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SYNOPSIS PRSENTATION 13/06/2016

SYNOPSIS PRSENTATION 13/06/2016. Phenotypic and Genotypic Detection of Plasmid Mediated Quinolone Drug Resistance and Virulence Factors in ESBL Producing Klebsiella spp. From the Clinical Isolates. Candidate: Jitendra Chandra Devrari PhD Scholar Reg. No: 155/Jan 2015 Dept. of Microbiology

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SYNOPSIS PRSENTATION 13/06/2016

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  1. SYNOPSIS PRSENTATION13/06/2016

  2. Phenotypic and Genotypic Detection of Plasmid Mediated Quinolone Drug Resistance and Virulence Factors in ESBL Producing Klebsiella spp. From the Clinical Isolates

  3. Candidate: Jitendra Chandra Devrari PhD Scholar Reg. No: 155/Jan 2015 Dept. of Microbiology Yenepoya Medical College Mangalore. Guide: Dr. Vidya Pai Professor & Head Dept. of Microbiology Yenepoya Medical College Mangalore

  4. Contents: • Introduction • Review of literature • Social Relevance of study • Aim and Objectives • Material and Methods • Work plan • Performa of data collection • Statistical analysis • Time plan • Budget • References

  5. Abbreviation: • ESBL : Extended Spectrum Beta Lactamases • PMQR : Plasmid Mediated Quinolone Resistance • MHB : Muller Hinton Broth • MHA : Muller Hinton Agar • MIC : Minimum Inhibitory Concentration • MSHA : Mannose Sensitive Hemagglutination • MRHA : Mannose resistant hemagglutination • CLSI : Clinical Laboratory Standards Institute

  6. Introduction: • The Klebsiella species are gram negative, rod shaped, non-motile bacteria and belongs to the family Enterobacteriaceae that are found in the environment and also present as the normal gut flora in humans. • Genus Klebsiella subdivided into several species: K. pneumoniae, K. oxytoca,K. ozaenae, and K. rhinoscleromatis. (Podschun et al., 1998) • Klebsiella spp. causes opportunistic infections such as – • UTI • pneumonia • soft tissue infection • Septicemia and meningitis.(Yu VL et al., 2007)

  7. Contd… • Virulence factors (Dhingra KR et al., 2008, Foysal MJ et al., 2013)which are expressed by the organism such as- • Capsule • Fimbriae (pili) • Lipopolysaccharides • hypermucoviscosity, etc. • Fimbriae are non flageller, filamentous projection on the bacterial surface. It is a first step in the infectious process of microorganisms as it must come as close as possible to host mucosal surfaces and maintain this proximity by attaching to the host cell (adherence). • K. pneumoniae produces two major fimbrial adhesion organelles, type 1 and type 3 fimbriae. (Stahlhut SG et al., 2009)

  8. Contd… • MagA (mucoviscosity associated gene A) is chromosomal gene which plays an important rolein serious infection of Klebsiella such as septicemia,bacteremia, and pneumonia as well as lung and liverabscesses. • RmpA (regulator of the mucoid phenotype) rmpA/rmpA2 is responsible for regulating the synthesis of the extracellular polysaccharide capsule. • A new virulent hypermucoviscous  strains associated with magA and rmpA genes have mainly emerged in Asia. (Cubero M et al., 2015)

  9. Contd… • Agents with high intrinsic activity against K pneumoniae should be selected for severely ill patients such agents include third-generation cephalosporins, carbapenem, aminoglycoside and quinolone. (Weisenberg SA et al., 2009) • Resistance against quinolone group of drugs, widely used for their treatment is mainly acquired by intra or inter species exchange of transferable plasmid encoded antibiotic resistant genes QNR gene, aac(6’)Ib-cr, qepA gene. (Oktem IMA et al., 2008) • Quinolone resistance is usually caused by mutations in the chromosomal genes for DNA gyrase and DNA topoisomerase IV, the targets of quinolone. (Drlica K et al., 1997; Tran JH et al., 2002)

