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P17 Prevalence and characterization of extended spectrum-β-lactamase-producing Klebsiella pneumoniae in Saudi Arabia
S32
Poster Presentations
high affinity, mainly due to the size of this site, which shows better accommodation for moxifloxacin. Conclusions: Insufficient specificity of ciprofloxacin/levofloxacin binding and the emergence of ParC/GyrA double mutants before the introduction of moxifloxacin suggest that ciprofloxacin and levofloxacin may have driven drug selection pressure on both ParC and GyrA even though they preferentially bind ParC since non-specific binding facilitates the development of resistant mutations in the active site. The size of the binding site as well as the interactions of moxifloxacin with Asp37, Ser82, Asp80, His76, Lys74, Asp113 and His124. The majority of these residues present the potential for the formation of salt bridges causing tight ionic interactions that increase the binding specificity of moxifloxacin for this binding site. This rationalizes the lower extent of emergence of drug resistance that is observed with moxifloxacin compared to ciprofloxacin and levofloxacin. The role of the identified residues in this binding site as well as the impact that amino acid subsitutions may have in the binding site will be discussed in this presentation. P16 Targeting PfHsp90 in Plasmodium falciparum malaria: a strategy to reverse resistance D. Shahinas1 *, D. Pillai1 . 1 Ontario Agency for Health Protection and Promotion, Etobicoke, Canada Objective(s): (1) To define the structure of the ATP-binding domain of PfHsp90. (2) To create a medium-throughput in vitro and in vivo assay to screen for drug candidates that target PfHsp90. (3) To determine the capacity of PfHsp90 candidate drugs to reverse resistance using a synergy assay. Methods: (1) The ATP-binding domain of PfHsp90 was expressed in a pET28 vector using BL21 Codon+ cells. The protein was purified using Whatman DE52 anion-exchange and Qiagen Nickel Superflow columns. Crystals were obtained using the hanging drop method from a SIGMAAldrich basic crystallization screening library. (2) An in vitro binding assay using bis-ANS (SIGMA) was developed and validated with a malachite green activity assay obtained from Innova Sciences. (3) An in vivo assay using cultured parasites from patient isolates was developed based on SYBR green staining of the parasite. (4) In collaboration with Dr. Alessandro Dati at the high throughput screening center at Mount Sinai Hospital, we screened libraries of FDA approved drugs, natural and synthetic compounds for activity against PfHsp90. Results: (1) The ATP binding domain was successfully expressed, purified and crystallized. Initial structural analysis is ongoing. (2) An in vitro binding assay was used to screen for ATP domain binding and was validated using the malachite green activity assay which showed arrested activity of the ATPase domain with increasing concentrations of control drugs. (3) For synergistic testing, a P. falciparum screen assay was developed to screen previously approved drugs and natural compounds for PfHsp90 inhibition and parasite growth arrest. Sybr green staining of the parasite DNA was quantified with flow cytometry in order to determine the effectiveness of these compounds. Several of them had drug-like properties and exhibited promising IC50 curves that will be used as a basis of enhancing the specificity of the drugs. Conclusions: PfHsp90 is a critical chaperone in the expression of drug resistance markers. Our strategy aims to elucidate this protein, identify drug targets, and evaluate the capacity to reverse resistance in P. falciparum by effectively designing cocktails that synergistically target this resistance marker chaperone and essential metabolic proteins of the parasite. P17 Prevalence and characterization of extended spectrumb-lactamase-producing Klebsiella pneumoniae in Saudi Arabia A. Shibl1 *, A. Tawfik1 . 1 King Saud University, Riyadh, Saudi Arabia Background: This study was aimed to determine the prevalence of extended-spectrum b-lactamases (ESBLs) in Klebsiella pneumoniae from Riyadh area and to characterize the genetic basis of ESbLs in
K. pneumoniae. Also, determination of the predominant b-lactamase gene in these isolates was carried out. Methods: A total of 400 K. pneumoniae were isolated during 2007 from two hospitals in Riyadh were screened for production of ESBL using ESBL-E-strips and combined disk methods. PCR assay was used to detect blaTEM, blaSHV, and blaCTX-M genes. Results: Phenotypic characterization identified a high ESBL rate of 55%. ESBL-producing K. pneumoniae were PCR positive for TEM, SHV, and CTX-M b-lactamase genes with prevalence 97.3% 84.1%, and 34.1% respectively. Within CTX-M family, two groups of enzymes, CTX-M-1 and CTX-M-9 group likes, were found with prevalence of 60% and 40% respectively. Conclusions: This study confirms the high rate of ESBL in Riyadh and consequently, treatment options were further limited as most isolates produced ESBLs. This study demonstrates the worldwide spread of blaCTX-M genes in our clinical isolates. This is the first report in Saudi Arabia of presence of blaCTX-M gene. P18 High prevalence of metallo-b-lactamases-producing Pseudomonas aeruginosa from Saudi Arabia A. Shibl1 , A. Tawfik1 , H. Radwan1 , M. Al-Agamy1 *. 