|Year : 2018 | Volume
| Issue : 2 | Page : 100-105
Dissemination of multidrug-resistant, Class I and II integrons and molecular typing of CTX-M-producing Klebsiella pneumoniae
Alisha Akya1, Azam Elahi2, Roya Chegenelorestani2, Mahya Rezaee2
1 Department of Medical Microbiology, Nosocomial Infection Research Centre, School of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran
2 Nosocomial Infection Research Centre, Faculty of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran
|Date of Submission||31-Aug-2016|
|Date of Acceptance||18-Jan-2018|
|Date of Web Publication||19-Apr-2018|
Ms. Azam Elahi
Department of Medical Microbiology School of Medicine, Kermanshah University of Medical Sciences, Shirudi Shahid Blvd, Daneshgah Street, 67148-69914, Kermanshah
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Introduction: Klebsiella pneumoniae (K. pneumoniae) is an important opportunistic pathogen causes serious community and hospital-acquired infections, which is highly resistant to antibiotics. We aimed to determine the frequency of multidrug resistant (MDR) and molecular typing of clinical isolates of K. pneumoniae. Methodology: One hundred isolates of K. pneumoniae were collected from clinical samples in three general hospitals in Kermanshah. The antimicrobial susceptibility and extended-spectrum beta-lactamases (ESBL) production of isolates were determined using disk diffusion and combined disk methods, respectively. The blaCTX-Mgene, class I and II integrons were detected using polymerase chain reaction. The blaCTX-Mpositive isolates were selected for genotyping using pulsed-field gel electrophoresis (PFGE). Results: MDR phenotype was observed in 56% of isolates. The 40% of isolates were ESBL positive and 35 isolates contained blaCTX-M. Class I and II of integrons were detected in 50 (89.2%) and 39 (69.6%) of MDR isolates, respectively. PFGE patterns of K. pneumoniae blaCTX-Mpositive isolates indicated 19 clusters (X1-19) with different genotype patterns. Conclusions: The study findings highlight the concern of circulating MDR strains of K. pneumoniae with blaCTX-Mand class I and II integrons in Kermanshah hospitals. The presence of integrons among isolates may facilitate the spread of new resistance genes in this bacterium. Therefore, surveillance for the spread of MDR strains of this bacterium is recommended in hospitals.
Keywords: Extended-spectrum beta-lactamases, integron, Klebsiella pneumoniae, pulsed-field gel electrophoresis
|How to cite this article:|
Akya A, Elahi A, Chegenelorestani R, Rezaee M. Dissemination of multidrug-resistant, Class I and II integrons and molecular typing of CTX-M-producing Klebsiella pneumoniae. Int J App Basic Med Res 2018;8:100-5
|How to cite this URL:|
Akya A, Elahi A, Chegenelorestani R, Rezaee M. Dissemination of multidrug-resistant, Class I and II integrons and molecular typing of CTX-M-producing Klebsiella pneumoniae. Int J App Basic Med Res [serial online] 2018 [cited 2019 Feb 21];8:100-5. Available from: http://www.ijabmr.org/text.asp?2018/8/2/100/230523
| Introduction|| |
Klebsiella pneumonia e (K. pneumoniae) is an important nosocomial pathogen with the potential of causing severe morbidity and mortality, particularly in intensive care units, surgical wards and among pediatric patients., Hospital-associated factors, including mechanical ventilation, catheterization, parenteral nutrition, and lengthy hospitalization have been identified as the risk factors for K. pneumoniae infections. This bacterium is one of the most prevalent agents of nosocomial infections with multidrug-resistant (MDR) characteristics.,
The role of integrons is very crucial in the spread and assemblage of resistant genes in pathogenic bacteria. The presence of integrons among Gram-negative bacteria has been increasingly reported worldwide. Integrons are genetic features that contain gene cassettes transferable to other mobile genetic elements such as plasmids in the bacterial genome. Over recent years and following the widespread use of the broad-spectrum beta-lactam antibiotics, outbreaks of infections caused by extended-spectrum beta-lactamase producing K. pneumoniae have been widely reported throughout the world.,, The resistance of this bacterium to third-generation cephalosporins was first described in 1983 and since then have been widely reported worldwide., The prevalence of extended-spectrum beta-lactamases (ESBLs) among bacteria is a serious alarm since the majority of them are multiresistant. The surveillance of local dissemination of resistant strains of bacteria, in particular among hospital environments has become an important epidemiological tool to control infection. Study the bacterial genotypic homology can provide a better understanding of sources and dissemination patterns of K. pneumoniae infections.
