Comparative Assessment of b-lactamases Produced by Multidrug Resistant Bacteria
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1 Comparative Assessment of b-lactamases Produced by Multidrug Resistant Bacteria Juhee Ahn Department of Medical Biomaterials Engineering Kangwon National University October 23, 27
2 Antibiotic Development
3 Antibiotic Resistance Threats Highly Resistance Pathogenic Bacteria World Spreading Treatment failure to the last resort of medicine for gonorrhoea (third generation cephalosporin antibiotics) has been confirmed in at least countries (Australia, Austria, Canada, France, Japan, Norway, Slovenia, South Africa, Sweden and the United Kingdom of Great Britain and Northern Ireland). Resistance in Klebsiella pneumoniae common intestinal bacteria that can cause life-threatening infections. Resistance in Escherichia coli to one of the most widely used medicines for the treatment of urinary tract infections is very widespread. Antibiotic resistance is present in every country. Antimicrobial resistant-microbes are found in people, animals, food, and the environment. They can spread between people and animals, and from person to person. Poor infection control, inadequate sanitary conditions and inappropriate food-handling encourage the spread of antimicrobial resistance.
4 Emergence of CR Klebsiella pneumoniae 68 % % % % 4 % 4 % WHO, Antimicrobial Resistance Global Report 24
5 Klebsiella pneumoniae Klebsiella pneumoniae has become an emerging nosocomial pathogen Infectious diseases by : pneumonia, urinary tract infection, bacteremia, cholecystitis, osteomyelitis, meningitis, and thromobophlebitis Antibiotic resistant has been rapidly increased worldwide The increased resistance to β-lactam antibiotics in K. pnuemoniae is mainly caused by production of extended spectrum β-lactamases (ESBL). Infection with ESBL producing leads to the failure of the disease treating. Moreover, various types of β-lactamases accelerate the emergence of multidrug-resistant.
6 Antibiotic Resistance Mechanisms Modification of membrane Permeability Activation in efflux pump systems Target site modification Enzymatic inactivation - b-lactamase production (Hydrolysis of b-lactams) Alteration in metabolic pathway
7 Efflux Pump Activity EtBr accumulation assay Sensitive strain Sensitive strain Resistant strain w/o PAbB Resistant strain with PAbN EP activity (Low fluorescence intensity) Decrease in EP activity EPI-insensitive EP systems - Competitively expel substrates - Specific substrates: chloramphenicol, tetracycline, norfloxacin, nalidixic acid, and levofloxacin
8 Lactamase activity (mmol/min/ml) b-lactamase Activity Nitrocefin hydrolysis assay 4 a 3 2 b c WT-KP CI-KP CA-KP Low b-lactamase activity - PBPs with low affinity for b-lactamases High b-lactamase activity - Production of b-lactamases
9 Classification of b-lactamase Ambler Classification Class A Class D Class C Class B SHV TEM CTX-M KPC OXA AmpC CMY FOX MOX IMP VIM SPM Ampicillin, cephalothin, penicillins, 3 rd G cephalosporins, extended-spectrum cephalosporins, carbapenems Cloxacillin, extended-spectrum cephalospotins, carbapenems Cephamycins, 3 rd G cephalosporins All b-lactams ESBLs AmpC BLs Metallo BLs
10 Experimental Approach Relationship between b-lactamase production and resistance phenotype to understand the resistance mechanisms in WT-KP, CIP-KP, clinical isolates Antibiotic susceptibility testing (with β-lactamase inhibitors) BLIs: BLI-48, clavulanic acid, sulbactam, and tazobactam Dual disc diffusion assay (with β-lactamase inhibitors) PCR assay intersection A A I Blank disc Inhibitor disc antibiotic diffusion inhibitor diffusion synergy effect zone
11 b-lactamase Inhibitors Structurally similar to β-lactam antibiotics Competitive inhibitor of b-lactamases Irreversible inhibition of b-lactam antibiotics Co-administrated with b-lactam antibiotics Clavulanic acid + amoxicillin/ticarcillin Sulbactam + ampicillin/cefoperazone Tazobactam + piperacillin b-lactamases b-lactamase inhibitors
12 Antibiotic Susceptibility Assay MICs (μg/ml) of strains were determined in presence and absence of β-lactamase inhibitors * CO, control; BLI-48, BL; clavulanate, CA; sulbactam, SB; tazobactam, TB. ** AMP, ampicillin; CFT, cefotaxime; CFX, cefoxitin; CFZ, ceftazidime; CFA, ceftriaxone; CEP, cephalothin; IMI, imipenem; MER, meropenem; PIP, piperacillin. S, susceptible; I, intermediate; R, resistant.
