Background: Resistance to contemporary broad-spectrum Î²-lactam antibiotics mediated by extended-spectrum Î²-lactamases (ESBLs) is increasing worldwide. Klebsiella pneumoniae, an important cause of nosocomial and community acquired urinary tract infections has rapidly become the most common ESBL producing organism. We examined ESBL production in urinary isolates of K. pneumoniae in relation to the presence of bla SHV, bla TEM and bla CTX-M genes. Methods: Antibiotic resistance of 51 clinical isolates of K. pneumoniae was determined to amoxicillin, amikacin, ceftazidime, cefotaxime, cefteriaxon, ceftizoxime, gentamicin, ciprofloxacin and nitrofurantoin by disc diffusion. Minimum inhibitory concentrations were also measured for ceftazidime, cefotaxime, cefteriaxon, ceftizoxime and ciprofloxacin. ESBL production was detected by the double disc synergy test and finally, presence of the bla SHV, bla TEM and bla CTX-M genes were shown using specific primers and PCR. Results: Disc diffusion results showed that 96.08 % of the isolates were resistant to amoxicillin followed by 78.43 % resistance to nitrofurantoin, 49.02 % to amikacin and ceftazidime, 41.17 % to ceftriaxone, 37.25% resistance to cefotaxime and ceftizoxime, and 29.42 % to gentamicin and ciprofloxacin. Both resistant and intermediately resistant organisms were resistant in MIC determinations. Twenty two isolates (43.14%) carried bla SHV, 18 (35.29%) had bla TEM and 16 (31.37%) harbored bla CTX-M genes. ESBL production was present in 14 isolates (27.45 %) of which, 3 did not harbor any of the 3 genes. Among the non- ESBL producers, 9 lacked all 3 genes and 2 carried them all. Conclusion: No relation was found between gene presence and ESBL expression

Eftekhar, F.

Golalipoor, M.

Mansoursamaei, N.

Rastegar, M.

Iranian Journal of Public Health

English

PubMed

Klebsiella pneumoniae is an opportunistic pathogen that causes a significant proportion of community and hospital acquired infection

F Eftekhar

M Rastegar

M Golalipoor

N Mansoursamaei

CiteSeerX

Detection of Extended Spectrum Β-Lactamases in Urinary Iso- lates of Klebsiella pneumoniae in Relation to BlaSHV, BlaTEM and

Background: Resistance to contemporary broad-spectrum b-lactam antibiotics mediated by extended-spectrum b-lactamases (ESBLs) is increasing worldwide. Klebsiella pneumoniae, an important cause of nosocomial and community acquired urinary tract infections has rapidly become the most common ESBL producing organism. We examined ESBL production in urinary isolates of K. pneumoniae in relation to the presence of blaSHV, blaTEM and blaCTX-M genes.Methods: Antibiotic resistance of 51 clinical isolates of K. pneumoniae was determined to amoxicillin, amikacin, ceftazidime, cefotaxime, cefteriaxon, ceftizoxime, gentamicin, ciprofloxacin and nitrofurantoin by disc diffusion. Minimum inhibitory concentrations were also measured for ceftazidime, cefotaxime, cefteriaxon, ceftizoxime and ciprofloxacin. ESBL production was detected by the double disc synergy test and finally, presence of the blaSHV, blaTEM and blaCTX-M genes were shown using specific primers and PCR.Results: Disc diffusion results showed that 96.08 % of the isolates were resistant to amoxicillin followed by 78.43 % resistance to nitrofurantoin, 49.02 % to amikacin and ceftazidime, 41.17 % to ceftriaxone, 37.25% resistance to cefotaxime and ceftizoxime, and 29.42 % to gentamicin and ciprofloxacin. Both resistant and intermediately resistant organisms were resistant in MIC determinations. Twenty two isolates (43.14%) carried blaSHV, 18 (35.29%) had blaTEM and 16 (31.37%) harbored blaCTX-M genes. ESBL production was present in 14 isolates (27.45 %) of which, 3 did not harbor any of the 3 genes. Among the non- ESBL producers, 9 lacked all 3 genes and 2 carried them all.Conclusion: No relation was found between gene presence and ESBL expression

N MansourSamaei

Directory of Open Access Journals

Detection of Extended Spectrum Β-Lactamases in Urinary Isolates of Klebsiella Pneumoniae in Relation to BlaSHV, BlaTEM and BlaCTX-M Gene Carriage