1 King Saud University, Riyadh, Saudi Arabia Objective: To determine the prevalence of metallo-b-lactamases (MBLs) among Enterobacteriaceae and P. aeruginosa and to investigate whether MBL genes have spread in MBL-producing isolates. Method: A total of 480 clinical isolates (135 P. aeruginosa and 345 Enterobacteriaceae) were screened for production of MBL. Antibiotic susceptibility testing was determined. Five MBL genes and class 1 integron were tested by PCR. Mapping of integron and matting out assay was carried out. Results: None of enterobacterial isolates were resistant to imipenem whereas 16.3% P. aeruginosa were resistant (32 mg/ml) and produce MBL. MBL-producing isolates were 100% resistant to b-lactams except aztreonam and ceftazidime, which showed resistance rate of 77.3% and 86.3% respectively. Only 31% and 22% of the MBL-producing isolates were resistant to ciprofloxacin and polymyxine B respectively while non-MBL producers were sensitive to polymyxin B. All MBL-producing isolates harbor blaVIM like gene and encoded on class 1 integron. Conclusion: High prevalence of imipenem resistant P. aeruginosa and all resistant isolates able to produce VIM-type MBL gene. This is the first study in Saudi Arabia to determine the prevalence and characterization of MBL genes. P19 Antibiotic resistance and serotype distribution in group B Streptococcus isolated in a maternity hospital in Kuwait S. Boswihi1 , N. Al Sweih1 , E. Udo1 *. 1 Kuwait University, Safat, Kuwait Objective: Recent reports from different countries document increasing prevalence of antibiotic resistance in Group B Streptococcus (GBS). The objective of this study was to screen GBS obtained patients at the Maternity hospital in Kuwait for antibiotic resistance and their serotypes. Methods: In total, 154 GBS isolates were collected between JulySeptember 2007 from mothers (149 isolates) and neonates (5 isolates). GBS serotypes were determined using latex agglutination test. Highlevel resistance to kanamycin was detected by the disk diffusion method with disks containing 200 mg kanamycin. MIC was determined by agar dilution or Etest. PCR was used to investigate the presence genes for resistance to Kanamycin (aph3 and ant4), tetracycline (tetK, tetM, tetL) and erythromycin (ermA, ermB, ermC, mrsA)). Results: The common serotype was serotype V (38.3%), followed by serotype III (19.5%), serotype Ia (10.4%), and serotype II (10.4%). Seventeen isolates (11.0%) were non typeable while serotypes IV (3.2%), VI (2.6%), VII (0.6%) and VIII (0.6) were less common. High-level kanamycin resistance (MIC > 1000 mg/ml) was detected in 74.7% of the isolates. Eighty nine, 34.4, 11.0 and 6.5 percent
Poster Presentations
high affinity, mainly due to the size of this site, which shows better accommodation for moxifloxacin. Conclusions: Insufficient specificity of ciprofloxacin/levofloxacin binding and the emergence of ParC/GyrA double mutants before the introduction of moxifloxacin suggest that ciprofloxacin and levofloxacin may have driven drug selection pressure on both ParC and GyrA even though they preferentially bind ParC since non-specific binding facilitates the development of resistant mutations in the active site. The size of the binding site as well as the interactions of moxifloxacin with Asp37, Ser82, Asp80, His76, Lys74, Asp113 and His124. The majority of these residues present the potential for the formation of salt bridges causing tight ionic interactions that increase the binding specificity of moxifloxacin for this binding site. This rationalizes the lower extent of emergence of drug resistance that is observed with moxifloxacin compared to ciprofloxacin and levofloxacin. The role of the identified residues in this binding site as well as the impact that amino acid subsitutions may have in the binding site will be discussed in this presentation. P16 Targeting PfHsp90 in Plasmodium falciparum malaria: a strategy to reverse resistance D. Shahinas1 *, D. Pillai1 . 1 Ontario Agency for Health Protection and Promotion, Etobicoke, Canada Objective(s): (1) To define the structure of the ATP-binding domain of PfHsp90. (2) To create a medium-throughput in vitro and in vivo assay to screen for drug candidates that target PfHsp90. (3) To determine the capacity of PfHsp90 candidate drugs to reverse resistance using a synergy assay. Methods: (1) The ATP-binding domain of PfHsp90 was expressed in a pET28 vector using BL21 Codon+ cells. The protein was purified using Whatman DE52 anion-exchange and Qiagen Nickel Superflow columns. Crystals were obtained using the hanging drop method from a SIGMAAldrich basic crystallization screening library. (2) An in vitro binding assay using bis-ANS (SIGMA) was developed and validated with a malachite green activity assay obtained from Innova Sciences. (3) An in vivo assay using cultured parasites from patient isolates was developed based on SYBR green staining of the parasite. (4) In collaboration with Dr. Alessandro Dati at the high throughput screening center at Mount Sinai Hospital, we screened libraries of FDA approved drugs, natural and synthetic compounds for activity against PfHsp90. Results: (1) The ATP binding domain was successfully expressed, purified and crystallized. Initial