- Email: [email protected]
P19 Identification of a novel aac(6′)-Iag associated with the blaIMP-1 integron in a multidrug-resistant Pseudomonas aeruginosa
Posters / International Journal of Antimicrobial Agents 42S2 (2013) S41–S159
In this study, we prepared antienterotoxin chicken IgY antibodies specific for each SE (SEA to SEE) without reaction to protein A, which enabled a drastic reduction in nonspecific reactions. ELISAs, lateral flow device (LFDs), and IgY-based immunopillar chips were developed for SE detection. Methods: His-tagged recombinant proteins (rSEs) were produced in E. coli and purified with Ni-resin chromatography. Each rSEs were immunized into chickens. Anti-rSEs IgYs were extracted from egg yolk and purified by affinity chromatography. The specificity and sensitivity were tested with ELISA, LFDs and immunopillar chips. SEA in milk and dairy products contaminated with S. aureus were also analyzed by ELISA and LFDs. Results: All the ELISAs developed were as sensitive as commercially available kits. The SEs in milk were successfully detected by the ELISAs, LFDs, and immunopillar chips without any sample pretreatment. The LFD could detect SEA even at the low concentration of 0.2 ng/ml within 15 min in milk without interference by milk components, with greater simplicity and rapidness than ELISA. The detection limit of the immunopillar chips for the SEs ranged from 0.01 to 0.1 ng/ml in milk; the SEs were detected within 12 min and specialized skills were not required. The direct ELISA and LFD assays were also found to be equally sensitive for contaminated milk, cafe´ au lait, ice cream, and yogurt. Conclusion: IgY is an excellent antibody for SEs and showed no nonspecific reaction with protein A. IgY is applicable for not only ELISA but also LFD and the immunopillar chip to achieve rapid, specific, simple, and highly sensitive detection of rSEs. Both ELISA and LFD showed good performance on contaminated dairy products, even in the presence of S. aureus . These techniques should be useful tools for screening SEs in milk and dairy products. P19 Identification of a novel aac(6')-Iag associated with the bla IMP-1 integron in a multidrug-resistant Pseudomonas aeruginosa K. Kobayashi1,2,3 *, I. Hayashi4 , S. Kayama1,2 , H. Ohge1,5 , A. Matsubara3 , M. Sugai1,2 . 1 Project Research Center for Nosocomial Infectious Diseases, Hiroshima University, 2 Department of Bacteriology, 3 Department of Urology, Hiroshima University Institute of Biomedical and Health Sciences, 4 Research Facility, Faculty of Dentistry, Hiroshima University, 5 Division of Infectious Diseases, Hiroshima University Hospital, Hiroshima, Japan E-mail address : [email protected] Introduction: Multidrug-resistant Pseudomonas aeruginosa (MDRP) is one of important bacteria that causes nosocomial infection. In a surveillance study from Dec 2006 to Apr 2008, we characterized nine MDRP strains isolated from four patients in a ward at the Hiroshima University Hospital, Japan. Pulsed-field gel electrophoresis (PFGE) of SpeI -digested genomic DNAs from the isolates suggested the clonal expansion of a single strain; however, only one strain, NK0009, was found to produce metallo-b-lactamase. Objectives: The aim of this study is to analyze the mechanism of antibiotic resistance in the strains of NK0009. Methods: The broth microdilution method was used to determine the MICs of antibiotics. PFGE, PCR, DNA sequencing, cloning, expression, conjugation experiment and thin-layer chromatography (TLC) were used to characterize the molecular mechanism of antibiotic resistance. Results: PCR and subsequent sequencing analysis indicated NK0009 possessed a novel class 1 integron, designated asIn 124, that carries an array of four gene cassettes: a novel aminoglycoside resistance gene aac(6')-Iag , bla IMP-1 , a truncated form of bla IMP-1 and a truncated form of aac(6')-Iag . The aac(6')-Iag encoded a 167-amino-acid protein that shows 40% identity with AAC(6')-Iz. Recombinant AAC(6')-Iag protein showed aminoglycoside 6'-N -acetyltransferase activity using TLC and MS spectrometric analysis. Kinetic studies using recombinant AAC(6')-Iag demonstrated that it modified amikacin, arbekacin, dibekacin, isepamicin, kanamycin, sisomicin, and tobramycin; but not gentamicin. A conjugation experiment and subsequent Southern hybridization with the gene probes for bla IMP-1 and aac(6')-Ig strongly suggested In 124is on a conjugal plasmid. Transconjugants acquired resistance to gentamicin and were resistant to virtually
