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Year : 2008  |  Volume : 26  |  Issue : 2  |  Page : 163-166
 

Detection of Enterobacteriaceae producing CTX-M extended spectrum β-lactamases from a tertiary care hospital in south India


1 School of Biotechnology, Chemical and Biomedical Engineering, VIT University, Vellore - 632 014, Tamil Nadu, India
2 Department of Microbiology, PSG Institute of Medical Sciences & Research, Coimbatore - 641 004, Tamil Nadu, India
3 Central Research Institute, Government of India, Kasauli - 173 204, Himachal Pradesh, India

Date of Submission11-Jun-2007
Date of Acceptance08-Oct-2007

Correspondence Address:
S Baby Padmini
School of Biotechnology, Chemical and Biomedical Engineering, VIT University, Vellore - 632 014, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.40534

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 ~ Abstract 

A total of 23 clinical isolates (15 Escherichia coli and 8 Klebsiella pneumoniae ), resistant to cefotaxime and ceftazidime recovered during 2002 and 2003, were investigated for production of CTX-M extended spectrum β-lactamase (ESBL) by phenotypic and molecular methods. The presence of ESBL was tested by NCCLS phenotypic confirmatory test using cephalosporin/clavulanate combination discs and E-test ESBL strips. Determination of MIC of cefotaxime and ceftazidime was done with and without the presence of clavulanic acid by agar dilution technique. Polymerase chain reaction revealed the presence of CTX-M type ESBLs in 19 isolates. Further sequencing resulted in identification of CTX-M-15 ESBLs. This is the first report identifying CTX-M type ESBL from clinical isolates of E. coli and K. pneumoniae from a tertiary care hospital in south India.


Keywords: CTX-M type ESBLs, Enterobacteriaceae, South India


How to cite this article:
Padmini S B, Raju B A, Mani K R. Detection of Enterobacteriaceae producing CTX-M extended spectrum β-lactamases from a tertiary care hospital in south India. Indian J Med Microbiol 2008;26:163-6

How to cite this URL:
Padmini S B, Raju B A, Mani K R. Detection of Enterobacteriaceae producing CTX-M extended spectrum β-lactamases from a tertiary care hospital in south India. Indian J Med Microbiol [serial online] 2008 [cited 2019 Nov 17];26:163-6. Available from: http://www.ijmm.org/text.asp?2008/26/2/163/40534


Resistance to extended spectrum cephalosporins can occur in  Escherichia More Details coli and Klebsiella pneumoniae via the production of extended spectrum β-lactamases that are capable of hydrolyzing the oxyiminocephalosporins and monobactams. [1] In recent years, a new family of plasmid-mediated CTX-M extended spectrum β lactamase (ESBL) called CTX-M has arisen and reported in the literature with increasing frequency from Europe, Africa, Asia, South America, and North America [2] . CTX-M type ESBLs show only 40% identity to TEM or SHV ESBLs, but they are closely related to β-lactamase of the Kluyvera spp. These ESBLs were named CTX-M type β-lactamases, owing to their high activity against cefotaxime. [2] However, unlike most CTX-Ms, some CTX-M variants, including CTX-M-15, CTX-M-16 and CTX-M-19, also hydrolyze ceftazidime efficiently, which may complicate their phenotypic recognition. [3]

In India, a multicentric study has reported 55-61% of ESBL prevalence among clinical isolates of Enterobacteriaceae from various hospitals. [4] Isolates producing ESBLs have not been characterized in most of the earlier studies except two studies from North India, which have reported CTX-M-15 β lactamases in isolates of Enterobacteriaceae . [5],[6] Although ESBL phenotypes have been reported from south India, there is no information on their molecular types. Hence, the present study was undertaken to characterize the β-lactamases in multidrug-resistant clinical isolates of Enterobacteriaceae by molecular techniques.