Golestan University of Medical Sciences Repository

Downloaded from http://journals.tums.ac.ir/ on Monday, June 11, 2012     Iranian J Publ Health, Vol. 41, No.3, 2012, pp.127-132                                                     Original Article   Detection of Extended Spectrum Β-Lactamases in Urinary Iso-lates of Klebsiella pneumoniae in Relation to BlaSHV, BlaTEM and BlaCTX-M Gene Carriage  *F Eftekhar ¹, M Rastegar ¹, M Golalipoor ², N MansourSamaei ³  ¹ Dept. of Microbiology, Faculty of Biological Sciences, Shahid Beheshti University G.C., Evin, Tehran, Iran  ² Dept. of Basic Sciences, Faculty of Science, Golestan University, Gorgan, Iran ³Dept. of Human Genetics, Faculty of Advanced Medical Technologies, Golestan University of Medical Sciences, Gorgan, Iran  (Received 11 Jun 2011; accepted 27 Dec 2011)     Introduction  Klebsiella pneumoniae is an opportunistic pathogen that causes a significant proportion of community and hospital acquired infections including urinary tract, pneumonia, septicemia and soft tissue infections (1). Extended-spectrum ß-lactam antibiotics have been widely used for treatment of serious Gram-negative infections since the 1980s. However, bacterial resistance has emerged quickly due to the production of ESBLs (2-6). These enzymes are capable of hydrolyzing extended spectrum β-lactam antibiotics such as ceftazidime, ceftriaxone, cefotaxime and aztreonam. ESBLs are derived from genes for the narrower-spectrum ß-lactamases (TEM-1, TEM-2 or SHV-1) by mutations that alter the amino acid Abstract Background: Resistance to contemporary broad-spectrum -lactam antibiotics mediated by extended-spectrum -lactamases (ESBLs) is increasing worldwide. Klebsiella pneumoniae, an important cause of nosocomial and community acquired urinary tract infections has rapidly become the most common ESBL producing organism. We examined ESBL production in urinary isolates of K. pneumoniae in relation to the presence of blaSHV, blaTEM and blaCTX-M genes.  Methods: Antibiotic resistance of 51 clinical isolates of K. pneumoniae was determined to amoxicillin, amikacin, cef-tazidime, cefotaxime, cefteriaxon, ceftizoxime, gentamicin, ciprofloxacin and nitrofurantoin by disc diffusion. Mini-mum inhibitory concentrations were also measured for ceftazidime, cefotaxime, cefteriaxon, ceftizoxime and ciproflox-acin. ESBL production was detected by the double disc synergy test and finally, presence of the blaSHV, blaTEM and blaCTX-M genes were shown using specific primers and PCR.  Results: Disc diffusion results showed that 96.08 % of the isolates were resistant to amoxicillin followed by 78.43 % resistance to nitrofurantoin, 49.02 % to amikacin and ceftazidime, 41.17 % to ceftriaxone, 37.25% resistance to cefo-taxime and ceftizoxime, and 29.42 % to gentamicin and ciprofloxacin. Both resistant and intermediately resistant organ-isms were resistant in MIC determinations. Twenty two isolates (43.14%) carried blaSHV, 18 (35.29%) had blaTEM and 16 (31.37%) harbored blaCTX-M genes. ESBL production was present in 14 isolates (27.45 %) of which, 3 did not harbor any of the 3 genes. Among the non- ESBL producers, 9 lacked all 3 genes and 2 carried them all.  Conclusion: No relation was found between gene presence and ESBL expression.    Keywords: Klebsiella pneumoniae, Urinary infection, Extended spectrum -lactamases, blaSHV, blaTEM, blaCTX-M.*Corresponding Author: Tel: +9821-29903208, E-mail address: f-eftekhar@cc.sbu.ac.ir Downloaded from http://journals.tums.ac.ir/ on Monday, June 11, 2012Eftekhar et al.: Detection of extended spectrum β-lactamases …  128 configuration around the enzyme active site (4, 5). They are typically encoded by plasmids that can be exchanged between bacterial species (7, 8). It is also reported that the predominant types of ESBLs in K. pneumoniae are SHV type followed by TEM and CTX-M (2, 4, 9, 10). The prevalence of ESBL producing K. pneumo-niae varies around the world and has been re-ported to be around 1-5% in European countries, 7.5% in North America, 13.3% in Europe, 22.4% in Asia Pacific Rim, 44% in Latin Amer-ica, 48.5% in Turkey, 51% in China, 71.4% in Mexico and 72% in India (4, 11-13). The preva-lence of ESBL producing K. pneumoniae in Iran is reported to range from 19.6 to 75% with SHV as the dominant type (14-18). We studied ESBL production in the urinary isolates of Klebsiella pneumoniae in relation to the presence of SHV, TEM and CTX-M -lactamase genes among the isolates.  