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Indian Journal of Medical Microbiology
Medknow Publications on behalf of Indian Association of Medical Microbiology
ISSN: 0255-0857 EISSN: 1998-3646
Vol. 26, Num. 4, 2008, pp. 365-368

Indian Journal of Medical Microbiology, Vol. 26, No. 4, October-December, 2008, pp. 365-368

Brief Communication

Molecular characterization of nosocomial CTX-M type β-lactamase producing Enterobacteriaceae from a tertiary care hospital in south India

Jemima SA, Verghese S

Department of Microbiology, International Centre for Cardio Thoracic and Vascular Diseases, A Unit of Frontier Lifeline, R-30-C, Ambattur Industrial Estate Road, Mogappair, Chennai-600 101, Tamilnadu
Correspondence Address:Department of Microbiology, International Centre for Cardio Thoracic and Vascular Diseases, A Unit of Frontier Lifeline, R-30-C, Ambattur Industrial Estate Road, Mogappair, Chennai-600 101, Tamilnadu
sam_jemi@yahoo.co.in

Date of Submission: 25-Nov-2007
Date of Acceptance: 31-Dec-2007

Code Number: mb08111

Abstract

CTX-M group of extended spectrum β lactamases (ESBLs) represents a rapidly emerging problem in many countries. The prevalence of nosocomial bla CTX-M-1 producing Enterobacteriaceae strains has not been reported earlier in Indian hospitals. This study describes molecular subtyping of nosocomial bla CTX-M producing strains of Enterobacteriaceae . Polymerase chain reaction with primers specific for bla CTX-M-1 coding genes was used to identify 95 Enterobacteriaceae strains producing bla CTX-M positive isolates. Of the 95 bla CTX-M producing isolates, 45 strains were positive for bla CTX-M-1 . bla CTX-M-1 was found to be most prevalent in Klebsiella strains.

Keywords: bla CTX-M , bla CTX-M-1, extended spectrum β lactamase, Enterobacteriaceae.

During the past decade, ESBLs of bla CTX-M type emerged in many countries of the world. [1],[2],[3],[4] The bla CTX-M type enzymes are a group of molecular class A extended spectrum beta lactamases that exhibit an overall preference for cefotaxime and ceftriaxone and a higher susceptibility to tazobactum than to clavulanate. [5] They were initially reported in the second half of 1980s, and their rate of dissemination among bacteria and in most parts of the world has increased dramatically since 1995. [6] Several different variants of bla CTX-M type enzymes have been identified till date.

The bla CTX-M genes are often carried on transferable plasmids. [7] We report the detection of bla CTX-M type enzyme CTX-M-1 gene in clinical isolates of Enterobacteriaceae isolates from a South Indian tertiary care hospital.

Materials and Methods

Bacterial strains
 A total of 600 phenotypically ESBL positive isolates were checked for the presence of bla CTX-M gene by PCR and 95 (15.83%) strains were found positive for the same. The 95 non repetitive Enterobacteriaceae strains included 25- E. coli , 22- Enterobacter spp. and 48- Klebsiella spp. These CTX-M positive isolates included, 30-isolates from urine samples, 26- from endotracheal secretions, 13 from pus aspirates, 10 from blood cultures, 8-sputum samples, 5-fluids and 3-central line tip cultures. Identification of these isolates was done based on colony morphology on blood agar, MacConkey agar and by standard biochemical reactions. All strains were collected from a tertiary care hospital in South India.

In vitro susceptibility testing for detection of ESBL production
In vitro susceptibility was determined using double disk synergy test (DDST), phenotypic confirmatory double disk test (PCDDT) as recommended by CLSI guidelines. [8] E. coli (ATCC-25922) and K. pneumoniae (ATCC-700603) were used as reference strains for quality control of in vitro susceptibility testing.

Molecular analysis
 PCR amplification of bla CTX-M and bla CTX-M-1 gene was carried out with specific primers using specific reaction parameters [Table - 1]. Bacterial strain producing the known β-lactamase- E. coli J53-pMG 267 (CTX-M-14) was used for quality control.

PCR was carried out in 50 µL volume with 20 pmol of each primer, 200 µM deoxynucleosidetriphosphates, 1.5 mM MgCl 2 and 0.5U Taq DNA polymerase (RBC-Bioscience) in the reaction buffer provided by the enzyme manufacturer using 1 µL of plasmid DNA as a template DNA.

