|Year : 2016 | Volume
| Issue : 2 | Page : 114-117
Extended spectrum beta-lactamase and AmpC producing Klebsiella pneumoniae: A therapeutic challenge
Sheevani Sheemar, Shashi Chopra, Gomty Mahajan, Jaspal Kaur, Yadwinder Singh Chouhan
Department of Microbiology, Punjab Institute of Medical Sciences, Jalandhar, Punjab, India
|Date of Web Publication||5-Jul-2016|
144, Gurjeet Nagar, Garha Road, Jalandhar - 144 022, Punjab
Klebsiella pneumoniae accounts for substantial proportion of nosocomial infections. Increasing multidrug resistance of these bacteria is posing a great threat to hospitalized patients as it results into limitation of therapeutic options. Therefore, the present study was undertaken with the following Aim and Objective :To know the burden of extended spectrum beta lactamase (ESBL) producing K. pneumoniae and coexistence of AmpC in these isolates using phenotypic methods in a newly established teaching hospital in Punjab, India. Materials and Methods: ESBL and AmpC screening and confirmation was done using phenotypic methods as per the Clinical and Laboratory Standards Institute (CLSI) guidelines in 200 clinical isolates of K. pneumoniae. Results: Out of 200 isolates of K. pneumoniae, 116 (58%) were ESBL producer and 84 (42%) were ESBL nonproducer. Of these 116 ESBL-positive isolates, 29 (25%) were coproducer of AmpC beta-lactamase. ESBL producing isolates were significantly more resistant to other group of antibiotics as compared to ESBL nonproducers. Discussion: High prevalence rate of ESBL producing K. pneumoniae was observed in our institute. MDR in these isolates was frequent, indicating the extensive injudicious use of antibiotics. Conclusion: Increasing prevalence of ESBL producing K. pneumoniae need to be checked to reduce the mortality and morbidity due to the infections caused by these strains.
Keywords: AmpC, extended spectrum beta-lactamase, Klebsiella pneumoniae
|How to cite this article:|
Sheemar S, Chopra S, Mahajan G, Kaur J, Chouhan YS. Extended spectrum beta-lactamase and AmpC producing Klebsiella pneumoniae: A therapeutic challenge. Trop J Med Res 2016;19:114-7
|How to cite this URL:|
Sheemar S, Chopra S, Mahajan G, Kaur J, Chouhan YS. Extended spectrum beta-lactamase and AmpC producing Klebsiella pneumoniae: A therapeutic challenge. Trop J Med Res [serial online] 2016 [cited 2019 Oct 17];19:114-7. Available from: http://www.tjmrjournal.org/text.asp?2016/19/2/114/185431
| Introduction|| |
Klebsiella pneumoniae (K. pneumoniae), a gram-negative pathogen, is notorious for causing nosocomial as well as community-acquired infections.  Acquisition of beta-lactamases such as extended spectrum beta-lactamases (ESBLs), AmpC, and other enzymes has strengthened the bacteria enormously. , ESBLs are plasmid mediated enzymes that are able to hydrolyze a wide variety of penicillin and cephalosporin including third-generation cephalosporin and monobactams.  However, the ESBL producing bacteria remain susceptible to cephamycins, beta-lactam plus beta-lactamase inhibitor combination, and carbapenems. Presence of AmpC (functional group 1 beta-lactamases) in the same strains renders them resistant to cephamycins as well as inhibitor combinations, further narrowing the treatment scope. 
Cross-transmission of ESBL and AmpC producing bacteria in hospital settings have been implicated for nosocomial infections worldwide making the detection of beta-lactamases of great importance for epidemiological purposes as well as for infection control purposes. Molecular detection of these enzymes is the gold standard in their diagnosis but lack of availability of molecular methods in routine diagnostic laboratories due to cost factor is a major limitation in the developing countries. In the absence of molecular techniques, phenotypic methods if performed using stringent precautions, and by standard methods provide an effective and reliable alternatives.
