|Year : 2013 | Volume
| Issue : 1 | Page : 28-31
Prevalence of extended-spectrum beta-lactamases, AmpC beta-lactamase, and metallo-beta-lactamase producing Pseudomonas aeruginosa and Acinetobacter baumannii in an intensive care unit in a tertiary care hospital
Varun Goel1, Sumati A Hogade2, SG Karadesai2
1 Clinical Microbiology Division, Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi, India
2 Department of Microbiology, Jawaharlal Nehru Medical College, Belgaum, Karnataka, India
|Date of Web Publication||28-Mar-2013|
Clinical Microbiology Division, Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi 110 029
Source of Support: None, Conflict of Interest: None
Background: Resistance to broad-spectrum beta-lactams, mediated by extended-spectrum beta-lactamases (ESBL), AmpC beta-lactamase, and metallo-beta-lactamase (MBL) enzymes, is an increasing problem worldwide. Aim of the study: This study was undertaken to detect ESBL, AmpC beta-lactamase and metallo-beta-lactamase producing Pseudomonas aeruginosa and Acinetobacter species from the endotracheal aspirates. Materials and Methods: A prospective study was performed over a period of 15 month in a tertiary care hospital. A total of 26 clinical isolates of P.aeruginosa and 40 of Acinetobacter species were tested for the presence of ESBL, AmpC beta-lactamase, and metallo-beta-lactamase enzyme. Detection of ESBL was done by the combined disk diffusion method as per Clinical and Laboratory Standards Institute (CLSI) guidelines, and MBL was detected by imipenem-Ethylenediaminetetraacetic acid (EDTA) combined disk method. Isolates showing reduced susceptibility to cefoxitin (30 μg) disk were considered 'screen positive' for AmpC beta-lactamases and selected for detection of plasmid-mediated AmpC by the AmpC disk test. Results : 42.30% isolates of P.aeruginosa were positive for ESBL while 53.85% were MBL producers. Among 39 isolates of Acinetobacter baumannii, 43.59% were AmpC producers while 48.72% were MBL-producing strains. Conclusion: The study emphasizes the high prevalence of multidrug-resistant P.aeruginosa and A.baumannii producing beta-lactamase enzymes of diverse mechanisms. Thus, proper antibiotic policy and measures to restrict the indiscriminative use of cephalosporins and carbapenems should be taken to minimize the emergence of this multiple beta-lactamase-producing pathogens.
Keywords: AmpC beta-lactamase, beta-lactamases, extended-spectrum beta-lactamases, metallo-beta-lactamase
|How to cite this article:|
Goel V, Hogade SA, Karadesai S G. Prevalence of extended-spectrum beta-lactamases, AmpC beta-lactamase, and metallo-beta-lactamase producing Pseudomonas aeruginosa and Acinetobacter baumannii in an intensive care unit in a tertiary care hospital. J Sci Soc 2013;40:28-31
|How to cite this URL:|
Goel V, Hogade SA, Karadesai S G. Prevalence of extended-spectrum beta-lactamases, AmpC beta-lactamase, and metallo-beta-lactamase producing Pseudomonas aeruginosa and Acinetobacter baumannii in an intensive care unit in a tertiary care hospital. J Sci Soc [serial online] 2013 [cited 2017 Apr 28];40:28-31. Available from: http://www.jscisociety.com/text.asp?2013/40/1/28/109691
| Introduction|| |
The incidence of nosocomial infections in critically ill patients is much higher than in general ward patients despite the immense advancement in therapeutic technologies. Severe nosocomial infections contribute to prolonged intensive care unit (ICU) stays, increased morbidity and mortality and of course, increased resource utilization. ,
The newer beta-lactamases, including extended-spectrum beta-lactamases (ESBL), AmpC beta-lactamases and metallo-beta-lactamases (MBL), have emerged worldwide as a cause of antimicrobial resistance in gram-negative bacteria.  The presence of ESBLs and AmpC beta-lactamases in a single isolate reduces the effectiveness of the beta-lactam-beta-lactamase inhibitor combinations, while MBLs and AmpC beta-lactamases confer resistance to carbapenems. Often, these enzymes are co-expressed in the same isolate.
