|Year : 2011 | Volume
| Issue : 3 | Page : 254-257
Molecular characterization of CTX-M β-lactamases among Klebsiella pneumoniae isolated from patients at Tehran hospitals
N Shoeib1, S Fereshteh1, FM Mehdi2, NV Sadat1, N Leila1
1 Department of Microbiology, Pasteur Institute, Tehran, Iran
2 Department of Microbiology, Tehran University of Medical Science, Tehran, Iran
|Date of Submission||23-Jan-2011|
|Date of Acceptance||06-Jun-2011|
|Date of Web Publication||17-Aug-2011|
Department of Microbiology, Pasteur Institute, Tehran
Source of Support: None, Conflict of Interest: None
Purpose: Plasmid-encoded CTX-M-group of extended-spectrum β-lactamases (ESBLs) represent a significant and rapidly emerging problem in most part of the world. The aim of the present study was to describe the prevalence of CTX-M producing Klebsiella pneumoniae at Tehran hospitals. Materials and Methods: Clinical isolates of K. pneumoniae (n=250) were collected from 10 hospitals of Tehran. Susceptibility to antimicrobial agents, MIC of cefotaxime and ESBLs production of collected isolates were detected. All ESBL-producing isolates were screened for bla CTX-M genes using PCR and DNA sequencing. Molecular typing of bla CTX-M harboring isolates was performed by Pulsed-field gel electrophoresis assay. Results: Of 250 K. pneumoniae clinical isolates, 102 isolates revealed ESBLs - phenotype. PCR assay and sequencing detected bla CTX-M genes in 71.5% (n= 73) of ESBL-producing isolates. The prevalence of CTX-M -I and CTX-M-III clusters among these isolates was 35.61% (n=26) and 21.9 % (n=16) respectively. Coexistence of CTX-M -I and CTX-M-III clusters was found among 42.5% (n= 31) of isolates. Of 102 isolates that were positive in the phenotypic confirmatory test (PCT), 29 isolates (28.4%) did not produce any amplicons in PCR for bla CTX-M gene. The results of PCR for CTX-M -II and CTX-M-IV clusters were also negative. Analysis of the 31 CTX-M producing K. pneumoniae isolates by PFGE typing showed 26 distinct patterns. Conclusions: The bla CTX-M genes are widespread among Iranian isolates of K. pneumoniae. PFGE demonstrated the high diversity of K. pneumoniae harboring bla CTX-M in our study.
Keywords: CTX-M, ESBLs, Klebsiella pneumoniae, Pulsed-field gel electrophoresis
|How to cite this article:|
Shoeib N, Fereshteh S, Mehdi F M, Sadat N V, Leila N. Molecular characterization of CTX-M β-lactamases among Klebsiella pneumoniae isolated from patients at Tehran hospitals. Indian J Med Microbiol 2011;29:254-7
|How to cite this URL:|
Shoeib N, Fereshteh S, Mehdi F M, Sadat N V, Leila N. Molecular characterization of CTX-M β-lactamases among Klebsiella pneumoniae isolated from patients at Tehran hospitals. Indian J Med Microbiol [serial online] 2011 [cited 2020 May 31];29:254-7. Available from: http://www.ijmm.org/text.asp?2011/29/3/254/83908
| ~ Introduction|| |
Extended-spectrum β-lactamases (ESBLs) are plasmid-encoded enzymes that mediate resistance to extended spectrum cephalosporins and aztreonam. These enzymes are most common in Gram-negative pathogens.,
The CTX-M β-lactamases as one of the most important ESBLs was first discovered in strains of Escherichia More Details coli isolated from patients in Germany in 1989.  In resent years, Cefotaximase (CTX-M) has been found in different genera of Enterobacteriaceae particularly in Klebsiella pneumoniae and E. coli.  The CTX-M enzymes efficiently hydrolyze cefotaxime, a broad spectrum cephalosporin. They can be inhibited by β-lactamase inhibitors such as clavulanic acid, sulbactam and tazobactam. 
CTX-M-producing strains have usually minimum inhibitory concentration (MIC) for cefotaxime in the resistant range >64μg/ml. It appears that the CTX-M types have approximately 40% amino acid similarity with TEM and SHV-type ESBLs, but these enzymes are closely related to β-lactamases of Kluyvera spp.  Over 60 different subtypes have been identified among the CTX-M group and these are grouped into five different clusters of CTX-M-I, CTX-M-II, CTX-M-III, CTX-M-IV, CTX-M-V based on their amino acid sequences (≤ 90% amino acid identity). , Recently, a new cluster designated as CTX-M-45 has also been introduced.  The members of each cluster exhibit > 94% amino acid similarity.  The blaCTX-M is usually located on plasmids, which are highly spread among nosocomial pathogens.  Pulsed-field gel electrophoresis (PFGE) is a powerful and useful technique for epidemiological tracking and tracing.  The aim of the present study was to describe the prevalence of CTX-M producing K. pneumoniae at Tehran hospitals. PFGE was then used to detect possible genetic relationships among the ESBL-producing isolates.
