Despite the availability of newer antibiotics, emerging antimicrobial resistance has become an increasing problem in many pathogens throughout the world. Infections caused by resistant pathogens result in significant morbidity and mortality, and contribute to escalating healthcare costs worldwide. The spectrum of antibiotic resistance of pathogenic bacteria has constantly changed year after year because of the wide application of antimicrobial drugs .
The production of extended spectrum β-lactamases (ESBLs) in Enterobacteriaceae is the most prevalent resistance mechanism to third-generation cephalosporins . ESBL-producing organisms represent a real problem in hospitals due to difficulty in diagnosis, limited treatment options, and an infection control challenge . The most common ESBL-producing organisms are Klebsiella species and Escherichia coli . ESBL-producing E. coli and K. pneumoniae have rapidly spread worldwide and pose a serious threat for healthcare-associated infection . Plasmids responsible for ESBL production frequently carry genes encoding resistance to other drug classes . Thus, ESBL-producing isolates are usually resistant to multiple groups of antibiotics [7,8].
Periodic epidemiological surveys of etiological agents and their antibiotic sensitivity patterns are essential for recognition of the most frequently encountered pathogens in a particular healthcare setting. Establishing clonality of these pathogens can aid in diagnosing an outbreak and in the identification of the source of infection. The incorporation of molecular methods for typing of nosocomial pathogens has assisted in efforts to obtain a more fundamental assessment of strain interrelationship . Pulsed-field gel electrophoresis (PFGE) allows DNA fingerprinting of bacterial strains and is the ‘gold standard’ for bacterial molecular typing .
The aim of this study is the molecular characterization of ESBL-producing E. coli and K. pneumoniae isolated at our institute to generate data regarding the interrelationship of these isolates using PFGE.
Materials and methods
Of all the clinical specimens (5976 specimens) (3048 blood, 1296 urine, 924 sputum, 312 pus, 396 other types of specimens) submitted for bacterial culture at the Microbiology Laboratory of the Pediatric Teaching Hospital (Cairo University, Egypt) from January 2008 to March 2010, a total of 156 E. coli and 300 K. pneumoniae isolates were retrieved.
The isolated microorganisms were identified by standard microbiological techniques, including Gram staining, colony characteristics, and biochemical properties.
Detection of extended spectrum β-lactamases production
Screening for ESBL production was carried out using double disc synergy test  and confirmation was conducted by combined disc method  and E-test [cefotaxime/cefotaxime+clavulanic acid strip and ceftazidime/ceftazidime clavulanic acid (TZ/TZL) strip] (AB BIODISK 2000, 75001448-M916, Sweden) based on the inhibitory effect of clavulanic acid on ESBLs .
Study of the clonal relationship of the nosocomial ESBL-K and ESBL-E by
(1) Biotyping: It was performed using API20 system (Bio-Mérieux, Marcy l'Etoile, France) ;
(2) Antimicrobial susceptibility testing (AST): AST was performed by Kirby–Bauer disc diffusion method according to Clinical and Laboratory Standards Institute (CLSI) guidelines . All isolates were tested by the standard disk diffusion method against β-lactam and non-β-lactam agents, including ampicillin, amoxicillin–clavulanic, third-generation cephalosporins, cefoperazone–sulbactam, cefepime, aztreonam, carbapenems, amikacin, gentamicin, cotrimoxazole, ciprofloxacin, and the results were also interpreted based on the CLSI guidelines. All antimicrobial disks used for susceptibility testing were obtained from BD BBL Sensi-Disc (Becton Dickinson, Sparks, Maryland, USA). E. coli, ATCC 25922, and K. pneumoniae, ATCC35657, were used for quality control processes as recommended by CLSI ;
(3) PFGE: PFGE was carried out for ESBL-E and ESBL-K isolates, which were phenotypically identical by biotyping and AST (10 isolates of the former and 29 isolates of the latter). PFGE was carried out according to a standard protocol using a CHEF-MAPPER System (Bio-Rad Laboratories, Hercules, California, USA) in 0.5X Tris Borate EDTA buffer. Agarose plugs containing bacterial DNA were prepared and processed for PFGE as described elsewhere . Restriction analysis of chromosomal DNA with XbaI (New England BioLabs, Beverly, Massachusetts, USA) was performed, and separation of DNA was carried out by using 1% pulsed-field gel agarose (Bio-Rad Laboratories, La Jolla, California, USA). A bacteriophage λ DNA ladder (Bio-Rad Laboratories, USA) was used as a size marker. The gels were run at 6.0 V/cm, with an increasing pulse time of 0.5–60 s and an angle of 120° at 14°C. The running time was 22 h. After ethidium bromide staining, cluster analysis and DNA banding patterns were compared using BioNumerics software program, version 2.5 (Applied Maths, St Martens-Latem, Belgium). The Dice coefficients were used for band similarity measurements to determine DNA relatedness. Isolates were considered to be genetically unrelated if difference was more than six bands, were considered to be related if they differed by three to six bands, and were considered to be genetically identical if their macrorestriction DNA patterns differed by fewer than three bands, and the Dice coefficient of correlation was 85% or greater. The results were interpreted according to the criteria published by Tenover et al. .
