Year : 2010 | Volume
: 53 | Issue : 1 | Page : 83--86
Plasmid profile of ESBL producing Gram-negative bacteria and correlation with susceptibility to β-lactam drugs
Jyoti Sharma, Pallab Ray, Meera Sharma
Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
Department of Microbiology, DR. HSJ Institute of Dental Sciences and Hospital Punjab University, Chandigarh-160 014
Background: Members of family Enterobacteriaceae can acquire resistance to extended spectrum beta lactams by a number of mechanisms; most important being the plasmid encoded extended spectrum beta lactamase (ESBL) and AmpC beta lactamase. This study has been designed to look for the presence of plasmids and their correlation with drug resistance. Methods: ESBL production was studied in different gram-negative bacteria and susceptibility testing of ESBL positive isolates was done for various beta lactams, cephalosporins and other commonly used drugs against them. Plasmid DNA isolation of all the ESBL positive strains was done by alkalilysis method. Finally the presence of plasmid was correlated with susceptibility to beta lactam drugs. Results: E. coli, K. pneumoniae, Enterobacter aerogenes and A. anitratus harbored multiple plasmids. One plasmid (M.W greater than 21,226 bp) was unanimously present in all the isolates. Conclusion: There is a strong correlation between the number of plasmids harbored by an isolate and resistance to various drugs tested.
|How to cite this article:|
Sharma J, Ray P, Sharma M. Plasmid profile of ESBL producing Gram-negative bacteria and correlation with susceptibility to β-lactam drugs.Indian J Pathol Microbiol 2010;53:83-86
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Sharma J, Ray P, Sharma M. Plasmid profile of ESBL producing Gram-negative bacteria and correlation with susceptibility to β-lactam drugs. Indian J Pathol Microbiol [serial online] 2010 [cited 2020 Sep 20 ];53:83-86
Available from: http://www.ijpmonline.org/text.asp?2010/53/1/83/59190
Today, bacterial pathogens are more complex than a decade or two ago. Not only have new resistance mechanisms like extended spectrum beta lactamase (ESBL) evolved, but isolates that produce multiple beta-lactamases are also frequently encountered posing a serious therapeutic problem in many parts of the world.  Such resistance has often been associated with plasmid-mediated b-lactamases. Resistance to the widely used fluoroquinolone class of antimicrobials occurs in 15-20% of more than16 000 bacteraemias caused by Escherichia coli in the UK per annum, and also other common Enterobacteriaceae such as Klebsiella spp. and Enterobacter spp. In many bacterial species there is a strong association between clinically significant fluoroquinolone resistance contingent on chromosomal mutation, and the carriage of plasmids including those that encode extended-spectrum b-lactamases. Co resistance to non-b-lactam antibiotics is also frequent either by the co transfer of resistance determinants in the same genetic elements (such as aminoglycoside resistance) or simply by the co relation of both resistance mechanisms, as occur with fluoroquinolones. 
This correlation between chromosomal and plasmid-mediated resistance occurs also in gram-positive species such as successful strains of methicillin-resistant Staphylococcus aureus and highly gentamicin-resistant Enterococcus faecalis. Moreover, as multiple strains of many species show this association it is apparent that clonal strain success is not the only factor involved, raising the question of what else is driving this relationship. The relative frequencies of these three situations: epidemic spread, plasmid transfer and anarchy- remain unclear. Hence this study was designed to look for different plasmids present in ESBL-producing strains and determine if these plasmids affect susceptibility to beta lactam drugs.
Material and Methods
ESBL production was studied in six gram-negative bacteria that included Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Enterobacter aerogenes, Acinetobacter anitratus and Alcaligenes fecalis. These six species were the most prevalent and constituted three-fourths of gram-negative isolates in the laboratory. Bacterial isolates were collected in the department of Medical Microbiology during May 2002 to Jan 2003, from pus, sputum and blood culture of patients admitted to Nehru Hospital, a tertiary care hospital attached to the Postgraduate Institute of medical education and research, Chandigarh, India. Two hundred and ninety isolates were screened to get ESBL producers among Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Enterobacter aerogenes, Acinetobacter anitratus and Alcaligenes fecalis. (25 each, except Alcaligenes fecalis, n is equal to15)
Three strains, E. coli J53 R1, E. coli C 600 PUD16 and K. pneumoniae ATCC 700603 were used as standard ESBL-positive strains. E. coli J53 R1 harbored TEM-ESBL and the remaining strains carried SHV-ESBL. E. coli ATCC 25922 was used as negative control for ESBL production.
Antibiotic Susceptibility Testing
Antibiotic susceptibility of the clinical isolates was determined by disc diffusion assay following NCCLS guidelines (National committee for clinical laboratory standards, 2000).  The strains were tested for susceptibility against amoxycillin (10μg), cephazolin (10μg), gentamicin (10μg), amikacin (10μg), cefotaxime (30μg), ceftazidime (30μg), ciprofloxacin (5μg), netilmicin (30μg) and piperacillin (100μg). All the strains were tested twice without any discrepancy between results. The strains were scored as sensitive, intermediate (moderately sensitive) or resistant according to NCCLS guidelines.