  10. Contd… • AAC(6’)Ib–cr gene a new Plasmid mediated drug resistance mechanism has been identified the based on enzymatic modification of some fluoroquinolones drugs (norfloxacin and ciprofloxacin). (Ruiz E et al., 2002) • QepA described flouroquinolone specific efflux pump protein. It is a new plasmid-mediated gene responsible for reduced FQ susceptibility such as ciprofloxacin, norfloxacin, and enrofloxacin. (Perichon B et al., 2007)

  11. Figure 1: Plasmid mediated drug resistance/ image https://www.google.co.in

  12. Review of Literature: • Klebsiella is emerging opportunity pathogens which is responsible for many infections like UTI, pneumonia, bacteremia, septicemia, soft tissue infection, which is considered as classical Klebsiella pneumoniae (cKP). • Another variant of Klebsiella spp. is emerging as a most dangerous pathogenous bread as hypervirulent Klebsiella pneumoniae (hvKP) which is responsible for pyogenic liver abscess, endophthalmitis, splenic abscess, meningitis. (Liu YM et al., 2014; Shon AS et al., 2013) • A study done in Taiwan showed that prevalence of rmpA and magA were 48% and 17% respectively. magA and rmpA gene are present most commonly in pyogenic liver abscess, endophthalmitis and pneumonia than patients without hypermucoviscosity. (Yu WL et al., 2006)

  13. Contd… • A study done in Iran showed prevalence of hypermucoviscosity (HV), out of 173 samples, 73 (42.19%) were positive for HV test and 100 (57.80%) were negative. (Amraie H et al., 2014) • ESBLs are able to hydrolyze oxyimino cephalosporins and generally inhibited by beta lactamase inhibitors like clavulanic acid, sulbactam, and tazobactam. Existence of ESBLs and quinolone resistance in the K. pneumoniae infection suggests that care should be taken for the choice of antibiotic therapy. (Raei F et al.,2014) • Plasmid Mediated Quinolone Resistant (PMQR) genes like qnr, aac(6)ib-cr, qepA produces drug resistance in ESBL producing E. coli and K. pneumoniae infections worldwide. (Seo MR et al, 2010; Limoncu MH et al., 2012)

  14. Contd.. • It has been proved that PMQR genes are associated with therapy failure in patients and also Co-transmission of PMQR, ESBLs, and plasmid-mediated AmpC genes are responsible for the production of multidrug resistance in the family Enterobacteriaceae. (Yang H et al., 2008; Lin CJ et al., 2012) • QepA gene along with the qnr family and aac(6’)Ib-cr, is the third recently detected plasmid-borne determinant of resistance to the fluoroquinolones. • Although these genes are usually responsible for low-level of resistance, but due to the mutational alteration these genes could present with the high level of resistance. (Perchon B et al., 2007)

  15. Contd… • Majority of the community isolates of K. pneumonia carried qnr and aac (6’)-Ib-cr genes which are responsible for quinolone and cephalosporin resistance. (Sayedpur MS et al., 2014) National Scenario: • Higher rate of ESBLs producing Klebsiella spp. were found to be associated with the urinary tract infection compared to the other clinical sites. Out of 233 isolates, 26.6% were ESBL producers, in that 38.5% Klebsiella spp were ESBL produced. (Khurana S et al , 2002) • A total of 173 (64%) of the isolates were found to be ESBL positive and 61 (23%) showed resistant to cefoxitin. ESBL was detected in 80 (62%) isolates of E. coli and 71 (73%) of Klebsiella spp. (Shingal S et al., 2005)

