Year : 2008 | Volume
: 51 | Issue : 2 | Page : 218--221
Sequence analysis of bla CTX-M-28 , an ESBL responsible for third-generation cephalosporin resistance in Enterobacteriaceae, for the first time in India
Jemima Kingsley, Susan Verghese
Department of Microbiology, International Centre for Cardio Thoracic and Vascular Diseases, A Unit of Frontier Lifeline, Chennai, Tamil Nadu, India
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, Tamil Nadu
The most common group of ESBLs not belonging to the bla TEM or bla SHV families were termed bla CTX-M , to highlight their ESBLs«SQ» greater activity against cefotaxime than against ceftazidime. The presence of nosocomial bla CTX-M-28 -producing Enterobacteriaceae strains has not been reported earlier in Indian hospitals. The sequences of bla CTX-M-28 gene from cephalosporin-resistant Enterobacteriaceae were analyzed. The structural gene encodes a 290 amino-acid protein, which is most related to the bla CTX-M β-lactamases. The conserved K-T-G was identified in the bla CTX-M-28 protein sequence, but significantly, two point mutations (N→T) and (F→S) were identified in the Y-G-N- and S-T-F-K-conserved motifs respectively. These point mutations were seen in all the three sequenced isolates
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Kingsley J, Verghese S. Sequence analysis of bla CTX-M-28 , an ESBL responsible for third-generation cephalosporin resistance in Enterobacteriaceae, for the first time in India.Indian J Pathol Microbiol 2008;51:218-221
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Kingsley J, Verghese S. Sequence analysis of bla CTX-M-28 , an ESBL responsible for third-generation cephalosporin resistance in Enterobacteriaceae, for the first time in India. Indian J Pathol Microbiol [serial online] 2008 [cited 2020 Nov 25 ];51:218-221
Available from: https://www.ijpmonline.org/text.asp?2008/51/2/218/41691
The CTX-M β-lactamases are a class of β-lactamases which have been more recently recognized to preferentially hydrolyze cefotaxime (CTX) and were initially reported in the second half of the 1980s.  Their rate of dissemination among bacteria in most parts of the world has increased dramatically since 1995.  Over the last decade, they have been identified in numerous countries, such as Africa, South America, Asia and Europe. 
CTX-M β-lactamases (cefotaximases) are a relatively novel family of plasmid-mediated extended-spectrum cephalosporins and have been classified under Ambler class A. These enzymes generally possess higher levels of hydrolytic activity against cefotaxime than against ceftazidime and are inhibited by clavulanic acid, sulbactam and tazobactam. They also provide resistance to cefepime.  On the basis of their amino acid sequences, the phylogenic study reveals 5 major groups of acquired CTX-M enzymes: (i) the CTX-M-1 group, (ii) the CTX-M-2 group, (iii) the CTX-M-8 group, (iv) the CTX-M-9 group and (v) the CTX-M-25 group.  Despite their structural diversity, most CTX-M β-lactamases have similar hydrolysis profiles, which include cefotaxime and ceftriaxone but not ceftazidime. 
Although ESBL-producing strains have been reported by several countries, very few reports of the CTX-M genotypes existing in south India have been published. Infections caused by ESBL-producing strains are increasing in frequency in India. However, only few polymerase chain reaction (PCR)-based surveys of ESBL-producing strains have been performed and reported until now in this country. The present prospective study was done in order to report the prevalence of ESBL-producing Enterobacteriaceae and to characterize the types of ESBLs produced by hospital-acquired isolates in south India. This is the first report from India to identify the presence of bla CTX-M-28 -producing Enterobacteriaceae .
Materials And Methods
A total of 416 clinical isolates were taken for the study. They included 111 Klebsiella sps, 117 Enterobacter sps and 188 E. coli . All 416 isolates were nonrepetitive and were collected from 382 patients from June 2005 to May 2007. Among these, 145 isolates were from urine samples, 116 were from endotracheal secretions, 70 were from pus samples, 23 were from sputum samples, 30 were from blood cultures, 10 were from fluid cultures, 3 were from tissue samples and 19 were from central venous line tips. All samples were collected under sterile conditions in sterile containers. All cultures were collected from inpatients admitted to the Adult Intensive Care Unit (AICU) and Pediatric Intensive Care Unit (PICU) of a tertiary care hospital in south India.
Antibiotic susceptibility testing and ESBL-production detection
ESBL production was detected for all collected 416 isolates by the double disc synergy test according to Clinical and Laboratory Standards Institute (CLSI) guidelines.  Antibiotic susceptibility of ESBL-producing strains was determined by the disc diffusion method according to the recommendations in CLSI guidelines.  The following commercial discs were tested: amoxicillin-clavulanic acid (20, 10 µg), cefoxitin (30 µg), cefotaxime (30 µg), ceftazidime (30 µg), gentamicin (10 µg), piperacillin-tazobactum (75, 10 µg), amikacin (10 µg) and imipenem (10 µg). The minimum inhibitory concentration (MIC) of ceftazidime and cefotaxime with or without clavulanic acid were determined by using serial twofold dilutions of antibiotics in Mueller-Hinton agar and inocula of 10 5 CFU/mL of bacteria were inoculated per spot.