Acinetobacter species and Pseudomonas aeruginosa are noted for their intrinsic resistance to antibiotics and for their ability to acquire genes encoding resistance determinants. Foremost among the mechanisms of resistance in both of these pathogens is the production of beta-lactamases and aminoglycoside-modifying enzymes. ,
The multidrug resistant (MDR) isolates that are present in the ICU and in the hospital environment pose not only therapeutic problems but also serious concerns for infection control management.  A local surveillance program is essential at each center, as the knowledge of local resistant patterns is vital for selecting appropriate agents for treating infections. So, the present study was undertaken to detect ESBL, AmpC beta-lactamase, and metallo-beta-lactamase producing P.aeruginosa and Acinetobacter species in our medical intensive care unit (MICU) settings.
| Materials and Methods|| |
The study was conducted in a tertiary care hospital with a 34-bed ICU during the period from January 2010 to April 2011. The approval of the institutional review board was obtained during the planning phase of the study and each patient (or his/her caregivers) gave informed consent prior to participation in the study. The organisms isolated by endotracheal aspirate from ventilator-associated pneumonia (VAP) patients were identified based on standard microbiological techniques. The susceptibility of the clinical isolates to some routinely used antibiotics was determined by the Kirby-Bauer disk diffusion method using Clinical and Laboratory Standards Institute (CLSI) standards: Cip-ciprofloxacin (5μg), Cn-cefoxitin (30μg), Cot-cotrimoxazole (1.25/23.75μg), Ak-amikacin (30μg), Ca-ceftazidime (30μg), I-imipenem (10μg), Pt-piperacillin-tazobactam (100/10μg), Ao-aztreonam (30μg), Do-doxycycline (30μg) (Hi-media Laboratories, Mumbai). Escherichia More Details coli ATCC 25922 and P.aeruginosa ATCC 27853 were used as quality control strains. 
Isolates showing reduced susceptibility to cefoxitin (30 μg) disk were considered 'screen positive' for AmpC beta-lactamases and selected for detection of plasmid-mediated AmpC by the AmpC disk test. The negative isolates were confirmed for AmpC production with the three-dimensional test. 
As per CLSI 2011 guidelines, when using the new interpretive criteria, routine ESBL testing is no longer necessary before reporting results (i.e. it is no longer necessary to edit results for cephalosporins, aztreonam, or penicillins to resistant). However, ESBL testing may still be useful for epidemiological or infection control purposes. All the isolates which 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. We used cefotaxime (30 μg), ceftazidime (30 μg), and ceftazidime/clavulanic acid (30/10 μg) disks for ESBL determination. Klebsiella pneumoniae ATCC 700603 (positive control) and Escherichia coli ATCC 25922 (negative control) were used for quality control of ESBL tests. 
Isolates showing reduced susceptibility to imipenem were selected for detection of MBL enzymes by imipenem-EDTA combined disk method. , P.aeruginosa ATCC 27853 was used as the control. For the combined disk test, two 10-μg imipenem disks were placed on the surface of an agar plate and 5-μl EDTA solution was added to one of them to obtain a concentration of 750 μg. The inhibition zones of imipenem and imipenem-EDTA were compared after 16-18 h of incubation in air at 35°C. An increase in zone size to ≥7 mm was taken as positive.
| Results|| |
Total 75 (91.46%) gram-negative organisms and 7 (8.54%) gram-positive organisms were isolated from 82 endotracheal aspirate samples. A total of nonduplicate 26 isolates of P.aeruginosa and 40 of Acinetobacter species were recovered from endotracheal aspirate cultures from VAP patients.
Among the gram-negative isolates, Acinetobacter species (48.78%) was the most commonly isolated pathogen followed by P.aeruginosa (31.71%). Other gram-negative organisms isolated were K. pneumoniae (4.89%), E. coli (3.66%), and Citrobacter freundii and Serratia marcescens (1.22%). Of 40 organisms belonging to the genus Acinetobacter, 39 isolates were of A.baumannii . Among gram-positive organisms, Staphylococcus aureus accounted for 8.54%. [Table 1] shows antibiotic sensitivity pattern of the isolates.