  16. Contd… • A study from Kurnool on the prevalence of ESBL-producing Klebsiella pneumoniae showed thatout of 100 isolates, 39% were resistant to 3rd-generation cephalosporins, in that 17 were ESBL producers. (Sarojamma V et al., 2011) • According to a study on ESBL producing Gram negative bacilli (GNB), out of 722 GNB isolates, 379 (52.49%) were ESBL producers, out of these 67% were Klebsiella species. In this study it was found that the presence of genes responsible for ESBL production was bla-CTX-M (82.5%), followed by bla-TEM (67.5%) and bla-SHV (57.5%). (Sharma M et al., 2013) • However, another study done in Amravati showed that among 100 ESBL positive Klebsiella strains, K. pneumoniae were 86%, K. oxytoca 13% and K. ozanae 1%. (Thosar MG et al., 2014)

  17. Contd… • These studies on frequency of ESBL producing strains among clinical isolates have been steadily increasing. • A study done in Kolkata showed out of 73 K. pneumoniae isolates, 97 % were resistant for beta lactam and 76.7% were quinolone drugs respectively. QnrA gene was detected in 37 % and QnrB gene in 56 % of the Klebsiella isolates. QnrS was not present in any of the samples. (Tripathi A et al., 2012) • In a study from South India prevalence of PMQR mediated by qnrA and qnrB, aac(6’)–1b-crmutant gene showed multi drug-resistant K. pneumoniae isolates. Out of 23 ESBL isolates, qnr genes was found to be significant for qnrA (70%), qnrB (48%) and qnrSwas absent and 57% aac(6’)–1b-crgene was observed. (Magesh H et al., 2011)

  18. Contd… • According to a study done in JIPMER Pondicherry, all PMQR positive strain screened for the presence of qnr A, B, D, S and aac(6’)-ib cr. Their result showed that the highest prevalence at 64% while, qnrB and qnrS genes were present in 15% and 10% of the isolates respectively. None of the strains were positive for qnrA and qnrD. (Yugendran T et al., 2016) • Moreover, the majority of the literatures on qnr gene are on prevalence rates from around the world and reports on mechanism of drug resistance at the molecular level are very few. Future research should focus more the molecular mechanism of the PMQR genes and its encoded proteins.

  19. Social Relevance: • After this study will be able to treat the patients in a better & proper way in our hospital setup and with the effective drug regimen we can reduce the spreading of drug resistance in our locality. • This study will help the clinicians in the proper selection of antibiotics. • After this prospective study will be knowing the common virulence pathogens which is responsible for Klebsiella infection along with the genes which are commonly responsible for drug resistance of Klebsiella infection.

  20. Aim: This study aimed to detect virulence genes and the most common drug resistance mechanism in ESBL producing Klebsiella spp. from the clinical isolates.

  21. Objective: • To isolate the ESBL producing Klebsiella spp. from the clinical samples and phenotypically detect the fluoroquinolone drug resistance in clinical isolates by Kirby Bauer disk diffusion method. • To detect the MIC of fluoroquinolone drugs by Agar dilution method. • To determine the frequency of hypervirulent Klebsiella spp. in comparison with classical Klebsiella spp. from clinical isolates.

  22. Contd.. 4. Phenotypically detect the sensitivity of type1 fimbriae by hemagglutination assay. 5. To detect the biofilm production in Klebsiella spp. by Microtitre plate method. 6. To detect the presence of magA, rmpA, qnr (A and B), qepA and aac(6)-Ib-cr genes among fluoroquinolone resistant Klebsiella isolates.

  23. Material & Method: • Research Design: Prospective study • Study Setting: Klebsiella spp. are collected from Yenepoya Medical Hospital, Mangalore. • Ethical clearance: Will be obtained from the Yenepoya University, Mangalore.

  24. Sample size: The sample size is calculated using the “G* Power Software, version 3.1.9.2” with the effective size of 10%. Where, Population proportion, P0 =76% (According to reference article) Sample proportion, Pα = 85% Z value (fixed) = 1.96 Power (1-β) = 80% Level of significance (α) = 5% Sample size = 123 ESBL producing Klebsiella species.