 ~ Materials and Methods Top


A total of 23 ESBL-producing isolates of E. coli (15) and K. pneumoniae (8) obtained from clinical specimens of inpatients and outpatients of PSG Hospitals, Coimbatore, Tamil Nadu (during August 2002 to August 2003) were included for the study. Antibiotic susceptibility testing was performed on Mueller-Hinton agar with the following antibiotic discs: cefotaxime (30 μg), ceftazidime (30 μg), piperacillin-tazobactam (100 μg/10 μg), imipenem (10 μg), meropenem (10 μg), ciprofloxacin (5 μg), amikacin (30 μg), gentamicin (10 μg), and co-trimoxazole (1.25-23.75 μg).

Detection of ESBL

ESBL detection was performed as recommended by NCCLS confirmatory procedure using cefotaxime (30 μg) and ceftazidime (30 μg) discs alone and in combination with clavulanic acid. MICs were determined for cefotaxime and ceftazidime with and without the presence of clavulanic acid by agar dilution techniques on Mueller-Hinton agar and results are interpreted according to NCCLS criteria. [7] Escherichia coli ATCC 25922 (β-lactamase negative) and K. pneumoniae ATCC 700603 (ESBL positive) strains were used as controls through out the study.

Confirmation of ESBL was also done by E-test ESBL strips (AB Biodisc, Solna, Sweden), and the test was performed in accordance with the guidelines of the manufacturer. Double-ended strips containing gradient of cefotaxime (CT) or ceftazidime (TZ) at one end and cefotaxime or ceftazidime plus clavulanic acid (CTL and TZL) at the other end were tested in parallel. The presence of ESBL was confirmed by the appearance of phantom zone below CT or deformation of TZ inhibition ellipse or when clavulanate caused a more than or equal to three doubling concentration decrease (ratio of ≥8)in the MIC values of cefotaxime and ceftazidime.

Transconjugation

The transmissibility of cefotaxime resistance was tested by mating each ESBL strain with E. coli recipient J53 Azi r and transconjugants were selected on trypticase soy agar plates containing 250 μg/ml of sodium azide and 10 μg/ml of cefotaxime.[8]

Isoelectric focusing

β-lactamases were characterized by isoelectric focusing using Pharmacia PhastSystem [9] of minigel with a pH range of 3-9. Clinical strain of K. pneumoniae 481880 producing β-lactamase with isoelectric points (pI s) 5.4 (TEM-1), 7.6 (SHV-I), and 8.2 (SHV-2) was used as pI standard.

PCR and DNA sequencing

To establish the molecular nature of ESBLs involved, PCR was performed for SHV and CTX-M-specific genes using specific primers. [10] SHV genes were amplified with primers - YS1 ATT TGT CGC TTC TTT ACT CGC and YS2, TTT ATG GCG TTA CCT TTG ACC and CTX-M genes amplified with the following primers. The primers were CTX 1 ATG TGC AGY ACC AGT AAR GTK and CTX 2 TGG GTR AAR TAR GTS ACC AG, where Y T R K S represents the standard nucleotide combinations. Y is C or T, R is A or G, K is T or G, S is C or G, the ambiguity designed to accommodate sequence variation in the largest number of known CTX-M type enzymes.

Purified amplicon products from few strains (6, 9 and 14) and a whole gene from a transconjugant (strain l7) were sequenced with the same primers as used for amplification using an ABI prism 3700 DNA analyzer (Applied Biosystem, CA, USA).


 ~ Results Top


Twenty-three clinical isolates included were obtained from clinical samples of urine (6), pus (10), sputum (3), endotracheal aspirate (3), and blood (1). Antibiotic susceptibility test results by disc diffusion method revealed very high susceptibility to piperacillin-tazobactam (100%), imipenem (100%), and meropenem (100%) followed by amikacin (82.6%). Resistance to cefotaxime, ceftazidime, gentamicin, ciprofloxacin, and co-trimoxazole was found to be 100, 100, 91, 82.6, and 82.6%, respectively. The results of phenotypic and molecular characterization of 23 isolates are summarized in table.

In NCCLS phenotypic confirmatory test using cephalosporin/clavulanate combination discs, all the strains showed enhanced susceptibility to ceftazidime and/or cefotaxime in the presence of clavulanic acid, a typical finding for an ESBL producer. The isolates were also demonstrated >3 log 2, dilution reduction in MIC values of cefotaxime and ceftazidime in the presence of clavulanic acid by agar dilution and E-test ESBL tests, confirming the presence of ESBLs.