SPSS (Statistical Package for Social Sciences) version 14.0 (Chicago, Illinois, USA) was used in data analysis, frequency and percentage were calculated.
Of the 5976 clinical specimens submitted for bacterial culture from January 2008 to March 2010, a total of 156 E. coli and 300 K. pneumoniae were isolated.
Results of detection of ESBL production (Table 1)
ESBL production was detected in 134 (86%) of the 156 E. coli isolates and 192 (68.3%) of the 300 K. pneumoniae isolates.
Biotyping and AST results of ESBL producers (Table 1)
All isolates demonstrated resistance to oxyiminocephalosporins, whereas they remained in the susceptible range for carbapenems. The percentages of sensitivity of ESBL-E for ampicillin, amoxicillin–clavulanic, third-generation cephalosporins, cefoperazone–sulbactam, cefepime, aztreonam, imipenem, and meropenem were 5.5, 20.1, 35.1, 40, 51.1, 41.3, 100, and 95, respectively, and those of ESBL-K for the same antibiotics were 1, 8.4, 13, 25, 22.9, 18, 91.2, and 87.8, respectively. Antimicrobial susceptibility testing to non-β-lactam agents, including amikacin, gentamicin, cotrimoxazole, and ciprofloxacin, for ESBL-E were 75.7%, 79.4%, 19.7%, and 100%, respectively, and for ESBL-K were 66.5%, 42%, 24.3%, and 31%, respectively. Only ten (7.5%) of the 134 ESBL-E and 29 (15%) of the 192 ESBL-K proved to be identical by biotyping and AST.
Results of genotyping of phenotypically identical ESBL-E and ESBL-K isolates using PFGE (Table 2 and Figs 1 and 2)
Of the biotyped E. coli and K. pneumoniae isolates, a total of eight and 13 different PFGE patterns were obtained, respectively. Among the E. coli isolates, only two clones were detected by PFGE. Samples 2 and 3 (b pattern) showed 92% similarity on Dice coefficient, whereas samples 4 and 5 (c pattern) showed 89.6% similarity on Dice coefficient. The remaining isolates showed different genotypes by PFGE band analysis. Among the K. pneumoniae isolates, seven clones encompassing 23 isolates were detected by PFGE: A (two isolates), E (nine isolates), G (four isolates), H (two isolates), K (two isolates), L (two isolates), M (two isolates), whereas the remaining isolates represented unique PFGE types. Thus, the clonal isolates contributed to 40 and 79.3% of the phenotypically identical ESBL-E (two clone types) and ESBL-K (seven clone types).
Thus, PFGE results showed that there were few isolates demonstrating DNA relatedness and many of the isolates showed unique, unrelated PFGE profiles and were unlikely to be considered as the cause of an epidemic. This clearly indicates that most ESBL-producing isolates were sporadic and that multiple clones were widespread in the institute.