Therefore, the present study was undertaken to know the burden of ESBL producing K. pneumoniae and coexistence of AmpC in these isolates using phenotypic methods in an urban hospital cum medical institute in Punjab, India. Comparative analysis of antimicrobial susceptibility pattern of ESBL producers and ESBL nonproducers was also done to study the variation in resistance amongst these two groups.
| Materials and Methods|| |
A total of 200 consecutive, nonrepeat clinical isolates of K. pneumoniae obtained from various clinical specimens sent to the microbiology laboratory of a hospital in Jalandhar, Punjab (India) from June 2012 to May 2013, were included in the study. The identification of the isolates was done with standard biotyping methods. 
Antimicrobial susceptibilities of the isolates to different antimicrobial agents (in μg), namely, cefoperazone (CPZ) (75), ceftazidime (CAZ) (30), cefotaxime (CTX) (30), aztreonam (ATM) (30), ceftriaxone (CRO) (30), cefepime (30), cefoxitin (30), co-trimoxazole (1.25/23.75), amikacin (30), gentamicin (30), ciprofloxacin (5), chloramphenicol (30), piperacillin (100), piperacillin + tazobactam (100/10), amoxicillin + clavulanic acid (AMCA) (20/10), imipenem (10), ceftazidime + clavulanic acid (CAZ/CA)(30/10), cefoperazone + sulbactam (50/50), netilmicin (30), nitrofurantoin (NFT) (100) were determined by the standard disk diffusion (SDD) method using commercially available disks (HiMedia Lab Pvt. Ltd, Mumbai) and were categorized as sensitive, intermediate, and resistant as per the Clinical and Laboratory Standards Institute (CLSI) guidelines wherever applicable. 
Quality control was achieved by using standard strain of Escherichia coli (E. coli) ATCC 25922.
The CLSI has developed screening tests for identifying the ESBL producing Klebsiella species. According to the CLSI guidelines, strains showing zones of inhibition of ≤ 22 mm for CAZ, ≤ 27 mm for CTX, and ≤ 25 mm for CRO were selected for confirmation tests of ESBL. 
Standard double-disk synergy test (DDST) was performed using disks of 30 μg each of CAZ and CTX along with AMCA (20 μg/10 μg). The disks of CAZ and CTX were placed at a distance 20 mm from that of AMCA and incubated at 37°C overnight. The organism was considered a potential ESBL producer if the zone of inhibition around the CAZ and CTX disk showed distinct shape/size potentiating toward amoxiclav disk. ,
Phenotypic confirmation of ESBL
Combined disk diffusion method was used as recommended by the CLSI. 
A CAZ disk and combined CAZ/CA disk (30/10 µg) were applied to the surface of the inoculated plate. Increase in zone diameter by 5 mm with antimicrobial agent tested in combination with clavulanic acid (CA) versus its zone when tested alone was taken as an indication of ESBL producing isolates. Quality control was achieved using standard strains of E. coli ATCC 25922 and K. pneumoniae ATCC 700603.
Cefoxitin disk (30 μg) was used for screening of AmpC producers. Zone of inhibition of less than 18 mm was taken as a potential AmpC producer.
AmpC disk test
lawn culture of E. coli ATCC 25922 was done on Mueller-Hinton agar (MHA) plate, and then cefoxitin disk and sterile disk of Whatman filter paper number 1 were placed adjacent to each other. Sterile disk was smeared with test strain and the plate was incubated at 37°C for 24 h. Distortion of zone of inhibition confirmed the presence of AmpC.
Statistical analysis was done using Chi-square test wherever applicable.
| Results|| |
Out of 200 isolates of K. pneumoniae, 116 (58%) were ESBL producer and 84 (42%) were ESBL nonproducer. Of these 116 ESBL positive isolates, 29 (25%) were coproducer of AmpC beta lactamase while 5 of 200 (2.5%) were pure AmpC producer. Overall prevalence of AmpC was found to be 17% (34/200). The categorization was done on the basis of criteria mentioned in [Table 1].
