpC producers were not detected in 21 out of 70 isolates. The coexistence of AmpC and ESBL was reported in 48 out of 70 isolates (68.71%) [Table 2]. Both sputum and pus showed equal number of chromosomal AmpC detection. ESBL was observed in 67 out of 70 isolates (95.71%).
All the AmpC-producing Pseudomonas aeruginosa isolates were multidrug-resistant [Figure 2]. The maximum numbers of ESBL and AmpC enzymes were detected from samples from inpatients. All the 70 ceftazidime-resistant isolates showed resistance to cefepime that showed derepressed AmpC mutant.
|Figure 1: ESBL and AmpC detection by CTX/CX and CIAT disk diffusion tests|
Click here to view
| Discussion|| |
This study was aimed to study the prevalence rates of beta-lactamases among various clinical isolates of Pseudomonas aeruginosa. Ceftazidime is a third-generation cephalosporin used frequently for the treatment of infections caused by Pseudomonas aeruginosa. Ceftazidime resistance is mainly mediated by production of beta-lactamases such as ESBL, MBL, and occasionally AmpC beta-lactamases. Besides production of various beta-lactamases, other mechanisms such as the lack of drug penetration due to mutation in porins and loss of certain outer membrane proteins and efflux pumps can also contribute to resistance to beta-lactams. The resistance to ceftazidime is increasing at an alarming rate, complicating the clinical management of patients infected with such resistant isolates. In our study, 70 isolates out of 116 were ceftazidime-resistant (60.34%). Ceftazidime resistance was relatively higher among the isolates from inpatients compared to those from outpatients. Similar results (65%) were observed in in a study done by Uma et al. In a study from Iran,  73.4% of Pseudomonas aeruginosa isolates were observed to be resistant to ceftazidime.
ESBLs, which are generally widespread among members of' Enterobacteriaceae, are also increasingly found in Pseudomonas aeruginosa. The resistance is due to hydrolysis of beta-lactam ring of beta-lactam antibiotics by the action of beta-lactamase enzymes. Other mechanisms of drug resistance to the beta-lactam group of antibiotics are loss of outer membrane protein, production of class AmpC beta-lactamases, and altered target sites. These ESBL enzymes are inhibited by beta-lactamase inhibitors clavulanic acid and sulbactam. The rates of ESBL production in various clinical isolates of Pseudomonas aeruginosa among ceftazidime-resistant isolates in our study was 95.71%. Similar high production of ESBL was observed in a study by Ahmad et al.  (61.6%), while studies by Dutta et al.  and Uma et al.  showed 27.33% and 19.4%. ESBL, respectively. This varied ESBL production could be due to irregular usage of antibiotics or due to environmental influence. Horizontal gene spread is considered to be responsible for the high frequency of ESBLs detected in Pseudomonas aeruginosa.
AmpC beta-lactamases are largely unknown due to difficulties in the phenotypic detection and often misidentified as ESBLs. In our further phenotypic observational study, though cefoxitin resistance was observed in all 116 isolates, inducible AmpC was observed in only 36 out of 70 isolates. Cefoxitin resistance in AmpC nonproducers could be due to some other mechanisms, lack of permeation of porins as one of the resistance (Manchanda et al.).  Observation by Upadhyay et al.  revealed the presence of inducible AmpC producer as 7%, whereas in our study, inducible ampC producers by both the phenotypic methods (CTX/CX and CIAT) were 51.42%. CIAT is a simple test that can be used to confirm the presence of known as well as new inducible ampC enzymes. (Vlademiret et al.).  In this present study, we used imipenem, a potent inducer of AmpC genes together with ceftazidime to detect inducible AmpC beta-lactamases (Vlademir et al.). In a study by Upadyay et al., the CIAT test was able to detect maximum number of blaPDC-harboring isolates. Though molecular study was not done in our study, we found that both CTX/CX and CIAT were equally effective in detection of inducible AmpC deduction. Black's  study reported that AmpC disk test based on filter paper disks impregnated with EDTA was a highly sensitive, specific, and convenient means of detection of plasmid-mediated AmpC beta-lactamases in organisms lacking a chromosomally mediated AmpC beta-lactamase. The test accurately distinguished between cefoxitin insusceptibility caused by AmpC production and non beta-lactamase mechanism such as reduced outer membrane permeability (porin mutations) as observed by the Black study. Coproduction of ESBLs did not interfere with the detection of AmpC beta-lactamases. The coexistence of AmpC and ESBL were observed in 48 out of 70 isolates, whereas Upadyay et al. reported 4 out of 120 isolates. Our study showed higher prevalence of ESBL and AmpC. The only beta-lactams active against co-AmpC and ESBL producers are carbapenems.  However, recently resistance to carbapenems has been increasing, which is mostly due to the production of MBL. Carbapenem resistance was observed in 30 out of 70 ceftazidime-resistant samples. Maximum resistance was noted in all inpatients from sputum samples.
| Conclusion|| |
High prevalence of ESBL and chromosomal AmpC was observed in Pseudomonas aeruginosa. The potential limitation of this study is that molecular epidemiologic analysis and characterization of ESBL and AmpC were not carried out. Routine screening for ESBL and AmpC production need to be done for all. As many studies have not been done on Pseudomonas aeruginosa for AmpC and ESBL detection, the present study emphasizes the early detection of these beta-lactamase-producing isolates in a laboratory by routine antibiotic susceptibility testing, especially for such nosocomial pathogens. Furthermore, strict antibiotic policies and measures to limit indiscriminate use of cephalosporins and carbapenems in the hospital environment should be undertaken to minimize the emergence of this multidrug resistance.
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Conflicts of Interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]