ESBL in Iraq-PhD thesis

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Dr. Alaa H. Al-Charrakh, Ph.D thesis, Baghdad University Bacteriological and Genetical Study on Extended-Spectrum B-Lactamases of Klebsiella isolated from Middle Euphrates region, Iraq

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Bacteriological and Genetical Study on Extended-Spectrum B-Lactamases of Klebsiella isolated from Middle Euphrates region, Iraq A Ph.D. thesis by Alaa H. Al-Charrakh :

Bacteriological and Genetical Study on Extended-Spectrum B-Lactamases of Klebsiella isolated from Middle Euphrates region, Iraq A Ph.D. thesis by Alaa H. Al-Charrakh

Taxonomy of genus Klebsiella :

Taxonomy of genus Klebsiella Klebsiella species are differentiated according to their biochemical reactions into six species; K. pneumoniae, K. oxytoca, K. terrigena ,K. ornithinolytica , K. planticola , and K. trevisanii. Recently, in 2001, The data from sequence analyses along with the previously reported biochemical and DNA- DNA hybridization data support the division of the genus Klebsiella into two genera. The name Raoutella is proposed as a genus name of the species ; K. terrigena ,K. ornithinolytica , K. planticola , and K. trevisanii.

Bacterial resistance to β-lactam antibiotics :

Bacterial resistance to β-lactam antibiotics 1-Hydrolysis of the drug by β-lactamase enzyme. 2- Reduced permeability of the outer membrane. 3- Decreased affinity of the target penicillin-binding proteins (PBPs).

Extended-spectrum β-lactamases:

Extended-spectrum β -lactamases Most ESBLs arise by mutations in genes for common plasmid-mediated β-lactamases (TEM-1, SHV-1,OXA-1) that alter the configuration of the active site so as to increase the affinity and hydrolytic activity of the enzyme for oxyimino β -lactams.

Mécanisme d ’action des -lactamases :

Mécanisme d ’action des  -lactamases Les -lactamases hydrolysent le pont amide du cycle -lactame de l ’antibiotique pour donner un acyl-enzyme qui sera ensuite dégradé en acide inactif.

Classification des -lactamases :

Classification des -lactamases De nombreuses classifications existent, qui prennent comme critère: le phénotype: classification utilisée en routine qui sépare les enzymes en pénicillinases, céphalosporinases, -lactamases à large spectre et -lactamases à spectre étendu la nature du site actif (classification d ’Ambler) la séquence d ’acide aminé les caractéristiques physiques l ’origine (plasmidique ou chromosomique) Les classifications d ’Ambler et de Bush-Jacob y -Medeiros sont aujourd’hui les plus pertinentes.

Classification d ’Ambler:

Classification d ’Ambler Classe A: Enzymes à sérine Actives sur les pénicillines et à un moindre degré sur les céphalosporines Inhibées par l ’acide clavulanique Médiation chromosomique ou plasmidique Comprend les -lactamases de type TEM, SHV, CTX-M Autres enzymes de la classe A (souvent inductibles) Classe B: Métallo-enzymes comportant un atome de zinc divalent Actives sur les céphalosporines et les carbapénèmes Insensibles à l ’acide clavulanique Médiation chromosomique ou plasmidique Inhibées par l ’EDTA

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Classe C: Enzymes à sérine; céphalosporinases Actives sur les céphalosporines (C1G ± C2G et C3G) Insensibles à l ’acide clavulanique A médiation chromosomique le plus souvent Classe D: Enzymes à sérine Actives sur les pénicillines et plus particulièrement les pénicillines M Plus ou moins sensibles à l ’acide clavulanique Comprend les enzymes de type OXA

ESBL detection methods :

ESBL detection methods

Test de double diffusion(1):

Dépôt des disques d ’antibiotiques. Incubation 18 à 24 heures à l ’étuve à 37°C. Test de double diffusion(1) AMC: amoxicilline + ac.clavulanique CAZ: ceftazidime CTX: céfotaxime FEP: céfépime AZT: aztréonam AZT 2 cm 3 cm CAZ CTX AMC 2 cm FEP 2 cm

Test de double diffusion (2):

Test de double diffusion (2) Interprétation: Il y a présence d ’une BLSE ou «bouchon de champagne » si la zone d ’inhibition de la pousse bactérienne est élargie entre le disque d ’AMC et le disque de Céfépime ou Céftazidime (effet de synergie) ou d ’AZT. CTR Acide clavulanique céfépime

Materials and Methods:

Materials and Methods

-A total of 298 samples (89 environmental and 209 clinical ) were collected during the period from January to July 2003 from different sites in Middle Euphrates area.:

- A total of 298 samples (89 environmental and 209 clinical ) were collected during the period from January to July 2003 from different sites in Middle Euphrates area.