In vitro susceptibility shown to various third-generation cephalosporins in confirmed ESBL producing K. pneumoniae is depicted in [Table 2]. Maximum false sensitivity was observed in case of CAZ (7/116) followed by CTX (5/116), CRO (4/116), cefpodoxime (4/116), while only one isolate showed false sensitivity to CPZ.
|Table 2: Sensitivity result by disk diffusion method (third-generation cephalosporins) among the confirmed ESBL positive isolates |
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Maximum number of ESBL producing K. pneumoniae was isolated from intensive care unit (ICU) (92.5%), followed by medicine, surgery, orthopedics, pediatrics, obstetrics and gynecology wards with isolation rate of 75%, 71.88%, 57.14%, 20%, and 18.75%, respectively [Table 3].
|Table 3: Ward-wise distribution of ESBL producing Klebsiella pneumoniae |
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Susceptibility pattern of ESBL producers and ESBL nonproducers showed that ESBL producers show significantly high resistance (P < 0.01) to different class of antimicrobial agents as compared to ESBL nonproducers [Table 4].
|Table 4: Comparative analysis of resistance pattern of ESBL-positive and ESBL-negative K. pneumoniae |
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Carbapenem resistance was observed in 13 isolates out of 116 (11.21%) ESBL producers and resistance was common to both meropenem and imipenem. All the carbapenem resistant isolates were from ICU.
| Discussion|| |
The present study was undertaken to know the trends of ESBL and AmpC producing K. pneumoniae in indoor patients of an urban hospital. High isolation rate (58%) of ESBL producing K. pneumoniae was observed in our institute. Prevalence rates of 4-80% of ESBL producing K. pneumoniae had been reported in various studies from India. ,,, The high rate of ESBL producing Klebsiella species at our institute could be due to the fact that it is a teaching institute and referral hospital and most of the complicated resistant cases referred here present with a history of extensive usage of antibiotics. An indiscriminate use or abuse of antibiotics especially cephalosporins is a known risk factor for acquisition of ESBLs.  A very high rate (~92%) of ESBL production in isolates from ICU samples was observed in our study as compared to other studies. ,,,, However high prevalence rate of 80% of ESBL producing K. pneumoniae in ICU patients was also reported by another study from Punjab.  It is perhaps due to the prolonged stay of patients in hospitals and the extensive usage of invasive devices in these patients. Intensive infection control practices and judicious use of antimicrobial agents are the need of the hour to reduce the prevalence of resistant pathogen in the ICUs.
Twenty nine isolates were found to be coproducer of AmpC. In these isolates, the CLSI-recommended methods would have failed to detect the ESBL production due to masking effect of AmpC enzyme as it is resistant to inhibition by clavulanic acid. Therefore, inclusion of cefoxitin disk as an indicator of AmpC and confirmation of the same by AmpC disk test is of great assistance to know the accurate incidence of ESBLs as well as AmpC beta lactamases.
Resistance to three or more antimicrobials of different classes was significantly more frequent (P < 0.01) in ESBL-positive isolates as compared to the ESBL-negative Klebsiella isolates as also reported by other studies. ,,, ESBLs are encoded by plasmids that may carry resistance genes to other antimicrobial groups and explains the multidrug resistance (MDR) of ESBL producing organisms have become an important clinical issue due to their resistance to multiple antibiotics. Thus, antibiotic options to check infections caused by these organisms are extremely limited.
Resistance to carbapenems in ESBL producers has further engraved the situation.Increased prevalence of MDR ESBLs has resulted into selective pressure on carbapenems. Consequently excessive usage of carbapenems has led to the development of resistance against carbapenems as well, limiting the treatment option. Treatment of these MDR strains is a therapeutic challenge for the clinicians and pharmaceuticals.
| Conclusion|| |
Unchecked growth of resistant strains of Klebsiella poses a real threat to medical fraternity and patient's health. It calls for strict adherence to antibiotic policies to curtail injudicious use of antibiotics. The occurrence of multiple beta lactamases among bacteria not only limits the therapeutic options but also poses diagnostic challenge to the microbiologist. Clinical and laboratory doctors need to join hands to curb the growing menace of MDR ESBL and AmpC producing strains of Klebsiella. It will help to check the increasing prevalence of the multi drug resistant Klebsiella and will also reduce the rates of mortality and morbidity due to the infections caused by these strains.
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Conflicts of interest
There are no conflicts of interest.
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