Various methods for bacterial genotyping have been developed using different molecular techniques. However, Pulsed-Field Gel Electrophoresis (PFGE) has been widely used as a standard method for K. pneumoniae typing., In this method, only relatively major genetic events can result in changes of fingerprinting patterns. Given the spread of multidrug resistance strains of K. pneumoniae in our region, study the Molecular typing of isolates can provide a better view of bacterial dissemination. We aimed to determine the frequency of MDR and molecular typing of clinical isolates of K. pneumonia e blaCTX-M positive.
| Methodology|| |
This descriptive cross-sectional study was performed on 100 available and nonduplicate isolates of K. pneumoniae. They were collected during 11 months (2012 and 2013) of an outbreak among hospitalized patients in three general hospitals (Imam Khomeini, Taleghani and Imam Reza) in Kermanshah. The isolates were from patients admitted in Kermanshah hospitals and no extra charges or procedures were imposed on the patients for this study. All relevant medical ethics were considered in this study.
All isolates were identified by bacteriological and biochemical tests  followed by confirmation using API 20 E Kit according to the manufacturer's instructions, and results were interpreted using API 20 E V4.1 identification software (biomerieux, France).
Antibiotic susceptibility test
Antimicrobial susceptibility testing for 15 antibiotics from nine different antibiotic categories was carried out by disk diffusion method on Mueller Hinton Agar (Merck, Germany) according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI). The antibiotics tested were ampicillin (10 μg), cefazolin (30 μg), gentamicin (10 μg), tobramycin (10 μg), cefotaxime (30 μg), ceftriaxone (30 μg), ceftazidime (30 μg), cefpodoxime (10 μg), azetronam (30 μg), ertapenem (10 μg), imipenem (10 μg), meropenem (10 μg), pipracilin-tazobactam (100/10 μg), ciprofloxacin (30 μg), and co-trimoxazole (30 μg) (Mast Group, UK). MDR was defined as acquired nonsusceptibility to at least one agent in three or more antimicrobial categories.
Extended-spectrum beta-lactamases Phenotypic confirmatory test
Phenotypic confirmatory test was performed using combination disk method according to the CLSI recommendations. In this method, cefotaxime (30 μg) and ceftazidime (30 μg) alone and in combination with clavulanic acid (10 μg) were used. If the inhibited zone diameter increased ≥5 mm for either antimicrobial agents in combination with clavulanic acid it was considered phenotypic positive for ESBL.K. pneumonia e ATCC 700603 was used as a positive control and E. coli ATCC 25922 was used as a negative control.
Polymerase chain reaction amplification
The bacterial genome was extracted by boiling method and used as DNA template for polymerase chain reaction (PCR). The DNA of ESBL producing isolates was targeted for the blaCTX-M, however PCR amplification of Class I and II integrons was carried out on DNA of MDR isolates using the specific primers (SinaClon, Iran) listed in [Table 1].,, The amplified products were visualized by ethidium bromide stained 1% agarose gel under ultraviolet (UV) transilluminator (Bio-Rad, USA).
Pulsed-field gel electrophoresis
The clonal relatedness between blaCTX-M positive isolates was investigated by pulsed-field gel electrophoresis. PFGE was carried out according to a previously described protocol with some modifications.K. pneumonia e isolates and Salmonella More Details enterica serovar Braenderup H9812 (As DNA marker) genome were digested with 20U of Xba I (Fermentas, Lithuania). After Xba I digestion of bacterial genomes, they were loaded into a 1% Low electroendoosmosis agarose (Merck, Germany). Electrophoresis was performed using a CHEF MAPPER apparatus (Bio-Rad, USA) at 14°C for 22 h. The following conditions were used for electrophoresis: initial switch time, 5 s; final switch time, 35 s; included angle, 120°; voltage gradient, 6 V/cm; ramping factor, linear. The gels were stained by ethidium bromide and visualized under UV light using Gel Doc apparatus (Bio-Rad, USA).
The DNA fragment patterns were analyzed using Gelcompar II version 6.6 software (Applied maths, Belgium). The Dice coefficient was used to calculate similarities, and the unweighted paired group method based on the average linkages was used for cluster analysis. A cluster of isolates was defined to include all isolates with ≥80% similarity of their DNA patterns according to the Tenover's criteria.