  25. Methodology: Sampling Criteria: • Inclusion Criteria: All Klebsiella spp. isolates from bacteriology laboratory are included. • Exclusion Criteria: Other genera of Enterobacteriaceae will be excluded. Sample Collection: • Klebsiella spp. will be collected from the Microbiology laboratory of Yenepoya Medical College Hospital. (The specimens will be delinked from the patients).

  26. PHENOTYPIC DETECTION METHOD : • ESBL detection method :(CLSI guidelines 2016) By combination disc diffusion test (CDDT): • CDDT will be process by using both ceftazidime (30 𝜇g) disc alone and in combination with clavulanic acid (30 𝜇g/10 𝜇g). Discs will be placed 25mm apart from each other. • A positive result will be indicated by an inhibition zone size difference of ≥ 5 mm between the combination disc compare with the cephalosporin disc only. • Antibiotic susceptibility testing:for Quinolone antibiotics By Kirby-Bauer disk-diffusion method as per CLSI guidelines. • Antibiotic disks required: Nalidixic acid (30µg), Ciprofloxacin (5µg), Sparfloxacin (5µg) and Moxifloxacin (5µg).

  27. Kirby Bauer Disc diffusion method: (CLSI guidelines 2016) Allow the Muller Hinton Agar(MHA) plate and disc cartriadge to come to room temperature before use. Prepare the test inoculum and compare with the 0.5 McFarland standard . Lawn culture the bacterial suspension evenly in 3 planes onto the surface of the MHA plate, using a cotton swab. Rim the edge of the plate. Place the antibiotics discs over the MHA plate with the help of sterile forcep.

  28. Contd… Press the discs to make a firm attachment between the discs and the agar. Incubate the MHA plate overnight at 35⁰C. Check the zone of inhibition and interpretate the result of individual antibiotic disc as Sensitive (S), Intermediate (I) and resistant (R).

  29. Figure 2: Antibiotic sensitivity test https://www.google.co.in

  30. MIC Detection method (Agar Dilution):(CLSI guidelines 2012, M07-A9) Bacterial inoculation: • Take 3-4 identical bacterial colonies and inoculate into Muller Hinton Broth (MHB). • Incubate at 35˚C for 4-6 hours. • Adjust the turbidity of MHB broth to 0.5 McFarland standard. Stock solution: • Prepare intermediate antimicrobial agent solutions by making successive dilutions using the dilution format described in M1002 Table 6A. Then, add one part of the antimicrobial solution to nine parts of molten agar.

  31. Agar Dilution Plates technique for MIC: Arrange the tubes containing the adjusted and diluted bacterial suspensions (107 CFU/ml) in order in a rack. Mark the agar plates for orientation of the inoculum spots. Apply an aliquot of each inoculum to the agar surface with standardized loops (with 1 mm pins that delivers 0.1 to 0.2 μl). Inoculate a growth-control plate first (no antimicrobial agent) and then starting with the lowest concentration, inoculate the plates containing the different antimicrobial concentrations.

  32. Contd… Allow the inoculated plates to stand at room temperature until the moisture in the inoculum spots has been absorbed into the agar, i.e. until the spots are dry, but no more than 30 minutes. Invert the plates and incubate at 35±2°C for 16 to 20 hours. After incubation Place the plates on a dark, nonreflecting surface to determine the MIC. Record the MIC as the lowest concentration of antimicrobial agent that completely inhibits growth, disregarding a single colony or a faint haze caused by the inoculum.

  33. Hemagglutination Assays:(Duguid JP et al., 1979) • The hemagglutination test will be carried out on sterile microtitre plate. • 100 µl of the bacterial suspension will be added to each well, followed by an equal volume of a 3% suspension of erythrocyte in PBS. Wells containing only suspension of erythrocytes will be utilized as a negative control. Plate will be incubate at 4˚C for 1 hour. • The presence of small pallet of RBCs at the bottom after incubation will be consider as negative result while that containing an even sheet of RBCs across the well be consider as positive.