S47
all aminoglycosides, suggesting that the In 124conjugal plasmid also possesses a gene conferring resistance to gentamicin. Conclusion: We identified and characterized a novel aminoglycoside acetyltransferase gene, aac(6')-Iag in a bla IMP-1 integron cassette in a MDRP strain. P20 Preliminary study of blood culture bottle culture of pleural fluid and ascites K. Machida1 *, M. Nagao1 , T. Higuchi1 , M. Tanaka1 , S. Ichiyama1 . Department of Clinical Laboratory, Kyoto University Hospital, Kyoto, Japan E-mail address : [email protected]
1
Objectives: Pleural infection and peritonitis are serious infection which occur with comparatively high frequency. Some reports show that standard laboratory culture is positive in less than 60% for pleural infection, 42–65% for peritonitis. In general, standard laboratory culture is performed by using some agar plates. On the other hand, implementing bacterial culture of pleural fluid and ascites by using blood culture bottles may increase the culture positivity. Thus, some studies suggest that blood culture bottles should be used. In this study, we evaluated whether using blood culture bottles increases the culture positivity of pleural fluid culture and ascites culture. Methods: We included samples which were obtained more than 10 ml for this study. Between May, 2011 and February, 2012, 580 pleural fluid samples and 801 ascites fluid samples were submitted to our laboratory. Total of 149 pleural fluids and 192 ascites were enrolled eventually. For standard laboratory culture, sample was centrifuged 3000rpm for 10 min, the sediment was used for Gram staining and inoculation onto blood agar, Drigalski lactose agar and Thioglycollate broth (standard method). Blood agar and Drigalski lactose agar were incubated at 35°C for 48 h aerobically. Thioglycollate broth was incubated at 35°C for up to five days. The remaining (0.5–10 ml, 6.7 ml on average) was inoculated into Bact/Alert blood culture bottle and incubated by Bact/Alert 3D system (Sysmex biomerieux, Tokyo, Japan) for up to five days (bottle method). FN bottle (Sysmex biomerieux, Tokyo, Japan) was used between May, 2011 and September, 2011. FA bottle (Sysmex biomerieux, Tokyo, Japan) was used between October, 2011 and February, 2012. All of these procedures were performed at Clinical Laboratory of Kyoto University Hospital. Results: Pleural fluid: The frequency of microbial isolation was 11/149 (7.4%) with standard method and 16/149 (10.7%) with bottle method. Positive concordance rate was 90.9%, negative concordance rate was 95.7%. As to the number of detected species, 9 out of 16 (56.2%) bottle culture positive cases were exactly matched. Ascites: The frequency of microbial isolation was 73/192 (38.0%) with standard method and 74/192 (38.5%) with bottle method. Positive concordance rate was 87.7%, negative concordance rate was 91.6%. As to the number of detected species, 42 out of 74 (56.8%) bottle culture positive cases were exactly matched. Conclusion: In our study, there was no significant difference in the culture positivity between two methods. However, according to the fact that there is a difference of the detected organisms between two methods, weconsidered that the culture positivity can be increased further by a combination of two methods. P21 Effects of antibiotics in immunomodulatory gene expression of LPS-stimulated human polymorphonuclear leukocytes T. Ubagai1 *, S. Nagakawa1 , T. Ueda1 , R. Nakano1 , H. Kikuchi1 , Y. Ono1 . Medicine, Teikyo University, Tokyo, Japan E-mail address : [email protected]
1
Introduction: Several antibiotics that have good intracellular distribution properties exert immunomodulatory effects. However, the effects of these antibiotics on immunomodulatory gene expression of human polymorphonuclear leukocytes (PMNs) have not been completely elucidated. Objectives: We analyzed gene expressions levels of lipopolysaccharide (LPS)-stimulated PMNs by using antibiotics such as fosfomycin