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● The bacterial isolates were recovered from the samples and identified to the level of subspecies using biochemical and morphological tests. Screening test for β- lactam resistance Preliminary screening of klebsiella isolates resistant to β-lactam antibiotics was carried out using pick and patch method on Muller-Hinton agar supplemented with Amp and Amx ( each alone ). Detection of β- lactamase production Rapid iodometric method was performed for all bacterial isolates that were resistant to β- lactam antibiotics. Antimicrobial disk susceptibility tests The resistance of the bacterial isolates to antimicrobial agents was determined using disk diffusion method and all bacterial isolates that were positive in β- lactamase production test, were tested.

Detection of ESBL production:

Detection of ESBL production Three methods were performed: 1- Disk approximation method. 2- NCCLS screening method: This method includes two main procedures : A- Screening test: - Disk diffusion method on Muller- Hinton agar with antibiotic disks (Ceftazidime, Cefotaxime, and Ceftriaxone ) was performed. - Each bacterial isolate should be considered a potential ESBL- producer, if the i nhibition zone (mm) were as follows: Ceftazidime < 22 mm ,Cefotaxime < 27 mm,Ceftriaxone < 25 mm B- Phenotypic confirmatory test : Using disk diffusion method, both ceftazidime and cefotaxime, alone and in combination with clavulanic acid ( 30μg/10μg ) were tested. - Inhibition zone of ≥ 5 mm increase in diameter for either antimicrobial agent tested in combination with clavulanic acid versus its zone when tested alone confirms an ESBL - producing isolate.

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3- Detection of ESBLs with clavulanate: Minimum inhibitory concent. of cefotaxime, ceftazidime, and ceftriaxone with and without clavulanic acid (4μg/ml) were determined,using agar dilution method. The test was considered positive (ESBL-production), if the MIC against the tested β-lactam antibiotic was reduced ≥ 8-fold by clavulanate. Determination of MICs of ESBL-producing isolates: ● The two-fold agar dilution susceptibility method was used for determination of MICs of number of β- lactam antibiotics. ● Agar dilution end points were recorded according to NCCLS documentations.

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Isolation of plasmid DNA ● Three ESBL–producing and two klebocin -producing Klebsiella isolates were selected and detected for their plasmid profiles. ● Plasmid DNA was extracted using alkaline lysis method described by Pospich and Neumann (1995 ). Agarose gel electrophoresis DNA contents of standard strain as well as plasmid profile of ESBL-producing isolates and of klebocin- producing isolates were determined using agarose gel electrophoresis . Bacterial conjugation ● Two ESBL -producing isolates and two klebocin- producing isolates were selected ( as donor cells ) for studying bacterial conjugation with the standard strain E. coli J53 ( as recipient cell ). ● The transconjugants were tested for their ability to produce β- lactamase enzyme, ESBL ,as well as for their antibiotic resistance and MICs to β-lactam antibiotics. ● The transconjugants resulted from conjugation between klebocin- producing strains and the standard strain E. coli J53, were detected for their ability to produce bacteriocin.

Results and Discussion:

Results and Discussion

Isolation and Identification of Bacterial Isolates:

Isolation and Identification of Bacterial Isolates It was found that 84 isolates belonged to K. pneumoniae and 4 isolates were K. oxytoca . All known subspecies of K. pneumoniae were isolated for the first time in the present study: K. pneumoniae subsp. pneumoniae (87 %) K. pneumoniae subsp. ozaenae (9.5 %) K. pneumoniae subsp. rhinoscleromatis (3.5 %).

Primary Screening of β-Lactam Resistant Isolates :

Primary Screening of β-Lactam Resistant Isolates Results showed that 65 Klebsiella strains (73.8 %) were resistant to both ampicillin and amoxicillin. Detection of β- Lactamase Production - Results showed that 38 isolates (58.5 %) gave positive results with this test. This ratio indicated that enzymatic resistance was prevalent in Klebsiella isolates. Results showed that 26 isolates (68.4%) gave rapid reaction within few seconds and this indicated that these β-lactamases were constitutive.

