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Identification of Clonal Clusters of Klebsiella Pneumoniae Isolates

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Identification of Clonal Clusters of Klebsiella Pneumoniae Isolates American Journal of Infectious Diseases 5 (2): 74-82, 2009 ISSN 1553-6203 © 2009 Science Publications Identification of Clonal Clusters of Klebsiella pneumoniae Isolates from Chennai by Extended Spectrum Beta Lactamase Genotyping and Antibiotic Resistance Phenotyping Analysis 1C. Kamatchi, 1H. Magesh, 2Uma Sekhar and 1Rama Vaidyanathan 1Department of Industrial Biotechnology, Dr. M.G.R. Educational and Research Institute, E.V.R. Periyar Salai, Maduravoyal, Chennai-600 095, Tamil Nadu, India 2Department of Microbiology, Sri Ramachandra University, Porur, Chennai Abstract: Problem statement: An increased resistance to antibiotics has been reported in Klebsiella pneumoniae , an opportunistic gram negative pathogen belonging to Entrobacteraceae due to the evolution and spread of plasmid encoded extended spectrum beta lactamases and other genes conferring cross-resistance to other antibiotics. This is of concern due to the increasing cost of antibiotic treatment and the spread of multidrug resistance to more pathogenic microorganisms. This study was undertaken to analyze the extent of the problem, to identify the most prevalent MDR isolates of Klebsiella pneumoniae in Chennai and to identify new plant based drugs. Approach: About 188 clinical isolates of Klebsiella pneumoniae were collected from Chennai during the period Nov. 2007- Oct. 2008 and their resistance to different groups of antibiotics were analyzed. These isolates were further characterized by molecular studies to identify the ESBL genes conferring the resistance phenotype. Plant extracts were tested against the MDR isolates to identify new treatment methods. Results: Our results showed that the combination therapy of clavulanic acid with cephalosporins and fluroroquinolones-norfloxacin and ciprofloxacin were the most effective antibiotics for treatment of Klebsiella pneumoniae infections. However resistance to clavulanic acid was increasing among the isolates (17%). All the isolates harbored a plasmid containing one or more of the genes for ESBL resistance. Plasmid isolation from the Isolates followed by ESBL PCR genotyping showed that CTX- M was present in 75 % isolates and TEM in 73% isolates either alone or in combination with the other ESBL genes. SHV ESBL type was present only in 42% of isolates. We identified 4 presumptive clonal spreads which were likely to be prevalent in India by clustering based on ESBL genotypic and antibiotic resistance. The isolates with both SHV and CTX were correlated with the most pathogenic and clavulanic acid resistant isolates. The extracts of Hemidesmus indicus and Terminalia arjuna had a significant antimicrobial activity against the MDR isolates. Conclusion: There was an increase in the spread of the ESBL genes and clavulanic acid resistance in the clinical isolates of Klebsiella pneumoniae in Chennai. Our data also supported a higher incidence of CTX-M genotype in India though the SHV genotype was associated with the most resistant forms of ESBL. Future research with separation of plant extracts to identify bioactive principles will pave the way for newer plant based antibiotics. Key words: Klebsiella pneumoniae , ESBL, TEM, SHV, CTX-M, multi-drug resistant, Hemidesmus indicus , Terminalia arjuna INTRODUCTION spread worldwide and pose a serious threat for health care-associated infections[1] . Increasingly the ESBL Klebsiella pneumoniae is an opportunistic Klebsiella pneumoniae are also showing co-resistance pathogen that causes a significant proportion of to quinolones and aminoglycoside antibiotics [5,6] present hospital-acquired urinary tract infections, pneumonia, in plasmids which can be transferred by conjugation to septicemias and soft tissue infections [1] . Studies have other strains. shown that Extended-Spectrum Beta-Lactamase The resistance to antibiotics can be of the (ESBL)-producing Klebsiella pneumoniae have rapidly following types (1) Drug inactivation by degradation or Corresponding Author: Rama Vaidyanathan, Department of Industrial Biotechnology, Dr. M.G.R. Educational and Research Institute, E.V.R. Periyar Salai, Maduravoyal, Chennai-600 095, Tamil Nadu, India 74 Am. J. Infect. Dis., 5 (2): 74-82, 2009 modification enzymes such as beta lactamaces and family that protects DNA gyrase from quinolone aminoglycoside transferases, (2) Alteration of the drug inhibition has also been reported [9] . target (3) Emergence of a bypass pathway not inhibited Other groups of antibiotics that is used is amikacin, by the drug (4) Reduced membrane permeability for the which inhibits protein synthesis by binding to the 30S drug (5) Drug efflux from the cells. Resistance due to ribosomal subunit to prevent the formation