Data were recorded and entered into an Excel file. Statistical analyses were performed using SPSS software (version 16). Variables were analyzed using Chi-square test. A P< 0.05 was set as the statistical significance of all analyses. Simpson's Index of Diversity (D value) was calculated using equation.
| Results|| |
The clinical samples tested included urine (n = 54, 54%), burn (n = 15, 15%), respiratory tract secretions (n = 15, 15%), and others (blood, wound, and ascitic fluid) (n = 16, 16%). They were collected form 59 (59%) female and 41 (41%) male with the average age of 39.5 ± 2.26 years old.
The antibiotic resistance of isolates is presented in [Figure 1]. Resistance to most antibiotics except carbapenem was significantly higher in ESBL producing isolates (P = 0.001). MDR and ESBL production were 56% and 40%, respectively. Among isolates, the highest prevalence of ESBL was in ICU (35.9%) and burn ward (26.4%). Forty (71.4%) were MDR isolates which of them ESBL producer. Class I and II integrons were found in 89.2% and 69.6% MDR isolates, respectively. In 35 isolates (62.5%) both class I and II genes were present. The blaCTX-M was found in 35% of isolates. Gene cassette was detected at 100% of MDR isolates.
|Figure 1: Antibiotic susceptibility of nosocomial isolates of Klebsiella pneumoniae measured by disk diffusion|
Click here to view
A significant relationship was detected between blaCTX-M and Class II integron (P = 0.039). As well as a significant association was revealed between blaCTX-M with resistance to cefotaxime (P = 0.001), ceftriaxone (P = 0.001), ceftazidime (P = 0.005), cefpodoxime (P = 0.005), and aztreonam (P = 0.001).
According to the DNA fingerprinting of isolates, 19 clusters (X1 − 19) were differentiated which included 10 clones and 9 unique clusters [Figure 2] and [Table 2].
|Figure 2: Phylogenetic relationship among isolates of extended-spectrum beta-lactamases-producing strains of Klebsiella pneumoniae using pulsed-field gel electrophoresis data. The strains were clustered using UPGMA. CTX-M: blaCTX-Mgene, MDR: Multidrug Resistant, intI: Integron class 1, intII: Integron class 2|
Click here to view
|Table 2: The distribution of clones (with at least 2 isolates) among hospital wards|
Click here to view
The ICU isolates were from respiratory tract secretions and urine samples, and infectious ward were from wound and blood samples. Cluster numbers from X11 to X19 each one had a unique genotype and the majority of their strains (>50%) were from the infectious ward. The Simpson's diversity index for the isolates was 0.9603.
| Discussion|| |
The increasing prevalence of clinical MDR isolates has been associated with higher morbidity and mortality rates. The rate of MDR among K. pneumoniae isolates in the present study is similar to previous research results reported. However, resistance to carbapenems is still low and therefore, this group of antibiotics is effective against K. pneumoniae isolates.
Plasmid-mediated ESBLs have been found more frequently in K. pneumoniae strains than in other Enterobacteriaceae species. Integrons associated ESBL genes, in isolates of K. pneumoniae, suggesting that the genetic mobile structures harbouring them are widespread. Our results indicated that the rate of ESBL-producing isolates of K. pneumoniae was high which may reflect the dissemination of resistant genes in hospitals. This is consistent with the previous research results that showed the ESBL-producing isolates were more common among hospitalized patients more likely exposed to antimicrobial agents such as third generation cephalosporins. In Asia, the prevalence of ESBL-positive K. pneumoniae isolates varies within different regions. The ESBL frequency in neighboring countries of Iran varied from 31% to 85%. Although in most of the studies, the ESBL prevalence was higher than our study.,,,
blaCTX-M has been found to be widely disseminated among clinical Enterobacteriaceae such as Escherichia More Details coli and K. pneumoniae. The prevalence of CTX-M type producing K. pneumoniae in Asia is variable among countries. The higher prevalence of blaCTX-M among our isolates indicates the more dissemination of this class of ESBL in our region. We observed the production of ESBLs in about 71.4% of our MDR isolates and all isolates contained blaCTX-M were MDR which indicate the cluster of resistant genes. The rate of class I and II integrons in our isolates was consistent with the results of previous studies.,
Molecular typing indicated two distinct features among genotypic patterns of K. pneumoniae strains; first, the strains with genotypic diversity and the second strains with similar genotypes. The genotypic polymorphism can reflect the genetic diversity of isolates or the various origins of them. The presence of strains with a similar genotypic pattern in our isolates may show the dissemination of strains among hospital patients, in particular in intensive care and burn wards. The hospitalized patients such as burned patients are at increased risk of infection with various nosocomial pathogens  due to the destruction of the skin barrier, suppression of immune system, and invasive procedures. Strains with similar genotypic patterns from different hospitals may suggest the bacterial spread of patients transferring to different hospitals.