  34. Contd.. • Mannose sensitive hemagglutination will be detected by the absence of hemagglutination in a parallel set of test in which a drop of 2% D-mannose will be added to the red cells and a drop of broth culture. • Mannose resistance hemagglutination will be detected by the presence of hemagglutination of 3% ‘O’ group human RBCs in the presence 2% D-mannose.

  35. Hypermucoviscosity Test (HV): (Shon AS et al., 2013) K. pneumoniae isolates will be separated and cultivate on bloodagar medium (containing 5% sheep blood) and incubate at 37°C for 24 hrs. After this step, isolates will be investigate for HV using standard bacteriological loops through the bacterial colony. Colonies which will drawn ˃ 5 mm will be consider as positive.

  36. Biofilm Formation assay(Microtitre plate method):(Ghellai L et al., 2014) Isolates from fresh agar plates will be inoculate in Brain heart infusion with 1% glucose broth and incubate for 24 hour at 37˚C and dilute 1 in 20 with fresh medium. Individual sterile polystyrene 96 well flat bottom tissue culture plates will be filled with 200 µl aliquots of the diluted cultures and only broth serve as control. Tissue culture plates will be incubating for 24 hours at 37°C. After incubation each well will be removed by tapping the plates. Wells will be washed four times with 200 µl of phosphate buffer saline (PBS pH 7.2).

  37. Contd… Biofilm formation will be fixe with sodium acetate (2%) and stained with crystal violet (0.1% w/v) for 15 min. Excess stain will be rinse off by thorough washing with deionized water and plates were kept for drying. Adherent bacterial cells usually formed biofilm on all side wells and will be uniformly stained with crystal violet. Optical density (OD) of stained adherent bacteria will be determined with a micro ELISA auto reader at wavelength of 630 nm (OD 630 nm).

  38. GENOTYPIC DETECTION METHOD: Molecular Diagnosis: • DNA extraction: (Zamani A et al., 2007) Single colony will be taken from overnight growth culture and emulsified into 100 μl of phosphate buffer solution Prior to DNA extraction centrifuge all samples at 4500 rpm for 6 minutes Suspend pellets in 1 ml of 0.01 M TRIS HCL pH 7.4 Briefly treat samples with 50 μl proteinase K (100 mg/ml) and 150 μl of TE (1 mM EDTA/10 mM Tris, pH 7.5). The DNA extraction will be performed according to the manufacturer’s instructions

  39. Multiplex PCR: (Compain F et al., 2014) • The multiplex PCR will be performed using the following oligonucleotide as primers for drug resistant genes (qnr, aac(6)Ib-cr, qepA and magA , rmp). • The reaction will be performed under different conditions; 0.2 mM of each primer will be used. • Multiplex PCR will be carried out in a 25-µl volume using the Qiagen multiplex PCR kit, according to the manufacturer’s instructions. • After the thermal cycles the amplicons will be detected with the help of PCR Machine.

  40. Work Plan: Selection of Klebsiella species from all clinical isolates ESBL Detection (123 isolates) Classification as classical Detection of fimbriae Detection of biofilm AST for and hypervirulent type (Hemagglutination formation assay Quinolone (String test) assay) (Microtitre plate drugs method) Detection of magA and andrmp gene Resistant to Quinolone Sensitive to Quinolone (Multiplex PCR) drugs drugs

  41. Contd… Resistant to Quinolone drugs Check the MIC for Quinolone Detection of gene for drug resistant Quinolone drug resistant (Agar Dilution technique) (Multiplex PCR)

  42. Statistical analysis: • Data will be analyzed with the help of SPSS version 22. • Descriptive statistics like frequency (%) will be used. Chi square test will be used to find the association between two categorical variables. • P value is ˂ 0.05 will be considered statistically significant.