of an drug efflux can result in multi-drug resistance. These initiation complex with messenger RNA. Amikacin genes can be present in the chromosome or plasmid resistance in Klebsiella is due to the presence of aac within integrons which help in horizontal transfer of the (6')-Ib gene which encodes many variants of an aminoglycoside-acetyltransferase enzyme first cloned antibiotic resistance. [2] Beta lactam antibiotics disrupt the synthesis of the from pMET1 an MDR plasmid in Klebsiella . peptidoglycan layer of bacterial cell walls by mimicing Trimethoprim is an antimetabolite antibiotic which a portion of the bacterial peptidoglycan interfering with binds to dihydrofolate reductase and inhibit formation of tetrahydrofolic acid. Trimethoprim rsistance is the action of the transpeptidase enzymes. Continued use mediated by a mutant dydrofolate reductase gene which of cephalosporins have resulted in the emergence of is insensitive to Trimethoprim. resistance. The most common mechanism of resistance These antibiotic resistance genes present within to beta lactam antibiotics is the production of integrons giving a selective advantage to these isolates chromosomal or plasmid encoded beta-lactamase enzyme that catalyze the hydrolysis of beta-lactam C-N leading to the clonal spread these highly resistant bond of the antibiotics to give the corresponding beta- pathogens. In addition the integorns can also capture amino acid devoid of antibacterial activity [7] . Extended more resistance determinants. Therefore it is very Spectrum Beta Lactamases (ESBLs) are a group of important to identify the prevalent clones of resistant enzymes that can hydrolyze the isolates and characterize the plasmids present in the oxyiminocephalosporins such as cefotaxime, antibiotic resistant isolates. Regular monitoring of ceftazidime, cefepime). There are more than 200 antibiotic resistance patterns are essential in deciding subtypes of ESBL which are believed to have evolved the effective antibiotics for healthcare regimes. from a few common ancestral types. The most common ESBL enzymes belong to TEM, SHV, CTX-M and MATERIALS AND METHODS OXA types. From these ESBL enzymes, amino acid substitutions have lead to high spectrum of ESBL Bacterial isolates: One hundred eighty eight clinical subtypes with changes in isoelectric pH and substrate isolates of K lebsiella pneumonia collected from tertiary specificity. The ESBL genes occur in plasmids within care hospitals during Nov. 2007-Oct. 2008 and were integrons which aid in capturing genes conferring subjected to routine culture and antibiotic susceptibility resistance to other antibioitcs. testing. Each isolate was stored as a glycerol stock in Fluoroquinolones are powerful broad-spectrum the-20°C. The isolates of Klebsiella pneumonia were antimicrobial agents used for the treatment of a wide collected from different sources-urine-66 sputum and variety of community-acquired and nosocomial pus 115 and blood-7. infections including ESBL infections. The prototype Antibiotic susceptibility test: Antibiotic susceptibility quinolone is nalidixic acid and the other commonly [11] used fluroroquinolone derivatives include testing was performed according to standard methods ciprofloxacin, norfloxacin and levofloxacin. These on Mueller Hilton agar (Hi media).The antibiotics used −1 −1 chemicals inhibit topoisomerase II, a DNA gyrase (µg disk ) were ampicillin (30 µg mL ), piperacillin, and DNA topoisomerase IV to inhibit DNA Norfloxacin (10), Nalidixic acid (30), Ciprofloxacin replication. Fluoroquinolone resistance arises through (30), Levofloxacin (5), Chloramphenicol (30), specific mutations within the target proteins DNA Amikacin (30), Trimethoprim (30), Cefomandole (30), gyrase and topoisomerase IV, at position 83 or 87 Cefoperzone (75), Cefoxitin (30), Ceftazidime (30), −1 within DNA gyrase A and position 80 or 84 within Cefotaxime (30) and Amoxyclav (30 mcg disc ) were the ParC subunit of topoisomerase IV. Energy- used for isolates from samples collected. The results dependent efflux and porin loss have also been were interpreted as per the guidelines of the NCCLS [12] shown to confer a fluoroquinolone resistance phenotype control . in K. pneumoniae [8] . Plasmid-mediated resistance mechanisms to fluoroquinolone due to qnrA which Transconjugation experiments: Transconjugation encodes a 218 amino acid protein of the pentapeptide experiments were performed for all isolates which 75 Am. J. Infect. Dis., 5 (2): 74-82, 2009 showed resistant to amoxyclav. Transconjugation were sinensis, Holarrhena antidysentrica against ESBL [13] performed according to standard methods . Mating producing organisms. Plant extract were prepared as was performed with E. coli 1652 nalidixic acid resistant described by Ahmed et al .[16].Three hundred fifty grams as recipient strain.Transconjugation was selected on of dry
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