The calculated values for Simpson's diversity indicated the diversity of our strains. The D value is between 0 and 1, in which the 1 represents infinite diversity and 0 no diversity.
The genetic diversity of our isolates showed that most strains were genetically different, indicating the dissemination of resistant strains of K. pneumoniae. In the same way, several studies between 2008 and 2012 on K. pneumoniae isolates in Iran indicated the genotype diversity among isolates of this bacterium.,,,,, However, there are studies that have reported less diversity and more genotype similarity among isolates.,,, One explanation could be the fact that the most isolates in the above study were from limited sources in one hospital. The epidemiology of ESBL-producing K. pneumoniae is complex, and the genetic agents encoding the varied ESBL behave in different ways.
No distinct association was found between the resistant phenotypes and pulsotypes by cluster analysis of the 35 K. pneumoniae strains. As expected, there was no significant (P = 0.29) relation among strains in terms of association between genotypes with ESBL production and antibiotic resistance phenotypes. This issue can be explained by the fact that the most ESBL and antibiotic-resistant genes are carried on plasmids that are not mostly large enough to make a difference in PFGE patterns (40–50 kb).
| Conclusions|| |
The clonal diversity of isolates carrying resistance genes suggests the strain transmission may be responsible for the recent spread of K. pneumoniae infection in hospitals. The results of our study suggest dissemination of K. pneumoniae MDR strains with blaCTX-M and class I and II integrons in Kermanshah hospitals. Given the ability of integrons for spreading and expressing of newly acquired genes in bacteria, the high frequency of these gens in K. pneumoniae in our region is alarming. Therefore, it is crucial the spread of resistant genes, in particular, integrons, in K. pneumoniae regularly test and report in hospitals.
This work was performed in partial fulfillment of the requirements for Master's degree of Medical Microbiology of Azam Elahi, in Faculty of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran.. This work was funded by Kermanshah University of Medical Sciences, Kermanshah (Grant No. 92319).
Financial support and sponsorship
This work was financially supported by Kermanshah University of Medical Sciences, Kermanshah, Iran as the Master's degree thesis of Azam Elahi (Grant No. 92319).
Conflicts of interest
There are no conflicts of interest.
| References|| |
Branger C, Lesimple AL, Bruneau B, Berry P, Lambert-Zechovsky N. Long-term investigation of the clonal dissemination of Klebsiella pneumoniae
isolates producing extended-spectrum beta-lactamases in a university hospital. J Med Microbiol 1998;47:201-9.
Podschun R, Ullmann U. Klebsiella
spp. As nosocomial pathogens: Epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev 1998;11:589-603.
Peña C, Gudiol C, Tubau F, Saballs M, Pujol M, Dominguez MA, et al.
Risk-factors for acquisition of extended-spectrum beta-lactamase-producing Escherichia coli
among hospitalised patients. Clin Microbiol Infect 2006;12:279-84.
Biedenbach DJ, Moet GJ, Jones RN. Occurrence and antimicrobial resistance pattern comparisons among bloodstream infection isolates from the SENTRY Antimicrobial Surveillance Program (1997-2002). Diagn Microbiol Infect Dis 2004;50:59-69.
Blot S, Depuydt P, Vandewoude K, De Bacquer D. Measuring the impact of multidrug resistance in nosocomial infection. Curr Opin Infect Dis 2007;20:391-6.
Fluit AC, Schmitz FJ. Class 1 integrons, gene cassettes, mobility, and epidemiology. Eur J Clin Microbiol Infect Dis 1999;18:761-70.
Peña C, Pujol M, Ardanuy C, Ricart A, Pallares R, Liñares J, et al.
Epidemiology and successful control of a large outbreak due to Klebsiella pneumoniae
producing extended-spectrum beta-lactamases. Antimicrob Agents Chemother 1998;42:53-8.