| ~ Materials and Methods|| |
Bacterial strains and antibiotic susceptibility testing
Clinical isolates of K. pneumoniae (n=250) isolated from patients with nosocomial infections from 10 hospitals of Tehran during August 2006 to February 2009 were included in this study. Of these isolates, 184 (73.6%) were isolated from urine and the remainders were from sputum (n=24), wound (n=20), blood (n=10), wound aspirate (n=4), tracheae (n=3) and CSF (n=2). The isolates were identified using conventional bacteriological methods. The susceptibility of all isolates to 12 antimicrobial agents was determined using disk-diffusion assay and the results were interpreted according to the CLSI guidelines.  Disks (MAST, Merseyside, UK) impregnated with ciprofloxacin (5 μg), imipenem (10 μg), gentamicin (10 μg), amikacin (30 μg), cefotaxime (20 μg), ceftriaxone (30 μg), cefepime (30 μg), ceftizoxime (30 μg), ceftazidime (30 μg), piperacillin (100 μg), carbenicillin (100 μg), and piperacillin-tazobactam (110 μg) were used in drug susceptibility testing of isolates. MIC-values of cefotaxime were determined by microbroth dilution method and results of susceptibility testing methods were interpreted according to CLSI guidelines.  Pseudomonas aeruginosa ATCC 27853 used as quality control in this part of the study.
The phenotypic confirmatory test (PCT) were used for screening ESBL-producing isolates by disk diffusion method with disks-containing cefotaxime (30 μg), cefotaxime plus clavulanic acid (30/10 μg), ceftazidime (30 μg) and ceftazidime plus clavulanic acid (30/10 μg) as recommended by CLSI. 
Detection of bla CTX-M by PCR
Plasmid DNA was extracted according to the published method of Johnson and Woodford  and used as DNA template in PCR experiments for detection of bla CTX-M genes. The primers, expected sizes of amplicons and cycling conditions used as previously described.  The PCR products were sequenced and analyzed using BLAST program.
Molecular typing by pulsed-field gel electrophoresis
PFGE of the CTX-M producing isolates was performed as previously described by Poh et al.  Whole-cell DNA from isolates harboring bla CTX-M genes were digested with XbaI endonuclease (Fermentas, Sylvius, Lithuania). Thirty-one CTX-M-producing isolates that were positive for both CTX-M-I and CTX-M-III clusters with the high similarity antibiotic pattern were selected for PFGE analysis. Electrophoresis was carried out in a 1% agarose gels in 0.5ΧTBE buffer with CHEF-DR III Apparatus (Bio-Rad Laboratories, Richmond, CA). The pulse time was 2-30 s over 35 h at 200 V. The gel was stained for 20 min in ethidium bromide and photographed with UVP gel documentation (UVP, UK).
To facilitate comparison of DNA banding patterns, dendrograms were prepared using Gel Compare II software, and interpreted according to the guidelines established by Tenover et al.  Salmonella More Details braenderup H9812 used as molecular weight marker.
| ~ Results|| |
Bacteriology and antimicrobial susceptibilities of study strains of 250 K. pneumoniae isolates, 38% (n=95) and 36.4% (n=91) were resistant to cefotaxime and ceftazidime, respectively. The result of drug susceptibility testing for other antibiotics [Table 1].
|Table 1: Antimicrobial susceptibility pattern of all isolates an ESBL-producing|
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Prevalence and resistance phenotypes of ESBL-producing isolates
Forty one percent of isolates (n=102) were positive for production of ESBL in DDST. Of 184 strains that isolated from patients with urinary tract infection, 35.3% produced ESBLs and 33% of them were also resistant to piperacillin-tazobactam. Antimicrobial resistance pattern for ESBL-producing strains [Table 1].
The MICs of cefotaxime were variable among ESBL-producing isolates and ranged from 4 to 512 μg/ml. The MIC of cefotaxime against the ESBL producers was ≥ 64 μg/ml. Three isolates showed resistance to all antimicrobial agents, including imipenem. The MICs of cefotaxime and imipenem against these isolates were 512 and 256 μg/ml, respectively. The results of phenotypic confirmatory test (PCT) for detction of ESBL for these isolates were negative.
Distribution of different CTX-M subtypes
PCR assay detected bla CTX-M genes in 71.5% (n=73) of ESBL-producing isolates. The prevalence of CTX-M-I and CTX-M-III clusters among these isolates was 35.61% (n= 26) and 21.9 % (n=16) respectively and 31 isolates (42.5%) were positive for both CTX-M-I and CTX-M-III clusters. Of 102 ESBL producing isolates detected by PCT, 29 (28.43%) were negative in PCR test. The results of PCR for bla CTX-M-II and bla CTX-M-IV genes were negative in this research. Three isolates showed resistance to all antimicrobial agents [Table 1]. In these isolates blaCTX-M genes were not detected by PCR. Further analysis of these strains for Metallo β-Lactamases genes in PCR experiment (with the specific primers used) did not yield any results.  The sequence of the bla CTX-M-I gene was GenBank accession number HQ 105389.