Among the 23 isolates tested, transconjugants were obtained for 13 isolates and all the transconjugants were resistant to cefotaxime. β-lactamase bands were obtained for 19 of 23 isolates tested for β-lactamases by isoelectric focusing. All the isolates produced minimum of two β-lactamases with pI of 5.4 and additional β-lactamases with pI value of 8.2/8.5. Two strains ( K. pneumoniae ) displayed another band with a pI value of 7.6 [Table - 1].

Out of 23 isolates screened for bla CTX-M genes, 19 yielded positive amplicons and four isolates did not. Out of three isolates tested by PCR with SHV-specific primers, one isolate was positive (strain no. 24) for SHV gene and other two strains (strain no. 17 and 25) were negative. Sequence analysis from three transconjugants (strain no. 6, 9, and 14) and whole gene amplified from a transconjugant (strain no. 17) matched the sequence of CTX-M type enzyme in the GenBank Data base (AYO 13478) called UOE-1 or CTX-M-15.


 ~ Discussion Top


Among the "newer" ESBL families, the CTX-M type ESBLs have become widely dispersed in many parts of the world. [2] Antibiotic susceptibility test results of the above isolates illustrated an alarming trend of associated resistance to gentamicin (91%), co-trimoxazole (82.6%), and ciprofloxacin (82.6%). Such resistance has been reported in recent surveys from Canada, Italy, Spain, Greece, and UK. The bla CTX-M genes are found in association with genetic structures such as sul 1 type integrons, and this might explain the multidrug-resistant nature of organism producing these enzymes. This structure is genetically linked to class 1 integrons known to integrate antibiotic-resistant gene cassettes responsible for resistance to β-lactams, aminoglycosides, chloramphenicol, sulphonamides, and to a lesser extent rifampicin. [3]

The belief that organisms producing CTX-M enzymes display higher levels of resistance to cefotaxime than ceftazidime is not universal among all CTX-M producers. Majority of the isolates in our study conferred high-level resistance to cefotaxime (>64 μg/mL) as well as to ceftazidime (>32 μg/mL) as evidenced by MIC determination [Table - 1]. These findings correlated well with other studies. [5],[11],[12] Therefore, MIC determinations to cefotaxime and ceftazidime are not a reliable approach for identifying CTX-M type ESBLs. In our study, commercially available E-test ESBL test results were 100% in agreement with standard NCCLS phenotypic confirmatory test, although varied sensitivity (87-100%) and specificity (95-100%) of E-test ESBL for confirmation of ESBLs have been reported earlier. [13]

Transconjugation experiments expressed evidence of transfer of plasmid-mediated cefotaxime resistance in all the isolates. However, transconjugants have not been analyzed for plasmid content. Test based on isoelectric focusing and molecular detection of ESBL genes by PCR is more conclusive in defining ESBL productions. IEF results for β-lactamases detection using Pharmacia PhastSystem revealed multiple bands in most of the isolates. All the isolates had β-lactamases with pI value of 5.4 (TEM-1 enzyme). In addition, some isolates exhibited β-lactamases with a pI value of ~8.2 and others with pI value of 8.5 consistent with the presence of SHV type ESBLs and CTX-M ESBLs. Two isolates of K. pneumoniae displayed additional β-lactamase with a pI value of 7.6 (SHV-1 enzymes). As the PhastSystem procedure did not generate an accurate pI, the presence of β-lactamases was further confirmed by PCR screening using specific CTX-M and SHV primers.

Nineteen isolates were found positive for bla CTX-M genes by PCR using specific primers. ESBLs were not characterized in the remaining four CTX-M negative strains. One isolate was positive for both CTX-M and SHV gene. As majority of isolates (19/23) belonged to CTX-M type ESBLs, sequence analysis was performed only for a few strains. Amplified DNA from three isolates (strain no. 6, 9, and 14) and a whole gene from a transconjugant (strain no. 17) revealed the presence of CTX-M-15 type ESBLs.