structural analysis is ongoing. (2) An in vitro binding assay was used to screen for ATP domain binding and was validated using the malachite green activity assay which showed arrested activity of the ATPase domain with increasing concentrations of control drugs. (3) For synergistic testing, a P. falciparum screen assay was developed to screen previously approved drugs and natural compounds for PfHsp90 inhibition and parasite growth arrest. Sybr green staining of the parasite DNA was quantified with flow cytometry in order to determine the effectiveness of these compounds. Several of them had drug-like properties and exhibited promising IC50 curves that will be used as a basis of enhancing the specificity of the drugs. Conclusions: PfHsp90 is a critical chaperone in the expression of drug resistance markers. Our strategy aims to elucidate this protein, identify drug targets, and evaluate the capacity to reverse resistance in P. falciparum by effectively designing cocktails that synergistically target this resistance marker chaperone and essential metabolic proteins of the parasite. P17 Prevalence and characterization of extended spectrumb-lactamase-producing Klebsiella pneumoniae in Saudi Arabia A. Shibl1 *, A. Tawfik1 . 1 King Saud University, Riyadh, Saudi Arabia Background: This study was aimed to determine the prevalence of extended-spectrum b-lactamases (ESBLs) in Klebsiella pneumoniae from Riyadh area and to characterize the genetic basis of ESbLs in
K. pneumoniae. Also, determination of the predominant b-lactamase gene in these isolates was carried out. Methods: A total of 400 K. pneumoniae were isolated during 2007 from two hospitals in Riyadh were screened for production of ESBL using ESBL-E-strips and combined disk methods. PCR assay was used to detect blaTEM, blaSHV, and blaCTX-M genes. Results: Phenotypic characterization identified a high ESBL rate of 55%. ESBL-producing K. pneumoniae were PCR positive for TEM, SHV, and CTX-M b-lactamase genes with prevalence 97.3% 84.1%, and 34.1% respectively. Within CTX-M family, two groups of enzymes, CTX-M-1 and CTX-M-9 group likes, were found with prevalence of 60% and 40% respectively. Conclusions: This study confirms the high rate of ESBL in Riyadh and consequently, treatment options were further limited as most isolates produced ESBLs. This study demonstrates the worldwide spread of blaCTX-M genes in our clinical isolates. This is the first report in Saudi Arabia of presence of blaCTX-M gene. P18 High prevalence of metallo-b-lactamases-producing Pseudomonas aeruginosa from Saudi Arabia A. Shibl1 , A. Tawfik1 , H. Radwan1 , M. Al-Agamy1 *. 1 King Saud University, Riyadh, Saudi Arabia Objective: To determine the prevalence of metallo-b-lactamases (MBLs) among Enterobacteriaceae and P. aeruginosa and to investigate whether MBL genes have spread in MBL-producing isolates. Method: A total of 480 clinical isolates (135 P. aeruginosa and 345 Enterobacteriaceae) were screened for production of MBL. Antibiotic susceptibility testing was determined. Five MBL genes and class 1 integron were tested by PCR. Mapping of integron and matting out assay was carried out. Results: None of enterobacterial isolates were resistant to imipenem whereas 16.3% P. aeruginosa were resistant (32 mg/ml) and produce MBL. MBL-producing isolates were 100% resistant to b-lactams except aztreonam and ceftazidime, which showed resistance rate of 77.3% and 86.3% respectively. Only 31% and 22% of the MBL-producing isolates were resistant to ciprofloxacin and polymyxine B respectively while non-MBL producers were sensitive to polymyxin B. All MBL-producing isolates harbor blaVIM like gene and encoded on class 1 integron. Conclusion: High prevalence of imipenem resistant P. aeruginosa and all resistant isolates able to produce VIM-type MBL gene. This is the first study in Saudi Arabia to determine the prevalence and characterization of MBL genes. P19 Antibiotic resistance and serotype distribution in group B Streptococcus isolated in a maternity hospital in Kuwait S. Boswihi1 , N. Al Sweih1 , E. Udo1 *. 1 Kuwait University, Safat, Kuwait Objective: Recent reports from different countries document increasing prevalence of antibiotic resistance in Group B Streptococcus (GBS). The objective of this study was to screen GBS obtained patients at the Maternity hospital in Kuwait for antibiotic resistance and their serotypes. Methods: In total, 154 GBS isolates were collected between JulySeptember 2007 from mothers (149 isolates) and neonates (5 isolates). GBS serotypes were determined using latex agglutination test. Highlevel resistance to kanamycin was detected by the disk diffusion method with disks containing 200 mg kanamycin. MIC was determined by agar dilution or Etest. PCR was used to investigate the presence genes for resistance to Kanamycin (aph3 and ant4), tetracycline (tetK, tetM, tetL) and erythromycin (ermA, ermB, ermC, mrsA)). Results: The common serotype was serotype V (38.3%), followed by serotype III (19.5%), serotype Ia (10.4%), and serotype II (10.4%). Seventeen isolates (11.0%) were non typeable while serotypes IV (3.2%), VI (2.6%), VII (0.6%) and VIII (0.6) were less common. High-level kanamycin resistance (MIC > 1000 mg/ml) was detected in 74.7% of the isolates. Eighty nine, 34.4, 11.0 and 6.5 percent