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Figure (3-3) :Antibiotic resistance pattern of β-lactamase- producing Klebsiella pneumoniae isolates .

Detection of Extended-Spectrum β-Lactamases: :

Detection of Extended-Spectrum β-Lactamases: 1-Disk Approximation Method: Out of the 38 β-lactamase producing strains, only 4 (10.5 %) ESBL–producing isolates were detected. Figure (3-4) :Detection of ESBL production in K. pneumoniae 6C by disk approximation method. 1- Amoxicillin-clavulana2te disk (20/10 μg ) 2- Cefotaxime disk ( 30 μg ). 3- Ceftazidime disk ( 30 μg ). 4- Ceftriaxone disk ( 30 μg ).

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2- Screening & confirmatory tests of NCCLS: Results showed that only 6 (15.7 %) ESBL - producing isolates were detected by this method. 3- Detection of ESBLs by Clavulanate: Results revealed that 8 (21%) ESBL– producing strains were detected as they reduced the MICs of CTX, CAZ, and CTR, > 12 fold. “Determination of MIC with and without clavulanate” was the most accurate method in detection of ESBL- producing isolates comparing with the two other methods used. The disk approximation,was the least accurate method in detecting such enzymes.

MICs Determination of ESBL –producing Isolates: :

MICs Determination of ESBL –producing Isolates:

Isolation of Plasmid DNA: :

Isolation of Plasmid DNA:

Bacterial Conjugation :

Bacterial Conjugation Results reveal that conjugation frequency was relatively low and ranged from 5 x 10 -7 to 2 x 10 -6 . The transconjugants expressed their antibiotic resistance when they were able to grow in selective medium containing Amp and sodium azide. The acquisition of Amp resistance in recipient cell indicated that this property was plasmid-mediated.

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Transconjugants were detected for their ability to produce β-lactamase . Results revealed that all transconjugants were able to give positive result in β-lactamase production test . Results also showed that the transconjugants have acquired multiple resistance for different antibiotics compared with the original donor cells.

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The transconjugants were also detected for MICs to β-lactam antibiotics :

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• The transconjugants were also detected for their ability to produce ESBL using determination of MIC with and without clavulanic acid. Results revealed that both of transconjugants were ESBL-producers as they reduced the MICs of CTX, CAZ, and CTR, > 12 fold.

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• The transconjugants resulted from conjugation between klebocin -producing isolates and the standard E. coli J53, were detected for their ability to produce klebocin using cup assay method.

Conclusions :

Conclusions 1- More than 58% of K. pneumoniae strains have an enzymatic resistance to β-lactam antibiotics by production of β-lactamase enzymes. 2- Twenty one percent of β-lacatamse-producing Klebsiella strains are ESBL-producers . 3- Determination of MIC with and without clavulanate, was the most accurate method for detection of ESBL- producing strains and the disk approximation , was the least accurate method in detecting such enzymes. 4- ESBL-production trait, in clinical K. pneumoniae strains, is carried on self-transmissible conjugative plasmids which encoded resistance to β-lacatams and other non-β-lacatam antibiotics. 5- Brain Heart Infusion medium supplemented with 5 % glycerol and cup assay method, were the best medium and method used for detection of klebocin-producing isolates. 6- 62.5 % of Klebsiella strains isolated in the present study are klebocin-producers . 7- Klebocins of Klebsiella strains have a broad antimicrobial spectrum and are active, in addition to Klebsiella strains, on many pathogenic species of Gram-negative and some Gram-positive bacteria. 8- Klebocin production trait is carried on a large conjugative and self-transmissible plasmid which also encoded resistance to some β-lactams and other antibiotics.

Recommendations :

Recommendations 1- Study of prevalence of ESBL-producing Klebsiella strains in other areas of Iraq and prevalence of these enzymes in other species of Gram-negative and Gram-positive bacteria. 2- The use of recent techniques like analytical isoelectric focusing and polymerase chain reaction for detection and identification of ESBLs, and using pulsed-field gel electrophoresis and randomly amplified DNA polymorphism analysis for studying the epidemiology of extended-spectrum β-lactamase-producing Klebsiella pneumoniae . 3- Study of the epidemiology of Klebsiella pneumoniae in Iraq by typing the local isolates using standard klebocin-producing strains. 4- Studying the future aspects of the possibility of using klebocins of Klebsiella pneumonia e as alternative to the broad-spectrum antibiotics for treatment of infections caused by Klebsiella and some genera of enteric bacteria.

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