Although extensive molecular characterization was not done in all the isolates, our preliminary findings suggest the presence of CTX-M β-lactamases as predominant ESBLs in isolates of E. coli and K. pneumoniae . Identification of CTX-M-15 in four strains of Enterobacteriaceae by sequence analysis correlated well with the earlier findings from north India. [5],[6] In conclusion, this study documents the presence of CTX-M type β lactamase among the ESBLs in this tertiary care hospital in south India.


 ~ Acknowledgments Top


We gratefully acknowledge Dr. George A. Jacoby MD, Lahey Clinic, Burlington, MA, USA, for excellent technical support for molecular characterization of ESBL positive isolates. We also thank Dr. M.K. Lalitha for the valuable guidance in MIC determination and providing Klebsiella pneumoniae ATCC 700603 (ESBL positive control) for the study.

 
 ~ References Top

1.Bradford PA. Extended-spectrum β-lactamases in the 21st century; characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev 2001;14:933-51.  Back to cited text no. 1    
2.Bonnet R. Growing group of extended spectrum β-lactamases: The CTX-M enzymes. Antimicrob Agents Chemother 2004;48:1-14.  Back to cited text no. 2    
3.Pitout JD, Nordmann P, Laupland KB, Poirel L. Emergence of Enterobacteriaceae producing extended spectrum b-lactamases (ESBLs) in the community. J Antimicrob Chemother 2005;56:52-9.  Back to cited text no. 3    
4.Mathai D, Rhomberg PR, Biedenbach DJ, Jones RN. Evaluation of the in-vitro activity of six broad spectrum β-lactam anitmicrobial agents tested against recent clinical isolates from India; a survey of ten medical center laboratories. Diagn Microbiol Infect Dis 2002;44:367-77.  Back to cited text no. 4    
5.Karim A, Poirel L, Nagarajan S, Nordmann P. Plasmid-mediated extended-spectrum β-lactamase (CTX-M-3 like) from India and gene association with insertion sequence ISEcp1. FEMS Microbiol Lett 2001;201:237-41.  Back to cited text no. 5    
6.Ensor VM, Shahid M, Evans JT, Hawkey PM. Occurrence, prevalence and genetic environment of CTX-M b-lactamases in Enterobacteriaceae from Indian Hospitals. J Antimicrob Chemother 2006; 58 : 1260-3.  Back to cited text no. 6    
7.National Committee for Clinical Laboratory Standards. 2000. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M7-A5 and informational supplement M100-S10. National Committee for Clinical Laboratory Standards, Wayne, PA.  Back to cited text no. 7    
8.Jacoby GA, Han P. Detection of extended spectrum b-lactamases in clinical isolates of Klebsiella pneumoniae and Escherichia coli . J Clin Microbiol 1996;34:908-11.  Back to cited text no. 8    
9.Huovinen S. Rapid isoelectric focusing of plasmid mediated b-lactamases with Pharmacia PhastSystem. Antimicrob Agents Chemother l988;32:1730-2.  Back to cited text no. 9    
10.Jacoby GA, Vacheva-Dobrevsky R. Epidemiology of extended spectrum b-lactamases in Sofia, Bulgaria. Eur J Clin Microbiol Infect Dis 2003;22:385-8.  Back to cited text no. 10    
11.Poirel L, Naas T, Le Thomas I, Karim A, Bingen E, Nordmann P. CTX-M type extended spectrum b-lactamase that hydrolyzes ceftazidime through a single amino acid substitution in the omega loop. Antimicrob Agents Chemother 2001;45:3355-61.  Back to cited text no. 11    
12.Poirel L, Gniadkowski M, Nordmann P. Biochemical analysis of the ceftazidime-hydrolyzing extended spectrum b-lactamase CTX-M-15 and of its structurally related b-lactamase CTX-M-3. J Antimicrob Chemother 2002;50:1031-4.  Back to cited text no. 12    
13.Paterson DL, Bonomo RA. Extended spectrum β-lactamases: A clinical update. Clin Microbiol Rev 2005;18:657-86.  Back to cited text no. 13    



 
 
    Tables

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