13 BLI-48 ATCC 2337 ATCC 2337CIP Ampicillin Ab + Ab Cefotaxime Cefoxitin Ceftazidime Ceftriaxone BLI-48 Cephalothin Piperacillin
14 BLI-48 Increased susceptibility - 237: cefotaxime, cefoxitin, ceftazidime, ceftriaxone, cephalothin, and piperacillin (Hydrolysis inhibited by BLI-48) - 263: cefotaxime, ceftazidime, ceftriaxone, and piperacillin No susceptibility changes - 263: ampicillin, cefoxitin, and cephalothin Antb. ATCC 2337 ATCC 2337CIP BLI-48 BLI-48 BLI-48 BLI-48 BLI AMP CFT CFX CFZ CFA CEP PIP
15 Clavulanic acid ATCC 2337 ATCC 2337CIP Ampicillin Ab + Ab Cefotaxime Cefoxitin Ceftazidime Ceftriaxone Clavulanate Cephalothin Piperacillin
16 Clavulanic acid Increased susceptibility - ATCC2337, ATCC-CIP, and 272: ampicillin and piperacillin - 237: cefotaxime and piperacillin No susceptibility changes and 263: ampicillin, cefoxitin, and cephalothin Antb. ATCC 2337 AMP 6..4 CFT ATCC 2337CIP Clav. Clav. Clav. Clav. Clav CFX CFZ CFA CEP PIP
17 Sulbactam ATCC 2337 ATCC 2337CIP Ampicillin Ab + Ab Cefotaxime Cefoxitin Ceftazidime Ceftriaxone Sulbactam Cephalothin Piperacillin
18 Sulbactam Increased susceptibility - 272: ampicillin, cefotaxime, ceftazidime, ceftriaxone, cephalothin, and piperacillin No susceptibility changes - ATCC2337 and ATCC-CIP: ampicillin, cefotaxime, cefoxitin, ceftazidime, ceftriaxone, and cephalothin Antb. ATCC 2337 ATCC 2337CIP Sulb. Sulb. Sulb. Sulb. Sulb AMP CFT CFX CFZ CFA CEP PIP
19 Tazobactam ATCC 2337 ATCC 2337CIP Ampicillin Ab + Ab Cefotaxime Cefoxitin Ceftazidime Ceftriaxone Cephalothin Tazobactam Piperacillin
20 Tazobactam Increased susceptibility - 237, 263, and 272: ceftazidime and piperacillin No susceptibility changes - 237: ampicillin and cefoxitin - 262: ceftazidime, ceftriaxone, and cephalothin Antb. ATCC 2337 ATCC 2337CIP Tazo. Tazo. Tazo. Tazo. Tazo AMP CFT CFX CFZ CFA CEP PIP
21 Marker SHV AmpR-Act AmpC SME- CTX-M FOX/MOX Marker VIM SPM OXA-23 OXA-48 OXA-8 Genotypic Characteristics of BLs. kb ATCC kb.7 kb ATCC 2337-CIP Agarose gel electrophoresis of β-lactamases extracted from selected in this study Predominant b-lactamases: plasmid-mediated ESBLs and chromosomally-mediated AmpC
22 Summary The production of various types of β-lactamase is considered a major mechanism responsible for the resistance to β-lactam antibiotics The MIC values of β-lactams against ATCC 2337, ciprofloxacininduced ATCC 2337, 237, K. pneumoniae 263, and 272 corresponded with the production of β-lactamases obtained from the dual disc diffusion assay. The constitute class A (SHV) and inducible class C (AmpC and FOX/MOX) were confirmed in ATCC 2337, ciprofloxacin-induced ATCC 2337, and 272. The class A (SHV), class C(FOX/MOX), and class D(OXA-48) were confirmed in 263. The β-lactam resistance associated with the production of β- lactamases in strains can provide useful information for designing an infection control strategy in combination with appropriate β-lactamase inhibitors.
23 Thank you!
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