In this study, we prepared antienterotoxin chicken IgY antibodies specific for each SE (SEA to SEE) without reaction to protein A, which enabled a drastic reduction in nonspecific reactions. ELISAs, lateral flow device (LFDs), and IgY-based immunopillar chips were developed for SE detection. Methods: His-tagged recombinant proteins (rSEs) were produced in E. coli and purified with Ni-resin chromatography. Each rSEs were immunized into chickens. Anti-rSEs IgYs were extracted from egg yolk and purified by affinity chromatography. The specificity and sensitivity were tested with ELISA, LFDs and immunopillar chips. SEA in milk and dairy products contaminated with S. aureus were also analyzed by ELISA and LFDs. Results: All the ELISAs developed were as sensitive as commercially available kits. The SEs in milk were successfully detected by the ELISAs, LFDs, and immunopillar chips without any sample pretreatment. The LFD could detect SEA even at the low concentration of 0.2 ng/ml within 15 min in milk without interference by milk components, with greater simplicity and rapidness than ELISA. The detection limit of the immunopillar chips for the SEs ranged from 0.01 to 0.1 ng/ml in milk; the SEs were detected within 12 min and specialized skills were not required. The direct ELISA and LFD assays were also found to be equally sensitive for contaminated milk, cafe´ au lait, ice cream, and yogurt. Conclusion: IgY is an excellent antibody for SEs and showed no nonspecific reaction with protein A. IgY is applicable for not only ELISA but also LFD and the immunopillar chip to achieve rapid, specific, simple, and highly sensitive detection of rSEs. Both ELISA and LFD showed good performance on contaminated dairy products, even in the presence of S. aureus . These techniques should be useful tools for screening SEs in milk and dairy products. P19 Identification of a novel aac(6')-Iag associated with the bla IMP-1 integron in a multidrug-resistant Pseudomonas aeruginosa K. Kobayashi1,2,3 *, I. Hayashi4 , S. Kayama1,2 , H. Ohge1,5 , A. Matsubara3 , M. Sugai1,2 . 1 Project Research Center for Nosocomial Infectious Diseases, Hiroshima University, 2 Department of Bacteriology, 3 Department of Urology, Hiroshima University Institute of Biomedical and Health Sciences, 4 Research Facility, Faculty of Dentistry, Hiroshima University, 5 Division of Infectious Diseases, Hiroshima University Hospital, Hiroshima, Japan E-mail address : [email protected] Introduction: Multidrug-resistant Pseudomonas aeruginosa (MDRP) is one of important bacteria that causes nosocomial infection. In a surveillance study from Dec 2006 to Apr 2008, we characterized nine MDRP strains isolated from four patients in a ward at the Hiroshima University Hospital, Japan. Pulsed-field gel electrophoresis (PFGE) of SpeI -digested genomic DNAs from the isolates suggested the clonal expansion of a single strain; however, only one strain, NK0009, was found to produce metallo-b-lactamase. Objectives: The aim of this study is to analyze the mechanism of antibiotic resistance in the strains of NK0009. Methods: The broth microdilution method was used to determine the MICs of antibiotics. PFGE, PCR, DNA sequencing, cloning, expression, conjugation experiment and thin-layer chromatography (TLC) were used to characterize the molecular mechanism of antibiotic resistance. Results: PCR and subsequent sequencing analysis indicated NK0009 possessed a novel class 1 integron, designated asIn 124, that carries an array of four gene cassettes: a novel aminoglycoside resistance gene aac(6')-Iag , bla IMP-1 , a truncated form of bla IMP-1 and a truncated form of aac(6')-Iag . The aac(6')-Iag encoded a 167-amino-acid protein that shows 40% identity with AAC(6')-Iz. Recombinant AAC(6')-Iag protein showed aminoglycoside 6'-N -acetyltransferase activity using TLC and MS spectrometric analysis. Kinetic studies using recombinant AAC(6')-Iag demonstrated that it modified amikacin, arbekacin, dibekacin, isepamicin, kanamycin, sisomicin, and tobramycin; but not gentamicin. A conjugation experiment and subsequent Southern hybridization with the gene probes for bla IMP-1 and aac(6')-Ig strongly suggested In 124is on a conjugal plasmid. Transconjugants acquired resistance to gentamicin and were resistant to virtually