Materials and methods  Fifty one urinary isolates of K. pneumoniae were chosen from a collection of urinary isolates between March and August 2008 from two hos-pitals in Tehran (Taleghani and Imam Hussein). The isolates were tested for antimicrobial sus-ceptibility by the disc diffusion method accord-ing to the NCCLS guidelines (19). The antibi-otic discs were obtained from Padtan Teb (Te-hran, Iran) and the following antibiotics were used: amoxicillin (30 g), amikacin (30g), cef-tazidime (30g), cefotaxime (30g), cefteriaxon (30g), ceftizoxime (30g), gentamicin (10g), ciprofloxacin (10g), and nitrofurantoin (300g). Minimum inhibitory concentrations were also measured for 5 antibiotics using the broth mi-crodilution method and the NCCLS guidelines (20). The double disc synergy test (DDST) was used to detect ESBL production (21). Briefly, the or-ganisms were swabbed on to Mueller-Hinton agar plates (as performed for disc diffusion), an amoxiclav disc (20 + 10 g) was placed in the center of the plate and ceftizoxime (30g), cefo-taxime (30g), cefteriaxon (30g) discs were placed 15 mm away from the central disc. The plates were incubated at 37 oC for up to 24 h. An increase of > 5 mm inhibition zone for antibiot-ics around the amoxyclav disc compared to the cephalosporin discs alone was considered ESBL production. ESBL production was further con-firmed by the phenotypic confirmatory test (PCT) using ceftazidime and ceftazi-dime/clavlanic acid (22). Genomic DNA was prepared from overnight cultures grown on Lauria-Bertani broth [LB] (Hi-Media, Mumbai, India) by inoculating a sin-gle colony into 100 l of double distilled water and boiling for 10 min. An equal volume of chloroform: isoamyl alcohol (24:1 v/v) was added, mixed by end over end rotation and the mixture was centrifuged at 8000 x g for 15 min. The upper layer was then used in the PCR reac-tion mixtures (21). PCR amplification was carried out with specific primers for blaSHV (forward: 5’-TGG TTA TGC GTT ATA TTC GCC-3’, and reverse: 5’-GGT TAG CGT TGC CAG TGCT-3’) amplifying an 800 bp fragment (Fazapajouh, Tehran), blaTEM (forward: 5’-ATA AAA TTC TTG AAG ACG AA-3’, reverse: 5’-GAC AGT TAC CAA TGC TTA ATC-3’) amplifying an 850 bp fragment and blaCTX-M (forward; 5’-TGG TTA AAA ATC ACT ACT GCG-3’, reverse: 5’-ATT ACA AAC CGT CGG TGA C-3’) amplifying an 891 bp product (Takapou Zist, Tehran) (22-24). PCR was carried out in 25 µl volume reaction mix-tures containing 10 pM of each primer, 200 µM dNTP, 1.5 mM MgCl2, 1.5 µl of crude template DNA and 1 U Taq polymerase in the reaction buffer provided by the manufacturer (CinnaGen, Tehran, Iran). The following thermocycler pro-gram was carried out for PCR experiments: 4 min denaturation at 94 oC followed by 32 cycles of 1 min at 94 oC, 1 min at the annealing tem-perature (55 oC for blaSHV and blaCTX-M and 58 oC for blaTEM) and 1 min at 72 oC with a final extension period of 10 min at 72 oC. Downloaded from http://journals.tums.ac.ir/ on Monday, June 11, 2012Iranian J Publ Health, Vol. 41, No.3, 2012, pp.127-132  129 Results  The antibiotic susceptibility profile measured by disc diffusion is shown in figure 1.  As observed the highest rate of resistance was found for amoxicillin (96.08 %) and the lowest for gen-tamicin and ciprofloxacin (29.42 %). Among the broad spectrum -lactam antibiotics, the level of resistance ranged between 37.25 to 41.17 %. The MIC results showed 88.23% resistance to cefotaxime, 83.35% to ceftriaxone, 82.35% to ceftazidime, 50.98% to ceftizoxime and 47.06% to ciprofloxacin. These values were much higher compared to the resistance patterns obtained by disc diffusion. However, when the numbers of resistant and intermediately resistant organisms were compiled, there was total agreement be-tween the two phenotypic tests.  