Pulsed-field gel electrophoresis
Twenty-six distinct PFGE patterns (P1-P26) were obtained from 31 selected isolates harboring blaCTX-M [Figure 1]. Four isolates were identical and grouped in the pattern P21 and these were cultured from patients during 2006 to 2007 [Table 2]. Pattern P8 also consisted of two identical isolates that were isolated from patients in 2007. PFGE demonstrated that approximately 81% of isolated were clonally unrelated. As demonstrated in [Table 2], these isolates were resistant to at least two classes of antimicrobial agents such as fluoroquinolones and amino glycosides.
|Table 2: Antibiotic resistance patterns of K. pneumoniae isolates with pattern similarity of PFGE typing|
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|Figure 1: Dendrogram of pulsed– fi eld gel electrophoresis patterns of the CTX-M producing K. pneumoniae isolates.|
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| ~ Discussion|| |
Klebsiella spp. accounts for 5-7% of all hospital-acquired bacterial infections. The most common nosocomial infections caused by K. pneumoniae include urinary tract infections (UTIs), pneumonia, septicemia and soft tissue infection. , In recent years, CTX-M-type have become a global problem among clinical isolates of the family Enterobacteriaceae. Moreover hospitals outbreak of these enzymes have been reported in various countries of the world.  The prevalence of CTX-M type producing K. pneumoniae in Asia is variable among countries.  CTX-M-1 and CTX-M-9 clusters are the most prevalent CTX-M types in the Middle East.  The CTX-M-1 cluster has also been reported in different Russian and Lebanese hospitals.  In present study, 71.5% of the ESBL producing isolates at Tehran hospitals contained blaCTX-M genes in a 4-year period surveillance. Among them 42.5 % (n=31) belonged to both CTX-M-I and CTX-M-III clusters. We confirmed the presence of these genes by sequencing of the PCR products. Our data indicate that the laboratory detection of the organisms producing blaCTX-M genes is not well-defined. Thus, clinical microbiology laboratories should recognize the outbreaks of ESBLs in many different genera of bacteria. According to CLSI and European guidelines, CTX-M-producing strains, which are susceptible to ceftazidime in vitro, could therefore be resistance to it in vivo.  Thus, the widespread use of ceftazidime for treat infection caused by CTX-M producing isolates leads to spreading of CTX-M enzymes with high levels of hydrolytic activity against ceftazidime.
In our study three isolates showed resistance to all antimicrobial agents including cefotaxime and imipenem [Table 1]. No blaCTX-M genes were detected in these isolates by PCR. It seems that other mechanisms of antibiotics resistance such as enzymatic drug inactivation or efflux pump mechanism are probably involved in resistance of these isolates.  The carbapenems such as imipenem and meropenem have been suggested for the treatment of serious infections caused by multidrug-resistant Gram-negative isolates and for ESBL-producing strains. , The ESBLs are generally inactivated by β-lactamase inhibitors such as clavulanate, sulbactam and tazobactam.  In the present study, resistance to piperacillin was 80% among ESBL-producing isolates but 33% of them showed resistance to piperacillin-tazobactam [Table 1]. Our finding underscore that the piperacillin-tazobactam is inhibitor for ESBL. Moreover, a similar study demonstrated that piperacillin-tazobactam is a suitable alternative to carbapenems therapy in many type of nosocomial infections.  Since the genes encoding ESBLs are usually located on a large self-transferable plasmid, many of the ESBL-producing strains also are resistant to other classes of antibiotic drugs, such as fluoroquinolones, aminoglycosides and chloramphenicol.  This is the possible mechanism for 45 and 53% of our ESBL-producing isolates that were coresistant to amikacin and ciprofloxacin, respectively, and shows increase comparing to the previous report in 2004. These rates were 26 and 44% at that time.  PFGE typing revealed that about 81% of 31 CTX-M-producing K. pneumoniae isolates were clonally unrelated, thus the CTX-M-producing isolates have not serious clonally spread in Tehran hospitals. As demonstrated in [Table 2], most isolates with identifiably PFGE typing had the similar antibiotic resistance profiles. For example, two isolated belonging to cluster P8 (P8a and P8b) were multidrug-resistant. These isolates exhibited high level of resistance to ciprofloxacin and aminoglycosides [Table 2].
In conclusion, presence of CTX-M-producing K. pneumoniae is a significant challenge to infection management in Tehran hospitals and treatment by extended-spectrum β-Lactams may lead to the spread of resistance to antimicrobial drugs. It appears that a regular surveillance of resistant to β-Lactam antibiotics is necessary. Existence of isolates with resistance to cefotaxime and imipenem is alarming at Tehran hospitals and strategy for prevention and control of these strains are necessary.
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[Table 1], [Table 2]