Seventeen of the 30 cefoxitin insusceptible clinical isolates of A.baumannii yielded positive AmpC disk tests (56.67%), and the remaining 13 were negative. The negative isolates were confirmed as negative for AmpC production with the three-dimensional test.
ESBL was produced by 42.30% of P.aeruginosa while 17.95% of A.baumannii [Graph 1[Additional file 1]].
Presently, there is concern about the acquisition of plasmid-mediated MBLs active against carbapenems, penicillins, and cephalosporins. In our study, 14 isolates (53.85%) of P.aeruginosa and 19 (48.72%) of A.baumannii were plasmid-mediated MBLs enzyme producing strains detected by imipenem-EDTA disk method.
| Discussion|| |
Beta-lactamases have been grouped into four molecular classes, namely A, B, C, and D, based on the amino acids sequence homology according to Ambler classification. A, C, and D classes are called serine-beta-lactamases, and B class beta-lactamases are referred to as MBL. Newer beta-lactamases that hydrolyze cephamycins, oxyimino and zwitterionic cephalosporins, monobactams, or carbapenemsare of increasing concern because they restrict therapeutic options, cause treatment failures, and are increasing in occurrence. 
In this study, a high degree of co-resistance to ceftazidime and cefoxitin (100%) and ciprofloxacin and amikacin (72.72%) was observed in ESBL-positive isolates of P.aeruginosa. This is due to the coexistence of genes encoding drug resistance to other antibiotics on the plasmids which encode ESBL.  Our study reported very high incidence of ESBL among P.aeruginosa (42.31%), which contrasts an earlier study which showed 20.27% of ESBL production.  Typical ESBLs production was observed in 17.95% among A.baumannii. In other studies, ESBL production in Acinetobacter spp. has been found to range from 20% in India to 54.6% in Korea. 
AmpC beta-lactamases are cephalosporinases that are poorly inhibited by clavulanic acid. They can be differentiated from other ESBLs by their ability to hydrolyze cephamycins (cefoxitin, cefotetan), as well as other extended-spectrum cephalosporins.  Although the current CLSI guidelines do not describe any method for detection of isolates producing AmpC beta-lactamases, we followed the AmpC disk method to detect AmpC beta-lactamases. Seventeen (43.59%) of 39 isolates of A. baumannii and 4 P.aeruginosa isolates have shown production of AmpC beta-lactamase enzyme.
Isolates that co-produce both an ESBL and a high level of AmpC are becoming more common. With such pathogens, a positive CLSI (or equivalent) ESBL confirmatory test can usually be accepted as accurate.  In this study, we got 3 (11.39%) isolates of P.aeruginosa and 6 (15.38%) A.baumannii isolates were positive to both ESBL and AmpC disk test.
Acinetobacter are generally considered less virulent than P.aeruginosa and these have nonetheless become problem pathogens because of increasing resistance to commonly used antimicrobial agents. Unlike that of AmpC enzymes found in other gram-negative organisms, inducible AmpC expression does not occur in A.baumannii. More than 85% of isolates are susceptible to carbapenems, but resistance is increasing due to either IMP-type metalloenzymes or carbapenemases of the OXA type. 
Emergence of MBL-mediated resistance in India is of serious concern. Carbapenems are effective therapeutic agents against highly resistant pathogens such as P.aeruginosa and Acinetobacter species. Spread of this resistance among these pathogens and transfer to other gram-negative bacteria would seriously restrict therapeutic options. 
The potential limitation of this study is that molecular epidemiologic analysis and characterization of ESBL, AmpC and MBL types were not carried out. Our study reports the genes for ESBL, AmpC and MBL enzyme have spread from Enterobacteriaceae to non-Enterobacteriaceae specifically in the ICU setup. Early detection of these beta-lactamase-producing isolates in a routine laboratory could help avoid treatment failure, as often the isolates producing this enzyme show a susceptible phenotype in routine susceptibility testing. Furthermore, strict antibiotic policies and measures to limit the indiscriminative use of cephalosporins and carbapenems in the hospital environment should be undertaken to minimize the emergence of this multiple beta-lactamase.
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