Ducel G. Prevention of hospital-acquired infections with reference to burns. Burns Incl Therm Inj 1984;11:42-7.
Arlet G, Rouveau M, Casin I, Bouvet PJ, Lagrange PH, Philippon A, et al.
Molecular epidemiology of Klebsiella pneumoniae
strains that produce SHV-4 beta-lactamase and which were isolated in 14 French hospitals. J Clin Microbiol 1994;32:2553-8.
Li W, Raoult D, Fournier PE. Bacterial strain typing in the genomic era. FEMS Microbiol Rev 2009;33:892-916.
Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, et al.
Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: Criteria for bacterial strain typing. J Clin Microbiol 1995;33:2233-9.
Murray PR, Baron JL, Jorgensen JH, Landry ML, Pfaller MA, editors. Manual of Clinical Microbiology. Washington, D.C.: American Society for Microbiology; 2007.
Clinical and Laboratory Standards Institute/NCCLS (CLSI). Performance Standards for Antimicrobial Susceptibility Testing; Twenty-First Informational Supplement. Approveds Standard M100-S21. Vol. 31. Wayne, PA: Clinical and Laboratory Standards Institute; 2011.
Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al.
Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: An international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect 2012;18:268-81.
Freschi CR, Silva Carvalho LF, Oliveira CJ. Comparison of DNA-extraction methods and selective enrichment broths on the detection of Salmonella typhimurium
in swine feces by polymerase chain reaction (PCR). Braz J Microbiol 2005;36:363-7.
Edelstein M, Pimkin M, Palagin I, Edelstein I, Stratchounski L. Prevalence and molecular epidemiology of CTX-M extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae in Russian hospitals. Antimicrob Agents Chemother 2003;47:3724-32.
Upadhyay S, Hussain A, Mishra S, Maurya AP, Bhattacharjee A, Joshi SR. Genetic Environment of Plasmid Mediated CTX-M-15 Extended Spectrum Beta-Lactamases from Clinical and Food Borne Bacteria in North-Eastern India. PloS one 2015;10:e0138056.
Levesque C, Piche L, Larose C, Roy PH. PCR mapping of integrons reveals several novel combinations of resistance genes. Antimicrob Agents Chemother 1995;39:185-91.
Durmaz R, Otlu B, Koksal F, Hosoglu S, Ozturk R, Ersoy Y, et al
. The optimization of a rapid pulsed-field gel electrophoresis protocol for the typing of Acinetobacter baumannii, Escherichia coli and Klebsiella spp. Jpn J Infect Dis 2009;62:372-7.
Li B, Hu Y, Wang Q, Yi Y, Woo PC, Jing H, et al
. Structural diversity of class 1 integrons and their associated gene cassettes in Klebsiella pneumoniae isolates from a hospital in China. PLoS One 2013;8:e75805.
de Souza Lopes AC, Falcao Rodrigues J, de Morais Junior MA. Molecular typing of Klebsiella pneumoniae isolates from public hospitals in Recife, Brazil. Microbiological research 2005;160:37-46.
Graffunder EM, Preston KE, Evans AM, Venezia RA. Risk factors associated with extended-spectrum beta-lactamase-producing organisms at a tertiary care hospital. J Antimicrob Chemother 2005;56:139-45.
Feizabadi MM, Mahamadi-Yeganeh S, Mirsalehian A, Mirafshar SM, Mahboobi M, Nili F, et al
. Genetic characterization of ESBL producing strains of Klebsiella pneumoniae from Tehran hospitals. J Infect Dev Ctries 2010;4:609-15.
Tahanasab Z, Mobasherizadeh S, Moghadampour M, Rezaei A, Maleki N, Faghri J. High Prevalence of Multiple Drug Resistance among ESBLs-Producing Klebsiella pneumoniae Isolated from Hospitalized Patients in Isfahan, Iran. Journal of Medical Bacteriology 2016;5:29-38.
Sharif MR, Soltani B, Moravveji A, Erami M, Soltani N. Prevalence and Risk Factors associated with Extended Spectrum Beta Lactamase Producing Escherichia coli and Klebsiella pneumoniae Isolates in Hospitalized Patients in Kashan (Iran). Electronic physician 2016;8:2081-7.