Plasmid profile identification
The culture was inoculated into 5 mL of Luria-Bertanii (LB) broth and incubated overnight at 37°C. Three milliliters of the culture was centrifuged and the cells were pelleted. Plasmid DNA was extracted by boiling-lysis method.  Plasmid profile of each strain was checked on 0.7% agarose gel which was run at 5 volts/cm.
Polymerase chain reaction
Entire CTX-M-type gene was amplified for sequencing purposes by using forward primer CTX-M-1-F (5'-GGTTAAAAAATCACTGCGTC-3'; located at positions 3 to 22) and reverse primer CTX-M-15-R (5'-TTACAAACCGTCGGTGACGA-3'; located at positions 876 to 857) as described previously. 
Controls used were J53 pMG 267 E. coli (CTX-M-14) [Kindly donated by Dr. George A. Jacoby, USA].
Isoelectric focusing of beta-lactamase
The analytical isoelectric focusing was done using broad-range soluble ampholytes (Biorad) that resolve proteins with pI values between 3.8 and 9.2. The periplasmic β-lactamases were extracted by the procedure that has been previously described.  The proteins were then separated according to the isoelectric point, the gels were extruded and the zymograms were developed by using chromogenic cephalosporin as a substrate. Zymograms were developed based on the principle that the intact substrate molecule, which is yellow, turns to pink when the β-lactam bond is broken, so that focused bands with β-lactamase activity appear as pink on a yellow background.
Restriction fragment length polymorphism (RFLP)
The amplified CTX-M product (10 µL) was directly subjected to digestion with 9U Pst I and 4U Pvu II enzymes (Medox) in One-Phor-All Plus buffer [10 mM Tris acetate (pH 7.5), 10 mM magnesium acetate, 50 mM potassium acetate] for 4 hours at 37°C. The restriction fragments of the PCR products were analyzed by electrophoresis in 3.5% AmpliSize agarose (Bio gene, USA) after ethidium bromide staining.
Conjugation assays were carried out by the filter-mating procedure using the E. coli K-12 Nal r Rif r [Kindly donated by Dr. Claudine Quentin, France] mutant as the recipient. Transconjugants were selected on Muller Hinton agar containing nalidixic acid (100 µg/mL) and/or rifampicin (100 µg/mL) plus CTZ (2 µg/mL). Plasmid DNA was extracted by boiling-lysis method and analyzed by electrophoresis on 0.8% (wt/vol) agarose gels. PCR was carried out for the transconjugants with the plasmid as template for bla CTX-M .
Nucleotide sequence analysis
All ESBL-producing isolates presented similar plasmid profiles, pI points and restriction patterns. One from each ( E. coli , Enterobacter sps and Klebsiella sps) was selected for sequence analysis. The DNA sequence and deduced amino acid sequence of bla CTX-M-28 have been deposited in GenBank and the accession numbers assigned were the following: EU531510, EU531512 and EU531513.
Of the 416 isolates, a total of 188 (45.19%) isolates were phenotypically identified as ESBL producers. All 188 ESBL producers were selected for PCR amplification of CTX-M gene, which included 84 E. coli , 76 Klebsiella sps and 28 Enterobacter sps. Antimicrobial susceptibility tests by the disc diffusion method indicated strong synergy between amoxicillin-clavulanic acid and either cefotaxime or ceftazidime. All ESBL-producing strains were resistant to extended-spectrum cephalosporins, cefotaxime and ceftazidime (90% of the MICs were >32 µg/L) but remained susceptible to imipenem.
Detection and distribution of entire bla CTX-M gene
Among the 188 ESBL-producing clinical isolates, 85 strains (45% of the isolates) were positive for bla CTX-M gene by PCR. The amplified product was visualized at 874 bp in all 85 strains. They included 22 E. coli , 43 Klebsiella sps and 20 Enterobacter sps. All isolates producing β-lactamases also had a similar isoelectric focusing point with pI of 8.6. The sequence analysis of the three isolates (one each from E. coli, Klebsiella sps and Enterobacter sps) revealed 100% similarity with CTX-M-28 gene associated with the GenBank number CAD70280.
Restriction fragment length polymorphism (RFLP)
All 85 isolates were subjected to RFLP with Pst I and Pvu II enzymes. There was no restriction site found for Pst I , but two restriction sites were recognized by Pvu II enzyme. The restriction was seen at 469 bp and 625 bp. Since there were two recognition sites, three bands were visualized at 469, 249 and 156 bp. All 85 isolates showed similar restriction patterns.