Figure 2 shows the PCR amplification products of -lactamase genes. The results showed that 22 isolates (43.14%) carried the blaSHV gene, 18 (35.29%) had blaTEM and 16 (31.37%) harbored the blaCTX-M genes. The DDST results detected ESBL production in 14 isolates (27.45%) of which, 4 carried the blaSHV gene, 3 had blaCTX-M, 3 harbored blaTEM, 1 carried blaSHV-TEM and sur-prisingly 3 did not have any of the 3 genes tested. Of the 37 Organisms which did not pro-duce ESBL, 9 lacked all 3 genes, 7 carried blaSHV, 4 blaCTX-M, 3 blaTEM, 4 blaSHV-TEM, 4 blaSHV-CTX-M, 3 blaTEM-CTX-M and finally, 2 carried all 3. These results suggest that other types of ESBL may be responsible for the ESBL pheno-type.    020406080100120AMX FM AN CAZ CRO CTX CT CP GMRESISTANT INTERMEDIATE SENSITIVE  Fig. 1: Antibiotic susceptibility of 51 urinary isolates of Klebsiella pneumoniae measured by disc diffu-sion. AMX, amoxicillin, FM, nitrofurantoin, AN, amikacin, CAZ, ceftazidime, CRO, cefriaxone, CTX,  cefotaxime, CT, ceftizoxime, CP, ciprofloxacin, GM, gentamicin   Downloaded from http://journals.tums.ac.ir/ on Monday, June 11, 2012Eftekhar et al.: Detection of extended spectrum β-lactamases …  130 M    C 1- C1+   1      2     C2- C2+    3       4    C3- C3+     5       6500bp 800bp     Fig. 2:  PCR amplification of -lactamase genes in urinary isolates of K. pneumoniae. M, 100 bp DNA ladder, C-, negative control, C+, positive control, lanes 1-2, blaCTX-M  (891 bp), lanes 3- 4, blaTEM  (850 bp), lanes 5-6, blaSHV  (800 bp) amplification prod-ucts  Discussion  Klebsiella pneumoniae has rapidly become the most common ESBL producing organism, mak-ing its eradication difficult from high risk wards such as intensive care units. In our study, 27.45% of the urinary isolates of K. pneumoniae were ESBL producers. Despite the fact that a small number of isolates were tested, ESBL production among our isolates was higher than a study performed in Semnan, Iran where ESBL production in urinary isolates was reported to be 19.6% (17). On the other hand, our results showed a lower rate of ESBL production com-pared to most of the other studies carried out in Iran and other parts of the Middle or Far East (11-18). The prevalence rates of ESBL produc-tion are different in different countries or differ-ent parts of each country and are very much de-pendent upon antibiotic policies in every region.  The conventional disc diffusion method does not usually allow for routine differentiation of ESBL producing strains. Several methods have been devised to differentiate between ESBL and non-ESBL producers among which, the double disc diffusion test has been reported to be the most discriminating (25-26). Also, molecular detection methods such as PCR have been used for molecular differentiation of β-lactamase pro-ducing isolates. However, most of the studies performed, aim for the detection of the more commonly occurring β-lactamase genes such as the TEM and SHV family and it is not practical to screen for a large number of -lactamase genes. In a study carried out in Taiwan, a corre-lation was reported between phenotypic resis-tance to cefotaxime, ceftazidime and ceftriaxone in ESBL producing K. pneumoniae isolates and presence of blaSHV, blaCTX-M and blaVEB-1 like genes but not blaTEM (22). Our results showed no relation between presence of TEM, SHV and CTX-M -lactamase genes with -lactamase production or ESBL phenotype. Presence of ESBLs can be masked by the expression of chromosomal or plasmid mediated AmpC -lactamases. Also, ESBL producing strains with AmpC -lactamases can cause a false negative in ESBL detection (25-27). More importantly, expression of -lactamase genes depend upon the environmental conditions such as the pres-ence of antibiotics and gene presence shown by PCR does not necessarily indicate its expression.   Ethical considerations  Ethical issues (including plagiarism, informed Con-sent, misconduct, data fabrication and/or falsifica-tion, double publication and/or submission, redun-dancy, etc) have been completely observed by the authors.  Acknowledgments  The authors wish to thank Shahid Beheshti Re-search Council for providing a special grant to finance this research.     Downloaded from http://journals.tums.ac.ir/ on Monday, June 11, 2012Iranian J Publ Health, Vol. 41, No.3, 2012, pp.127-132  131 References 1. Podschun R, Ullman U (1998). Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods and pathogenicity factors. Clin Microbiol Rev, 11: 589-603.  2. Bradford PA (2001). Extended-spectrum β-lac-tamases in the 21st century: Characterization, epidmiology and detection of this important resistance threat. Am Soc Microbiol, 14: 933-51. 3. Paterson DL, Bonomo RA (2005). Extended-spectrum ß-lactamases: a clinical update. Clin Microbiol Rev, 18: 657–86. 4. 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