  43. TIME PLAN: TIMELINE

  44. Budget Requirements:

  45. References: 1. Podschun R and Ullmann U. Klebsiella spp. as Nosocomial Pathogens: Epidemiology, Taxonomy, Typing Methods, and Pathogenicity Factors. Clinical Microbiol Reviews. 1998; 11:589-603. 2. Yu VL, Hansen DS, Ko WC, Sagnimeni A, Klugman KP, Von Gottberg A et al., Virulence Characteristics of Klebsiella and Clinical Manifestations of K. pneumonia Bloodstream Infections. Emerging Infectious Disease. 2007; 13:986-93. 3. Dhingra KR. A Case of Complicated Urinary Tract Infection: Klebsiella pneumonia Emphysematous Cystitis Presenting as Abdominal Pain in the Emergency Department. West J Emerg Med. 2008; 9(3):171-73. 4. Fosyal MJ, Rahman MM and Prodhan MS. PCR Based Molecular Detection of the Gyr-B-2 Gene from the KlebsiellaSp. Isolated from patients who were suffering with Pneumonia and Urinary Tract Infection. Journal of clin and Dig Research. 2013; 7(1):23-25. 5. Cubero Ma, Grau I, Tubau F, Pallares R, Dominguez MA, Linares J et al., Hypervirulent Klebsiella pneumoniae clones causing bacteraemia in adultsin a teaching hospital in Barcelona, Spain. Clinical Microbiology and Infection. 2015. 6. Gerlach GF, Clegg S, and Allen BL. Identification and characterization of the genes encoding the type 3 and type 1 fimbrial adhesins of Klebsiella pneumoniae. Journal of Bacteriology. 1989; 171:1262-1270. 7. Ottow JC. Ecology, physiology, and genetics of fimbriae and pili. Annu. Rev. Microbiol. 1975: 29:79-108.

  46. Contd… 8. Stahlhut SG, Chattopadhyay S, Struve C, Weissman SJ, Aprikian P, Libby SJ et al., Population Variability of the FimH Type 1 Fimbrial Adhesin in Klebsiella pneumonia. Journal of Bacteriology. 2009; 191(6):1941-1950.Mulvey MA, Lopez-Boado YS, Wilson CL, Roth R, Parks WC, Heuser J et al., Induction and evasion of host defenses by type 1-piliated uropathogenicEscherichia coli. Science. 1999; 282:1494-1497. 10. Weisenberg SA, Morgan DJ, Espinal-Witter R and Larone DH, Clinical outcomes of patients with KPC-producing Klebsiella pneumoniae following treatment with imipenem or meropenem. DiagnMicrobiol Infect Dis. 2009; 64(2): 233–235. 11. Taneja N, Chatterjee SS, Singh M, Singh S and Sharma M. Pediatric urinary tract infections in a tertiary care center from north India. Ind J Med Res. 2010; 131:101-105. 12. Oktem IMA, Gulay Z and Bicmen M and Gur D. Qnr prevalence in extended spectrum beta lactamase-positive enterobacteriaceae isolates from Turkey. Jpn J Infect Dis. 2008; 61:13-17. 13. Drlica K and X Zhao. DNA gyrase, topoisomerase IV, and the 4-quinolones. Microbiol. Mol. Biol. Rev. 1997; 61: 377-392. 14. Tran JH and Jacoby GA. Mechanism of plasmid-mediated quinolone resistance. Proceeding of the National Academy of Science. 2002; 99:5638-5642. 15. Cavaco LM and Aarestrup FM. Evaluation of Quinolones for Use in Detection of Determinants ofAcquired Quinolone Resistance, Including the New TransmissibleResistance Mechanisms qnrA, qnrB, qnrS, and aac(6’)Ib-cr, in Escherichia coli and Salmonella entericaand Determinations of Wild-Type Distributions. Journal of clinical Microbiology. 2009; 47(9):2751-2758.