Restriction fragment length polymorphism (RFLP)
The amplified CTX-M product (10μL) was directly subjected to digestion with 9U Pst -I and 4U Pvu -II enzymes (Medox) in One-Phor-All Plus Buffer (10mM Tris acetate (pH 7.5), 10 mM magnesium acetate, 50 mM potassium acetate) for four hours at 37°C. The restriction fragments of the PCR products were analysed by electrophoresis in 3.5% agarose containing ethidium bromide (Bio gene, USA).

Conjugation assays
 Conjugation assays were carried out by the filter mating procedure using the E. coli K-12Nal r Rif r mutant as the recipient. Transconjugants were selected on MH agar containing rifampicin (100 µg/mL) plus ceftazidime (2µg/mL). Plasmid DNA was extracted by boiling-lysis method and analysed by electrophoresis on 0.8% (wt/vol) agarose gels. PCR was carried out for the transconjugants with plasmid as template for bla CTX-M .

Results

Phenotypic detection of ESBLs was carried out according to CLSI guidelines. Out of 1010 isolates screened for ESBL production, out of which 600 gave positive result. According to the guidelines mentioned a difference of 3-5 mm increase in zone diameter on either agent tested in combination with clavulanic acid versus its zone diameter when tested alone confirms the presence of ESBLs.

Among the 600 ESBL positive isolates, bla CTX-M was demonstrated [Figure - 1] in 95(15.83%) clinical strains. All 95 CTX-M positive isolates were subjected to RFLP. All isolates showed similar restriction patterns [Figure - 2] and they all grouped under CTX-M-1 group as previously described by Edelstein. [3]

Among the 95 CTX-M producing isolates, CTX-M-1 gene was positive [Figure - 3] in 45(47.3%) isolates. It included 9/25 E. coli isolates (36%), 8/22 Enterobacter species (36.3%) and 28/48 Klebsiella species (58.3%)

Plasmid analysis of the transconjugants showed that the beta lactamase gene was associated with the plasmid. Oxyimino-β-lactam antibiotic resistance was transferred to E. coli K-12. All 95 isolates demonstrated bla CTX-M by PCR.

Discussion

The present study documents the emergence of bla CTX-M-1 gene in clinical isolates from South India for the first time. The bla CTX-M-1 gene has previously been reported in France, Italy and Germany it was reported in the year 1989 in E. coli , P. mirablis and E. coli respectively. [6] In 2002, Dutour et al reported the spread of CTX-M 1 in France. [9] In Sweden real time PCR assays were developed for the detection of bla CTX-M-1 type beta lactamases. [10] The CTX-M-1 gene was also reported from E. coli strains in healthy pets in Portugal by Daniela et al in the year 2004. [11] In Germany, at the beginning of 1989, Bauernfeind et al reported on a clinical cefotaxime-resistant E. coli strains which produced a non-TEM, non-SHV ESBL, designated CTX-M-1, in reference to its hydrolytic activity against cefotaxime. [4] In Poland, Gniadkowski et al identified a variant of CTX-M-1, designated CTX-M-3, in different members of the family Enterobacteriaceae isolated in 1996. [12] In India, a variant of the CTX-M-3 enzyme, designated CTX-M-15, was reported from six unrelated members of the family Enterobacteriaceae ( E. coli -4, K.pneumoniae -1, E.aerogenes -1) isolated between April and May 2000. [6] Sekar et al reported that 44.4% of E. coli and 35.29% K.pneumoniae strains were found to be positive for bla CTX-M gene by PCR. [13]

In this study, of 600 ESBL positive isolates which were analysed for the presence of bla CTX-M gene by PCR, 95 (15.83%) isolates were positive for bla CTX-M . These 95 strains were further analysed for the presence of CTX-M-1 gene using primers specific for bla CTX-M-1 . Among the 95 CTX-M producing isolates, CTX-M-1 gene was positive in 45 (47.3%) of the isolates. They were found in 36% of E. coli isolates, 36.3% of Enterobacter spp. and 58.3% of Klebsiella spp. CTX-M.

A high prevalence of CTX-M-1 (58.3%) type gene was recorded in Klebsiella spp. when compared with E. coli (36%) and Enterobacter spp. (36.3%). The restriction patterns of all the CTX-M PCR products was similar, with restriction bands at 267 bp, 156 bp and 120 bp. Therefore, it can be concluded that all the strains belong to CTX-M-1 group as reported by Edelstein. [3] The remaining (55.7%) probably belong to the other CTX-M subtype in CTX-M-1 group. RFLP is a technique which can be used to identify the bla CTX-M into groups by its restriction patterns. However, only with the specific primer sequence the gene type can be identified.