ESBL detection was carried out following NCCLS-recommended method for screening and confirmation using cefotaxime and ceftazidime as substrates (National Committee for Clinical Laboratory Standards, 2000). 
Preparation of Plasmid DNA
Plasmid DNA was isolated from bacterial cells by alkalilysis method.  The DNA was stored at minus 20 0 C. The samples were run on 0.8% agarose gel and stained with ethidium bromide. The stained gel was examined under UV light to look for the presence of plasmid bands of particular size using a molecular weight marker, λ DNA hind III double digest.
The results were statistically analyzed by correlation analysis.
We collected 25 ESBL positive strains for all the organisms except A. fecalis for which we collected 15 ESBL producers only because of low isolation rate of the organism and low prevalence of ESBL production. The resistance pattern of ESBL producers given in [Table 1] and [Table 2] depicts resistance profile of ESBL non-producers. ESBL positive strains of E. coli, K. pneumoniae, Enterobacter aerogenes and A. anitratus showed more than 80% resistance to beta lactam antibiotics (Amoxycillin, cefazolin and cefotaxime).
Over all, ESBL producers showed 66-100% to gentamicin, 40-90% resistance to amikacin and 33-92% resistance to ciprofloxacin.
Plasmid DNA was extracted using alkalilysis method. All the isolates showed presence of plasmids. Multiple plasmids were seen in K. pneumoniae, E. coli, E. aerogenes and A. anitratus [Figure 1],[Figure 2],[Figure 3],[Figure 4]. Number of plasmids in these isolates ranged from one to 13. Majority of the strains in case of Pseudomonas aeruginosa and A. fecalis showed single plasmid only [Figure 5],[Figure 6]. One plasmid of greater than 21,226bp was present in all the isolates tested.
Statistical analysis reveals that organisms with more number of plasmids are resistant to more number of drugs. As the number of plasmids is increasing, resistance ratio (resistance to various antimicrobial) is also increasing. There was a positive correlation between the number of plasmid harbored by an isolate and resistance to various drugs tested. (correlation analysis) The relation is linear for organisms harboring multiple plasmids and is non-linear for organisms with single plasmid i.e. Pseudomonas aeruginosa and Alcaligenes fecalis.
In this study, ESBL producing strains not only showed high-level resistance to beta lactam antimicrobial agents but also exhibited resistance to other groups of antimicrobials like gentamicin (66-100%), ciprofloxacin (33-92%) and amikacin (40-96%) [Table 1]. We found that in comparison to ESBL non-producers, strains that harbor ESBLs are more resistant to b-lactam antibiotics and other groups of antimicrobial agents as well [Table 2]. Schwaber et al.  compared antimicrobial co resistance between ESBL-producing and ESBL-non producing Enterobacteriaceae to determine the impact of ESBL presence on the likelihood of resistance to antimicrobial classes in addition to β-lactams. Similar findings have been reported by Grover et al. Resistance to various antimicrobial agents is well correlated with the presence or absence of plasmids; we found a trend of increase in resistance ratio as the number of plasmids increase. Strains harboring multiple plasmids simultaneously exhibit co resistance to different classes of antibiotics. Association of plasmid-mediated quinolone resistance with ESBL is well documented by Poirel et al.  Another study by Kim et al. says that since ESBL producers express their b-lactamase genes from plasmids, these findings suggest that gene coding for ESBLs and resistance to other class of antibiotics may reside within the same plasmid and therefore be spread together. This means that resistance to two different kinds of drugs may be co selected by the use of either one or all of the antibiotics concerned could be a selective pressure for spreading such isolates. Plasmids encoding extended spectrum beta lactamases usually co transfer resistance to unrelated antibiotics. This results in complex epidemiological situations in which the emergence and spread of extended spectrum beta lactamases is not related to the selective pressure of third generation cephalosporins and other drugs such as the aminoglycosides.  Another study by Sirot et al.  indicated that resistance to beta lactams, aminoglycosides, chloramphenicol, tertacycline and sulphonamides were transferable at high frequency.
Plasmid profiling is also an important tool for epidemiological typing and has got diagnostic value as well. Multiple plasmids were seen in K. pneumoniae, E. coli, E. aerogenes and A. anitratus. Number of plasmids in these isolates range from one to 13. Majority of the strains, in case of Pseudomonas aeruginosa and A. fecalis, showed single plasmid only. One plasmid of more than 21,226bp was present in all the isolates tested. Since we did not perform any drug transfer experiment, it is difficult to assume that this is the plasmid encoding for β-lactam resistance. The strains of K. pneumoniae, E. coli, E. aerogenes and A. anitratus, containing multiple plasmids exhibited more resistance to various groups of antimicrobial agents as compared to strains (P. aeruginosa and A. fecalis) having single plasmid. These strains had relatively low level of resistance.
There is obvious need for a collaborative study on epidemiology of ESBLs to establish the role of plasmids in transfer of resistance to different antibiotics. Beta lactamase genes are often found on plasmid encoding resistance to aminoglycosides, sulfonamides, tetracyclines and other antibiotics. Hence the finding of unusual resistance to these agents should alert the laboratory to the need for further studies.
Financial assistance from PGIMER in terms of PGI fellowship is duly acknowledged.
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