  47. Contd… 16. Kunikazu Y, Wachino JI, Suzuki S, and Yoshichika A. Plasmid-Mediated qepA Gene among Escherichia coli Clinical Isolates from Japan. Antimicrobial Agents and Chemotherapy. 2008; 52(4):1564-1566. 17. Liu YM, Li BB, Zhang YY, Zhang W, Shen H, Li H et al., Clinical and Molecular Characteristics of Emerging Hypervirulent Klebsiella pneumoniae Bloodstream Infections in Mainland China. Antimicrobial Agents and Chemotherapy. 2014; 58 (9):5379-5385. 18. Shon AS, BajwaRajinder PS and Russo TA. Hypervirulent (hypermucoviscous) Klebsiella pneumoniae a new and dangerous breed. Virulence. 2013; 4(2):107-118. 19. Yu WL, Ko WC, Cheng KC, Lee HC, Ke DS, Lee CC et al., Association between rmpA and magA genes and clinical syndromes caused by Klebsiella pneumoniae in Taiwan. Clinical Infectious Disease. 2006; 42:1351-1358. 20. Amraie H, Shakib P, Rouhi S, Bakhshande N and Zamanzad B. Prevalence assessment of magA gene and antimicrobial susceptibility of Klebsiella pneumoniae isolated from clinical specimens in Shahrekord, Iran. Iranial journal of microbiology. 2014; 6(5):311-316. 21. Rosen DA, Pinkner JS, Walker JN, Elam JS, Jones JM, and Hultgren SJ. Molecular Variations in Klebsiella pneumoniae and Escherichia coli FimH Affect Function and Pathogenesis in the Urinary Tract. Infection and Immunity. 2008; 76(7): 3346-3356. 22. Raei F, Eftekhar F and Feizabadi MM. Prevalence of Quinolone Resistance Among Extended-Spectrum β -Lactamase Producing UropathogenicKlebsiella pneumoniae. Jundishapur Journal of Microbiology. 2014; 7(6):1-5.

  48. Contd… 23. Winn WC. In: Koneman’s color atlas and textbook of diagnostic Microbiology.6th ed. Philadelphia. Lippincott Williams and Wilkins; 2006. 24. Shah RK, Singh YI, Sanjana RK, Chaudhary N and Saldanha D. Study of extended spectrum β-lactamases (ESBLs) producing Klebsiella species in various clinical specimens: A preliminary report. Journal of College of Medical Sciences Nepal. 2010; 6(3): 19-23. 25. Tribuddharat C, Srifuengfung S and Chiangjong W. A Correlation between Phenotypes and Genotypes of Extended-Spectrum Beta-Lactamase (ESBL) Producing Klebsiella pneumoniae in a University Hospital, Thailand. Journal Infectious Disease Antimicrobial Agents. 2007; 24:117-23. 26. Yang J, Luo Y, Chi S, Wang W, and Han L. Diverse Phenotypic and Genotypic Characterization Among Clinical Klebsiella pneumoniae and Escherichia coli Isolates Carrying Plasmid-Mediated Quinolone Resistance Determinants. Microbial Drug Resistance. 2011; 17(3): 363-367. 27. Seo MR, Park YS and Pai H. Characteristics of Plasmid-Mediated Quinolone Resistance Genes in Extended-Spectrum Cephalosporin-Resistant Isolates of K. pneumoniae and Escherichia coli in Korea. Chemotherapy. 2010; 56: 46-53. 28. Limoncu MH, Erac B, Yurtman AN and Aydemir S. Plasmid mediated quinolone resistance mechanisms in ESBL positive Escherichia coli and Klebsiella pneumoniae strains at a Tertiary-Care Hospital in Turkey. Journal of Chemotherapy. 2012; 24(3): 144-149. 29. Yang H, Chen H, Yang Q, Chen M and Wang H. High Prevalence of Plasmid-Mediated Quinolone Resistance Genes qnr and aac(6)-Ib-cr in Clinical Isolates of Enterobacteriaceae from Nine Teaching Hospitals in China. Antimicrobial Agents and Chemotherapy. 2008; 52(12): 4268-4273.

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