Prevention of infection requires epidemiological investigations for identification of its sources and reservoirs and for differentiation between sporadic and epidemic cases. The development of molecular typing methods has provided clinical microbiologists with powerful tools to reveal the molecular identity of the isolates within the healthcare facilities . In this study, ESBL production was detected in 134 (86%) of the 156 E. coli isolates and in 192 (63.8%) of the 300 isolates of K. pneumoniae. Studies elsewhere  reported that the frequency of ESBL-producing K. pneumoniae has increased steadily in recent years with reports of 10–30% frequency among clinical isolates of K. pneumoniae. The same finding was demonstrated by Hsueh et al. , whereas Fiett et al.  identified ESBL in 100% of K. pneumoniae isolates. All isolates demonstrated resistance to oxyiminocephalosporins while they remained in the susceptible range for carbapenems. The percentages of sensitivity of ESBL-E for ampicillin, amoxicillin–clavulanic, third-generation cephalosporins, cefoperazone–sulbactam, cefepime, aztreonam, imipenem, and meropenem were 5.5, 20.1, 35.1, 40, 51.1, 41.3, 100, and 95, respectively, and those of ESBL-K for the same antibiotics were 1, 8.4, 13, 25, 22.9, 18, 91.2, and 87.8, respectively. AST to non-β-lactam agents, including amikacin, gentamicin, cotrimoxazole, and ciprofloxacin, for ESBL-E were 75.7%, 79.4%, 19.7%, and 100%, respectively, and for ESBL-K were 66.5%, 42%, 24.3%, and 31%, respectively. Only ten (7.5%) of the 134 ESBL-E and 29 (15%) of the 192 ESBL-K proved to be identical by biotyping and AST. Kiratisin et al.  found comparable results regarding the β-lactam agents and the non-β-lactams except for the sensitivity of ESBL-E to gentamicin and ciprofloxacin (33.7% and 21.9%, respectively) and that of ESBL-K to gentamicin (23.6%). By using PFGE for the phenotypically identical isolates, only two clones of E. coli were found, each of which consisted of two isolates that shared the same restriction profile, whereas the other six isolates showed different PFGE patterns, despite their similarity by phenotypic methods, indicating the poor discriminatory power of the latter and the importance of genotyping for confirmation of microbiological relatedness and the importance of genotyping for confirmation of microbiological relatedness, similarly Abbo et al.  found that outbreaks thought originally to have been caused by a single isolate (by the phenotypic methods) were in fact multiple unrelated strains by genotypic ones. The lack of reproducibility of many biotyping methods has also been previously reported by Reboli et al. . The most prevalent isolates of ESBL-K were of PFGE types E (nine isolates) and G (four isolates), which most probably represented two strains with relatively high epidemic potential, clonally spread in the Department of Neonatal Intensive Care Unit (NICU). Nosocomial spread of ESBL-producing K. pneumoniae was also reported by Fiett et al.  who documented ESBL-mediated resistance in a single medical center, caused by three related pulsotypes. In accordance with our results, Yu et al.  found that 67% of ESBL-producing K. pneumoniae isolates were contained in six groups as confirmed by PFGE, and Espinal et al.  also found two particular PFGE subtypes of ESBL-producing E. coli and K. pneumoniae clinical isolates from two hospitals of the Colombian–Caribbean region and concluded that both clonal and horizontal dissemination of resistance were observed . Therefore, PFGE revealed that the clonal isolates contributed to 40% of the phenotypically identical ESBL-E (two clone types) and 79.3% of ESBL-K (seven clone types). Results of Seputiene et al.  were very close to ours; their clonal isolates contributed to 50% of E. coli (seven clone types) and to 76% of K. pneumoniae (nine clone types) ESBL producers. These findings demonstrate the high diversity of E. coli and K. pneumoniae in our institute, and suggest that both horizontal gene transfer and clonal spread were responsible for the dissemination of the ESBL-producing strains. In another study, PFGE analysis carried out by Kiratisin et al.  demonstrated that there was no major clonal relationship among the typed ESBL producers in their hospital. PFGE results showed that there were few isolates demonstrating DNA relatedness and many of the isolates showed unique, unrelated PFGE profiles and were unlikely to be considered the cause of an epidemic. This clearly indicates that most ESBL-producing isolates were sporadic and that multiple clones were widespread in our institute. These results are similar to those of Kiratisin et al. .
The results of this study suggest the endemicity of ESBL-producing K. pneumoniae and E. coli strains and the dissemination of a plasmid rather than the occurrence of a clonal outbreak during the study period. Results also imply that colonized patients might act as the major epidemiological reservoirs for infection. Inadequate prevention of crosstransmission is thus the main determinant for the persistent ESBL-producing E. coli and K. pneumoniae. All this emphasizes the importance of adherence to infection control policies. Our results also show that phenotypic methods such as biotyping and antimicrobial susceptibility patterns are useful, simple, and cost-beneficial tests but with a weak discriminatory power. Molecular typing techniques are more powerful tools in epidemiological studies.
These results call for reinforcement of infection control practices to prevent nosocomial spread of ESBLs. Practices that are needed include cohorting, surveillance, contact precautions, accurate typing, switching to different classes of broad-spectrum antibiotics such as imipenem, and therapy with synergistic antibiotic combinations.
The authors are very grateful to Dr Pimentel and all the staff of the Molecular Laboratory and Disease Surveillance Programme in NAMRU-3 for their valuable help and the great experience.
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