Conjugation experiments were carried out on the Enterobacteriaceae isolates producing bla CTX-M ESBL. Transconjugants were selected on MacConkey and Muller Hinton agar plates supplemented with rifampicin and ceftazidime. 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. After transfer, the plasmid was isolated and PCR was repeated for bla CTX-M , which was demonstrated to be positive again for all 85 isolates.
Sequence analysis of the bla CTX-M-28 β-lactamase gene
The bla CTX-M-28 gene comprises an 874-bp, open reading frame, between the initiation codon ATG (positions 51 to 53) and the stop codon TAA (positions 871 to 873), which translates into a protein of 290 amino acids [Figure 1], showing the homology to β-lactamase enzymes. Some unique features and distinct biochemical properties were noted. A highly conserved motif K-T-G (lysine-threonine-arginine) at positions 236 to 238, believed to contribute to the function of the serine active site, was identified. The other two motifs serine-threonine-serine-lysine tetrad (S-T-S-K) were found at positions 72 to 75. A third motif tyrosine-glycine-threonine (Y-G-T) was found at positions 242 to 244, which differs from the corresponding motifs in all other enzymes by the presence of serine (S) instead of phenalyalanine (F) and threonine (T) instead of asparagines (N) respectively. This unique substitution may have significant biochemical effects. These three motifs included the conserved serine and lysine residues characteristic of β-lactamases possessing a serine active site.
Although several studies addressed the issue of the emergence of ESBL-producing Enterobacteriaceae worldwide, only few reports of genotypic characterization have been published from India till now. It has been mostly reported that resistance to β-lactam antibiotics is on the rise among clinical isolates in different Indian hospitals, expressing the need for an exhaustive research. The present report is the first report of isolation and characterization of bla CTX-M-28 gene from 3 strains of ESBL-producing Enterobacteriaceae isolated from a tertiary care hospital in south India.
A total of 188 nonrepetitive ESBL-producing isolates were isolated and identified from a tertiary care hospital in Chennai. The percentage of ESBL-producing strains was 45.19%, which was relatively high when compared to the report by Subha et al. (6.6%) in 2002.  Grover et al. in 2006 have reported from Delhi a percentage positivity of ESBLs to be 88% in their study,  which is much higher compared to our present report. Baby Padmini et al. from Coimbatore in 2004 have reported 40% and 41% ESBL positivity among K. pneumoniae and E. coli respectively in their study cohort, which is very similar to our findings. 
Molecular characterization revealed that bla CTX-M gene was prevalent in 45% of these ESBL producers.Sequencing of 3 isolates ( E. coli , Enterobacter sps and Klebsiella sps) showed that all 3 had the CTX-M-28 gene. Unlike other ESBLs that preferentially have three motifs, K-T-G, Y-G-N, S-T-F-K, here in the present study, point mutation was seen in two motifs. In S-T-F-K, the phenylalanine (F) had mutated into serine (S); and in Y-G-N, the asparagines (N) had mutated into threonine (T), which would increase the characteristics of β-lactamases in the serine active site.
The first detection of bla CTX-M-28 , an ESBL, was reported by Li Xu et al.  This enzyme differs from bla CTX-M-3 by a single nucleotide (A750-G750) within the PCR amplicon. A report on ESBL types in Enterobacteriaceae in Argentinean public hospitals found that CTX-M's accounted for roughly 70% of all ESBLs found, with similar findings being reported in studies conducted in Japan, China, United Kingdom and Spain. ,,,,
In India, a variant of the bla CTX-M-3 enzyme, designated bla CTX-M-15 , was reported from 6 unrelated members of the family of Enterobacteriaceae isolated between April and May 2000.  Sekar et al. reported that 44.4% of E. coli and 35.29% of K. pneumoniae strains were found to be positive for bla CTX-M gene by PCR.  The prevalence of bla CTX-M was also reported by Shahid et al. , with 72 (77.4%) of the 93 E. coli isolates being found to be CTX-M group -1 positive by PCR in the north Indian isolates.  Ankur Goyal et al. reported that prevalence of ESBLs in their hospital was 63.6% in E. coli strains and 66.7% in K. pneumoniae strains. They reported that on sequence analysis, all 20 CTX-M-1 clusters matched with CTX-M-3 subtype. 
Our finding emphasizes the increasing role of the bla CTX-M β-lactamases in antibiotic resistance worldwide and leads to consideration of empirical treatment for infections caused by coliforms, especially in patients compromised by underlying disease or immunological status. The carriage of ESBL genes is a novel finding; identification of bla CTX-M 28 among E. coli , Enterobacter sps a nd Klebsiella sps is uncommon; and this is the first report from India. In conclusion, the data presented here illustrates the complexity and extent of the spread of ESBL-producing Enterobacteriaceae in India. This result demonstrates the emergence and the presence of bla CTX-M-28 in our country.
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