A study on Prevalence of extended spectrum Beta- lactamases Gene (CtX-M, teM & shV) Producing Enterobacteriaceae Members Isolated from Different Clinical Specimens at Tertiary Care Hospital, Uttarakhand

Extended spectrum b-lactamases (ESBLs) are one of the major enzymes responsible for antimicrobial drug resistance in bacterial isolates. The objective of this study was to find out the ESBL genes (blateM, blashV and blaCtX-M) in Enterobacteriaceae. This study was conducted from November 2013 to October 2015. The identification of Enterobacteriaceae isolates & antimicrobial drug resistance was done by conventional standard microbial methods. Further genotypic detection of ESBL was done by multiplex PCR. Results: Among 942 Enterobacteriaceae isolates, 332 (35.24%) isolates was ESBL producers. We observed high prevalence of ESBL enzyme in Klebsiella pneumonia (59.09%). These isolate revealed high resistance to co-trimoxazole, fluoroquinolones and aminoglycosides. Out of 48 randomly selected isolates, ESBL genes were identified in 45 isolates which were found resistant to third generation cephalosporins. Single CTX-M gene noticed in 29 strains of Escherichia coli (E. coli), 02 strains of Klebsiella pneumoniae, and 01 strain of Proteus mirabilis. Occurrence of Combination of the genes e.g., (blateM+blaCtX-M), (blashV+blaCtX-M), (blateM+blashV+blaCtX-M) was detected in 08, 01 and 04 isolates respectively. Multiplex PCR in CTX-M carrying isolates revealed the presence of blaCtX-M group-1. The most common blaCtX-M group-1 was observed in all isolates of family Enterobacteriaceae. Antimicrobial drug resistance is a major problem of concern now a day. Studies like this will be helpful to knowing the burden of multi drug resistance as well as formulating antibiotics policy for a particular region.


INTRODUCTION
The β-lactamases are the groups of enzymes that open up the β-lactam ring by supplementing a water molecule to the β-lactam bond, and this disables the β-lactam antibiotics. A type of β-lactamases enzyme, extended spectrum beta-lactamases (ESBLs), is produced by Enterobacteriaceae which hydrolyze both the oxy-imino beta-lactams and monobactams, but do not have any effect on carbapenems and. cephamycins 1,2 . The prevalence of ESBLs differs among clinical isolates of various countries. 3 Recently prevalence of multiple ESBL genes in a single strain has further complicated the process of detection 4 . Being plasmid and transposon mediated, it rapidly spread to numerous species of bacteria. In the early 1960s, the first plasmid mediated beta lactamase in gram negative bacteria, TEM-1, was observed 5 . The next plasmid mediated beta-lactamase discovered was SHV-1 6 . The CTX-M type beta lactamases have become more important and groups in molecular class A β-lactamases, however most of the ESBLs are mutants of the TEM and the SHV enzymes. The CTX-M gene was 1st detected in E. coli strains in Germany and exhibit quickly spreaded to different countries [7][8][9][10][11] . The CTX-M gene have more than 450 diverse groups depending upon amino acids moiety and distributed into five groups: the CTX-M group 1 CTX-M group 2, CTX-M group 8, CTX-M group 9 and CTX-M group 25 12 . The CTX-M 15 gene was first detected in New Delhi, India. It refer to the CTX-M group-1 13 . Walsh et al. also described a more prevalence of bla TEM in Indian Enterobacteriaceae and remarkably all were reported as CTX-M group-1 14 . So It becomes essential to find out the prevalence of ESBL producing strains in a geographical area because it can help in judicious use of antibiotics and can guide the empirical therapy. In this consideration this study was planned in, Government Medical College and Dr. Susheela Tiwari Government Hospital, Haldwani to explore ESBL producing Enterobacteriaceae isolates from different clinical samples.

MAteRiAls ANd MethOds
The descriptive study was conducted during November 2013 to October 2015. The clinical Specimens were collected in central laboratory, microbiology section of Dr. Susheela

Detection of ESBL screening test
All enterobacteriaceae isolates were tested for resistance to aztreonam (30 μg) and the third generation cephalosporins namely cefotaxime (30 μg), ceftriaxone (30 μg) and ceftazidime (30 μg) by the Kirby-Bauer disc diffusion method. This was a part of the routine antimicrobial susceptibility testing. As per 2012 CLSI guidelines, resistance to at least one of the antimicrobials was taken as positive in the screening test for ESBL production 17 .

Phenotypic confirmatory disc diffusion test (PCddt)
The ceftazidime (30 μg) disc alone and in combination with clavulanic acid (Ceftazidime + Clavulanic acid, 30/10 μg disc) was applied onto a plate of Mueller Hinton Agar (MHA) for ESBL detection, which was inoculated with the test strain. The culture plates were incubated at 37 0 C for 16-18 hr and identified more than 5 mm zone of inhibition around the combination disc in comparison to the ceftazidime disc alone was deliberated to be a marker for ESBL production. e-test E-test was applied for further confirmation of ESBL isolates were made by evaluation of minimum inhibitory concentration (MIC). The

MICs were evaluated by E-test strips (Hi-Media Laboratories Pvt. Limited, Mumbai). Two ends of
The E-test strips were impregnated with cefotaxime (CTX) and cefotaxime + clavulanic acid (CTX+). The E-test strip was placed on the Mueller Hinton Agar (MHA) plate at centre and incubated aerobically at 37°C for 16-18 hours. The interpretation of MIC was considered as the value at the intersection of the growth ellipse with the strip. The ratio of the MIC value of cefotaxime to the MIC value of cefotaxime in combination with clavulanic acid was more than 8 or no zone was observed around cefotaxime but zone was obtained in cefotaxime and clavulanic acid combination was considered as an ESBL producer.