Cefotaxime is a very commonly used third generation cephalosporin in hospitals in India for community acquired infections like pneumonia, enteric fever and meningitis. This result suggests that the spreading of this plasmid among different E. coli , Klebsiella spp., Enterobacter spp. was important in the dissemination of the CTX-M-1 gene. In a hospital environment, plasmids could be transferred easily between patients through health care workers due to hand carriage and selection pressure. To conclude, we have found a high prevalence of bla CTX-M-1 β-lactamase in South Indian isolates while the prevalence of CTX-M-15 has been reported the world over. Thus, it indicates the need for a more detailed surveillance and epidemiological survey in this region.

References

1.Sophie B, Francois-Xavier W, Axel C, Martine V, Eric M, Karine P, et al. Clonal Emergence of Extended-Spectrum β-Lactamase (CTX-M-2)-Producing Salmonella enterica Serovar Virchow Isolates with reduced susceptibilities to ciprofloxacin among poultry and humans in Belgium and France (2000 to 2003) J Clin Microbiol 2006;44:2897-903.  Back to cited text no. 1    
2.Aroonwadee C, Fatima HM, John H, Jian-Hui X, Peter MH. Three Cefotaximases, CTX-M-9, CTX-M-13, and CTX-M-14, among Enterobacteriaceae in the People's Republic of China. Antimicrob Agents Chemother 2002;46:630-7.  Back to cited text no. 2    
3.Edelstein M, Pimkin M, Edelstein I, Narezkina A, Stratchounski L. High prevalence of nosocomial Escherichia coli and Klebsiella pneumoniae producing CTX-M-type extended spectrum β-lactamases in Russian hospitals. 12th European Congress of Clinical Microbiology and Infectious Diseases. Poster # P1398.  Back to cited text no. 3    
4.Markovska R, Schneider I, Keuleyan E, Bauernfeind A. Extended-spectrum β-lactamase (ESBL) CTX-M-15-producing Escherichia coli and Klebsiella pneumoniae in Sofia, Bulgaria. Clin Microbiol Infect 2004;10:752-5.  Back to cited text no. 4    
5.Bonnet R, Dutour C, Sampaio JL, Chanal C, Sirot D, Labia R, et al . Novel Cefotaximase (CTX-M-16) with Increased Catalytic Efficiency Due to Substitution Asp-2403Gly. Antimicrob Agents Chemother 2001;45:2269-75.  Back to cited text no. 5    
6.Bonnet R. Minireview: Growing Group of Extended-Spectrum β-Lactamases: The CTX-M Enzymes. Antimicrob Agents Chemother 2004;48:1-14.   Back to cited text no. 6    
7.George AJ, Luisa SM. Mechanisms of disease The New β -Lactamases N Engl J Med 2005;352:380-91.  Back to cited text no. 7    
8.Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing. 15th informational supplement: M100-S15 2005.  Back to cited text no. 8    
9.Dutour C, Bonnet R, Marchandin H, Boyer M, Chanal C, Sirot D, et al. CTX-M-1, CTX-M-3, and CTX-M-14 β-lactamases from Enterobacteriaceae isolated in France. Antimicrob Agents Chemother 2002;46:534-7.  Back to cited text no. 9    
10.Hong F, Christina L, Barbro OL, Goran H, Emma L, Åsa R, et al. Molecular Epidemiological Analysis of Escherichia coli Isolates Producing Extended-Spectrum β-Lactamases for Identification of Nosocomial Outbreaks in Stockholm, Sweden. J Clin Microbiol 2004;42:5917-20.  Back to cited text no. 10    
11.Daniela C, Patricia P, Laura B, Yolanda S, Jorge R, Carmen T. Detection of CTX-M-1 and TEM-52 β-lactamases in Escherichia coli strains from healthy pets in Portugal. J Antimicrob Chemother 2004;54:960-1.  Back to cited text no. 11    
12.Marek G, Ines S, Andrzej P, Renate J, Barbara M, Adolf B. Cefotaxime-Resistant Enterobacteriaceae Isolates from a Hospital in Warsaw, Poland: Identification of a New CTX-M-3 Cefotaxime-Hydrolyzing beta-Lactamase that is closely related to the CTX-M-1/MEN-1 Enzyme. Antimicrob Agents Chemother 1998;42:827-32.  Back to cited text no. 12    
13.Sekar B, Shwetha R, Arunagiri K, Menaka K, Lalitha P, Aparna V, Oommen PK. Detection and characterization of bla CTX-M gene by PCR-RFLP analysis among third generation cephalosporin resistant gram negative isolates. XXX National congress of Indian Association of Medical Microbiologists. Microcon 2006-p27.  Back to cited text no. 13    

Copyright 2008 - Indian Journal of Medical Microbiology

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