S47
all aminoglycosides, suggesting that the In 124conjugal plasmid also possesses a gene conferring resistance to gentamicin. Conclusion: We identified and characterized a novel aminoglycoside acetyltransferase gene, aac(6')-Iag in a bla IMP-1 integron cassette in a MDRP strain. P20 Preliminary study of blood culture bottle culture of pleural fluid and ascites K. Machida1 *, M. Nagao1 , T. Higuchi1 , M. Tanaka1 , S. Ichiyama1 . Department of Clinical Laboratory, Kyoto University Hospital, Kyoto, Japan E-mail address : [email protected]
1
Objectives: Pleural infection and peritonitis are serious infection which occur with comparatively high frequency. Some reports show that standard laboratory culture is positive in less than 60% for pleural infection, 42–65% for peritonitis. In general, standard laboratory culture is performed by using some agar plates. On the other hand, implementing bacterial culture of pleural fluid and ascites by using blood culture bottles may increase the culture positivity. Thus, some studies suggest that blood culture bottles should be used. In this study, we evaluated whether using blood culture bottles increases the culture positivity of pleural fluid culture and ascites culture. Methods: We included samples which were obtained more than 10 ml for this study. Between May, 2011 and February, 2012, 580 pleural fluid samples and 801 ascites fluid samples were submitted to our laboratory. Total of 149 pleural fluids and 192 ascites were enrolled eventually. For standard laboratory culture, sample was centrifuged 3000rpm for 10 min, the sediment was used for Gram staining and inoculation onto blood agar, Drigalski lactose agar and Thioglycollate broth (standard method). Blood agar and Drigalski lactose agar were incubated at 35°C for 48 h aerobically. Thioglycollate broth was incubated at 35°C for up to five days. The remaining (0.5–10 ml, 6.7 ml on average) was inoculated into Bact/Alert blood culture bottle and incubated by Bact/Alert 3D system (Sysmex biomerieux, Tokyo, Japan) for up to five days (bottle method). FN bottle (Sysmex biomerieux, Tokyo, Japan) was used between May, 2011 and September, 2011. FA bottle (Sysmex biomerieux, Tokyo, Japan) was used between October, 2011 and February, 2012. All of these procedures were performed at Clinical Laboratory of Kyoto University Hospital. Results: Pleural fluid: The frequency of microbial isolation was 11/149 (7.4%) with standard method and 16/149 (10.7%) with bottle method. Positive concordance rate was 90.9%, negative concordance rate was 95.7%. As to the number of detected species, 9 out of 16 (56.2%) bottle culture positive cases were exactly matched. Ascites: The frequency of microbial isolation was 73/192 (38.0%) with standard method and 74/192 (38.5%) with bottle method. Positive concordance rate was 87.7%, negative concordance rate was 91.6%. As to the number of detected species, 42 out of 74 (56.8%) bottle culture positive cases were exactly matched. Conclusion: In our study, there was no significant difference in the culture positivity between two methods. However, according to the fact that there is a difference of the detected organisms between two methods, weconsidered that the culture positivity can be increased further by a combination of two methods. P21 Effects of antibiotics in immunomodulatory gene expression of LPS-stimulated human polymorphonuclear leukocytes T. Ubagai1 *, S. Nagakawa1 , T. Ueda1 , R. Nakano1 , H. Kikuchi1 , Y. Ono1 . Medicine, Teikyo University, Tokyo, Japan E-mail address : [email protected]
1
Introduction: Several antibiotics that have good intracellular distribution properties exert immunomodulatory effects. However, the effects of these antibiotics on immunomodulatory gene expression of human polymorphonuclear leukocytes (PMNs) have not been completely elucidated. Objectives: We analyzed gene expressions levels of lipopolysaccharide (LPS)-stimulated PMNs by using antibiotics such as fosfomycin