Ghafourian S, Bin Sekawi Z, Sadeghifard N, Mohebi R, Kumari Neela V, Maleki A, et al
. The Prevalence of ESBLs Producing Klebsiella pneumoniae Isolates in Some Major Hospitals, Iran. Open Microbiol J 2011;5:91-5.
Canton R, Gonzalez-Alba JM, Galan JC. CTX-M Enzymes: Origin and Diffusion. Front Microbiol 2012;3:110.
Nematzadeh S, Shahcheraghi F, Feizabadi MM, Nikbin VS, Nasehi L. Molecular characterization of CTX-Mbeta-lactamases among Klebsiella pneumoniae isolated from patients at Tehran hospitals. Indian J Med Microbiol 2011;29:254-7.
Ahangarzadeh Rezaee M, Langarizadeh N, Aghazadeh M. First report of class 1 and class 2 integrons in multidrug-resistant Klebsiella pneumoniae isolates from northwest Iran. Jpn J Infect Dis 2012;65:256-9.
Rao AN, Barlow M, Clark LA, Boring JR, 3rd
, Tenover FC, McGowan JE, Jr. Class 1 integrons in resistant Escherichia coli and Klebsiella spp., US hospitals. Emerg Infect Dis 2006;12:1011-4.
Lari AR, Alaghehbandan R. The evaluation of nosocomial infection during 1-year-period in the burn unit of a training hospital in Istanbul, Turkey. Burns 2003;29:627.
Mayhall CG. The epidemiology of burn wound infections: then and now. Clin Infect Dis 2003;37:543-50.
Magurran AE. Meausuring Biological Diversity. oxford blackwell 2004.
Ashayeri-Panah M, Feizabadi MM, Eftekhar F. Correlation of Multi-drug Resistance, Integron and blaESBL Gene Carriage With Genetic Fingerprints of Extended-Spectrum beta-Lactamase Producing Klebsiella pneumoniae. Jundishapur J Microbiol 2014;7:e8747.
Ashayeri-Panah M, Eftekhar F, Ghamsari MM, Parvin M, Feizabadi MM. Genetic profiling of Klebsiella pneumoniae: comparison of pulsed field gel electrophoresis and random amplified polymorphic DNA. Braz J Microbiol 2013;44:823-8.
Lau YJ, Hu BS, Wu WL, Lin YH, Chang HY, Shi ZY. Identification of a major cluster of Klebsiella pneumoniae isolates from patients with liver abscess in Taiwan. J Clin Microbiol 2000;38:412-4.
Al-Marzooq F, Mohd Yusof MY, Tay ST. Molecular analysis of ciprofloxacin resistance mechanisms in Malaysian ESBL-producing Klebsiella pneumoniae isolates and development of mismatch amplification mutation assays (MAMA) for rapid detection of gyrA and parC mutations. Biomed Res Int 2014;2014:601630.
Tijet N, Sheth PM, Lastovetska O, Chung C, Patel SN, Melano RG. Molecular characterization of Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae in Ontario, Canada, 2008-2011. PLoS One 2014;9:e116421.
Dedeic-Ljubovic A, Hukic M, Pfeifer Y, Witte W, Padilla E, Lopez-Ramis I, et al
. Emergence of CTX-M-15 extended-spectrum beta-lactamase-producing Klebsiella pneumoniae isolates in Bosnia and Herzegovina. Clin Microbiol Infect 2010;16:152-6.
Christian NA, Roye-Green K, Smikle M. Molecular epidemiology of multidrug resistant extended spectrum beta-lactamase producing Klebsiella pneumoniae at a Jamaican hospital, 2000-2004. BMC microbiology 2010;10:27.
Mshana SE, Hain T, Domann E, Lyamuya EF, Chakraborty T, Imirzalioglu C. Predominance of Klebsiella pneumoniae ST14 carrying CTX-M-15 causing neonatal sepsis in Tanzania. BMC Infect Dis 2013;13:466.
Gouby A, Neuwirth C, Bourg G, Bouziges N, Carles-Nurit MJ, Despaux E, et al.
Epidemiological study by pulsed-field gel electrophoresis of an outbreak of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in a geriatric hospital. J Clin Microbiol 1994;32:301-5.
Birren BW, Lai E, Hood L, Simon MI. Pulsed field gel electrophoresis techniques for separating 1- to 50-kilobase DNA fragments. Anal Biochem 1989;177:282-6.
[Figure 1], [Figure 2]
[Table 1], [Table 2]