Metalo-β-laktamaze u kliničkih sojeva Pseudomonas aeruginosa otpornih na karbapenemske antibiotike [Metalo-β-lactamases in carbapenem-resistant clinical isolates of Pseudomonas aeruginosa]

Sardelić, Sanda (2010) Metalo-β-laktamaze u kliničkih sojeva Pseudomonas aeruginosa otpornih na karbapenemske antibiotike [Metalo-β-lactamases in carbapenem-resistant clinical isolates of Pseudomonas aeruginosa]. PhD thesis, Sveučilište u Zagrebu.

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Abstract

In the last two decades, metalo-β-lactamases of type VIM, IMP, GIM, SPM, and recently AIM are frequently described in Pseudomonas aeruginosa. Those enzymes hydrolyse all known β-lactams except aztreonam. The aim of this study was to determine clinical and epidemiological significance of P. Aeruginosa strains producing metallo-β-lactamases, their genetic similarity and distribution, relationship between serotypes and resistotypes and to check the reliability of laboratory methods in testing sensitivity of pseudomonas to antibiotics. In the period from 2001 to 2007, a total of 169 P. aeruginosa isolates with reduced susceptibility to carbapenems were collected from all clinical departments of a regional hospital. The sensitivity to antipseudomonal antibiotics was determined by disk diffusion and microdilution methods (for ceftazidime alone and with addition of clavulanic and boronic acid; for imipenem alone and with EDTA and phenantrolin). O-serotypes, resistotypes and PFGE-genotypes were determined for each strain and compared. Comparison of MICs to several antibiotics was used in estimation of efflux pumps' activity. Outer membrane proteins were detected by SDS-PAGE. Genes encoding metallo-β-lactamases - blaVIM and blaIMP were detected by PCR. By using several primers' pairs the whole integron containing MBL gene was amplified and sequenced. Clinical data obtained retrospectively from the patients survey sheet accompanying every isolate were statistically analyzed. In six isolates the presence of VIM-2 MBL was proved. Gene encoding the enzyme was located within the first gene cassette in so far undescribed integron called InCRO with 3277 bp. MBL-positive isolates belonged to two clones and were of serotype O11 and O12. Most of the MBL-negative P. aeruginosa isolates had derepressed AmpC, loss of OprD and increased activity of efflux pumps. Genotyping showed the dominance of two clones - one harboring multiresistant O12 P. aeruginosa, and other with prevailing O11 serotype. Patients with multiresistant pseudomonas isolates were hospitalized longer and had higher mortality compared to patients who had imipenem-reistant but not multiresistant isolates. Laboratory methods of testing the sensitivity of carbapenem-resistant P. aeruginosa to antibiotic are unreliable and subject to variations.

Abstract in Croatian

Posljednih 20-ak godina u Pseudomonas aeruginosa sve češće se opisuju stečene β-laktamaze tzv. metalo-β-laktamaze koje mogu biti VIM, IMP, GIM, SPM i AIM tipa, a hidroliziraju sve poznate β-laktame s izuzetkom aztreonama. Cilj ovog rada bio kliničko-epidemiološko značenje izolata P. aeruginosa koji posjeduju metalo-β-laktamaze, njihovu genetsku srodnost i proširenost, odnos serotipova i rezistotipova te provjeriti pouzdanost laboratorijskih metoda u testiranju osjetljivosti P. aeruginosa na antibiotike. U razdoblju od 2001. do 2007. godine prikupljeno je ukupno 169 izolata P. aeruginosa sa smanjenom osjetljivošću na karbapeneme s kliničkih odjela jedne regionalne bolnice. Svim izolatima određena je osjetljivost prema antipseudomonasnim antibioticima disk difuzijskom i mikrodilucijskom metodom (za ceftazidim i s dodatkom klavulanske i boroničke kiseline; za imipenem s dodatkom EDTA i fenantrolina), određen im je O-serotip te genetska srodnost uspoređena elektroforezom u pulzirajućem polju. Odnosom MIK-ova antibiotika procijenjena je aktivnost efluksnih crpki. Proteini vanjske membrane detektirani su SDS-PAGE elektroforezom. Geni koji kodiraju metalo-β-laktamaze blaVIM i blaIMP istraženi su lančanom reakcijom polimeraze. Nizom parova početnica amplificiran je i sekvenciran integron koji sadržava gen za metalo-β-laktamazu. Statistički su obrađeni klinički podatci bolesnika dobiveni retroaktivno iz anketnog listića koji je pratio svaki izolat pseudomonasa. U šest izolata dokazano je prisustvo VIM-2 metalo-β-laktamaze. Gen se nalazio u sastavu prve genske kazete u do sada neopisanom integronu lociranom na kromosomu nazvanom InCRO, ukupne veličine 3277 parova baza. MBL pozitivni izolati pripadali su dvama klonovima i serotipovima O11 i O12. Otpornost na karbapeneme ostalih izolata P. aeruginosa posljedica je hiperekspresije AmpC β-laktamaze, nepostojanja proteina vanjske membrane OprD i pojačanog rada efluksnih crpki. Usporedba genotipova P. aeruginosa ukazala je na dominaciju dva klona, multirezistentnog klona P. aeruginosa O12 i drugog, u kojem prevladava serotip O11. Bolesnici s multirezistentnim izolatima pseudomonasa dulje su hospitalizirani i imaju veći mortalitet u odnosu na bolesnike koji nemaju multirezistentne izolate. Laboratorijske metode testiranja osjetljivosti P. aeruginosa na antibiotike nepouzdane su i podložne varijacijama.

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