Quality Control
Klebsiella pneumoniae ATCC 700603 strain was used as the positive control whereas ATCC 25922 strain Escherichia coli were used as negative control.

Gene detection in members of family enterobacteriaceae Preparation of DNA template
Randomly selected 48 ESBL positive clinical isolates (confirmed by PCDDT) were subcultured on Teepol lactose agar. After overnight incubation, 3-4 discrete colonies of each isolate were inoculated in 100 µl mili Q water and then heated at 95°C for 5 min followed by immediate chilling at 4°C. Primers used for revealing of ESBL genes (TEM, SHV, CTX-M) are shown in Table 1.

Detection of bla CtX-M gene
The bla CTX-M gene areas in all isolates were determined with the primers as depicted by woodford et al. 18 Briefly, a total volume of 25 µl reaction mixture was used to amplify the bla CTX-M gene . The polymerase chain reaction (PCR) was performed in MJ-mini Bio-Rad thermal cycler with the cycling conditions. It consists of an early denaturation step at 94°С for 7 minutes and then 35 cycles of denaturation at 94˚С for 50 seconds, annealing at 50°С for 40 seconds and extension for 72°С for 60 seconds. The process was usually concluded with a final extension step at 72°С for 5 minutes. Gel electrophoresis with 2% agarose (Bangalore Genei, India) gel containing ethidium bromide was used to amplify PCR products.

The CTX-M gene groups detection by multiplex PCR
The isolates were expressed with the presence of CTX-M genogroups-1, 2, 8, 9 and 25 by using multiplex PCR with few adjustments. 18 The MJ-min bio red thermal cycler was used to perform PCR reaction mixture in 25μl (Table A). The PCR cycling conditions have denaturation step in beginning at 94°С for 5 minute, this is followed by 40 cycles of final denaturation at 94°С for 1 minute, annealing at 52°С for 60 second and extension at 72°С for one minute and with a last extension at 72°C for 10 minutes. Gel electrophoresis with 2% agarose (Bangalore Genei, India) gel containing ethidium bromide was used to screen the amplified product.
Identification of bla teM and bla shV genes were determined by PCR as adopted by woodford et al 18 . The polymerase chain reaction was conducted in a reaction mixture of 25µl. (Table 2) The PCR cycling conditions have initial denaturation step at 94° C for 3 min, then 30 cycles of final denaturation at 94° C for 30 sec, annealing at 64° C for 30 sec, extension at 72° C for 1 min with final extension at 72° C for 10 min.

RESULT AND OBSERvATIONS
A total of 1346 gram negative bacilli (GNB) were isolated from different, non-repeated clinical specimen received during this period of study. Out of these 1346 GNB isolates, 942 (78.08%) isolates were belongs to Enterobacteriaceae family. Among the ESBL producing Enterobacteriaceae,   (Fig. 1). Maximum numbers of ESBL producing Enterobacteriaceae were found from the urine. (Table 3) Among ESBL producing Enterobacteriaceae strains, more drug resistance were seen to co-trimoxazole (100%) followed by gentamicin (63.85%) and ciprofloxacin (45.83%). All these ESBL Enterobacteriaceae strains were sensitive to Amikacin (75.61%) and Imipenem (100%). Antibiotic Resistance pattern of ESBLs producing organisms is depicted in Table-

DISCUSSION
In this recent time, extended-spectrum ß-lactamases (ESBLs) are main global crisis in the

CONCLUSION
The ESBL producing Enterobacteriaceae isolates arising worldwide as described in previous studies in India. Antimicrobial drug resistance is a major global problem of concern now a day. An indiscriminate use of the higher antibiotics and hospitalization are the major risk factors. The present study concludes the existence of CTX-M beta-lactamase gene in singly or various beta-lactamase gene combinations. The occurrence of CTX-M has an harmful effect on public health such as dispersion of hospital-acquired infections to the community 25 . Studies like this will be helpful to knowing the burden of multi drug resistance as well as formulating antibiotics policy for a particular region.

ACkNOWLEDgMENT
We are thankful to Dr. Shahid & Mrs. Anuradha Singh, department of Microbiology, Jawaharlal Nehru Medical College & Hospital, Aligarh Muslim University (AMU), U.P. (INDIA) for guidance and support.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.

FUNDINg
None.

AUTHORS' CONTRIBUTION
HS drafted the Concepts, manuscript, compiled information from the literature, Clinical studies, and designed the figures and tables. U supervised and reviewed the manuscript, Data Acquisition, Data analysis and Statistical Analysis. VR drafted the manuscript and gathered information from the literature. NN assisted for statistical analysis and Manuscript Editing. S help in data Analysis All the data pertaining to this study is available with principal investigator, Dr. Hitendra Singh. So it would be produced anytime if it is asked by the editor.

ethiCs stAteMeNts
The protocol was approved by the Ethical committee of government medical college, Haldwani, Uttarakhand under the reference number 141-IEC/01/13.