The aims were to investigate the level of antibiotic-resistant bacteria in hospital and urban wastewater and to determine the similarity of isolates obtained from wastewater and hospitalized patients. Wastewater samples were collected in September 2013 and 2014. After identification using MALDI-TOF MS, beta-lactamase production was determined by relevant phenotypic tests. Genes responsible for the production of single beta-lactamase groups and Qnr proteins were established. The epidemiological relationship of the isolates from wastewater and hospitalized patients was determined by PFGE. A total of 51 isolates of enterobacteria were obtained. Overall, 45.1% of them produced broad-spectrum beta-lactamases. Genes encoding TEM, SHV, CTX-M, CIT, DHA and EBC types of enzymes and Qnr proteins were detected. No broad-spectrum beta-lactamase production was confirmed in the urban wastewater treatment plant. The most important finding was the detection of two identical isolates of K. pneumoniae in 2013, one from a patient's urinary catheter and the other from a wastewater sample.
- MeSH
- antibakteriální látky farmakologie MeSH
- Bacteria účinky léků enzymologie genetika MeSH
- bakteriální léková rezistence genetika fyziologie MeSH
- beta-laktamasy metabolismus MeSH
- mikrobiologie vody MeSH
- nemocnice * MeSH
- odpadní voda mikrobiologie MeSH
- regulace genové exprese enzymů MeSH
- regulace genové exprese u bakterií MeSH
- velkoměsta * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- velkoměsta * MeSH
A total of 78 bacterial strains with known β-lactamases were used to optimize a rapid detection system consisting of multiplex polymerase chain reaction and melting curve analysis to amplify and identify blaTEM, blaSHV, and blaCTX-M genes in a single reaction. Additionally, to evaluate the applicability of this method, 32 clinical isolates of Escherichia coli displaying an extended-spectrum β-lactamase phenotype from patients hospitalized at intensive care units were tested. Results were analyzed by the Rotor-Gene operating software and Rotor-Gene ScreenClust HRM Software. The individual melting curves differed by a temperature shift or curve shape, according to the presence of β-lactamase genes. With the use of this method and direct sequencing, blaCTX-M-15-like was identified as the most prevalent β-lactamase gene. In conclusion, this novel detection system seems to be a suitable tool for rapid detection of present β-lactamase genes and their characterization.
- MeSH
- beta-laktamasy genetika MeSH
- Escherichia coli genetika izolace a purifikace MeSH
- infekce spojené se zdravotní péčí mikrobiologie MeSH
- infekce vyvolané Escherichia coli mikrobiologie MeSH
- jednotky intenzivní péče MeSH
- lidé MeSH
- multiplexová polymerázová řetězová reakce MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Background. In 1928, the first antibiotic, penicillin, was discovered. That was the beginning of a great era in the development and prescription of antibiotics. However, the introduction of these antimicrobial agents into clinical practice was accompanied by the problem of antibiotic resistance. Currently, bacterial resistance to antibiotics poses a major problem in both hospital and community settings throughout the world. Methods and results. This review provides examples of modern genetic methods and their practical application in the field of extended-spectrum ß-lactamase detection. Since extended-spectrum ß-lactamases are the main mechanism of Gram-negative bacterial resistance to oxyimino-cephalosporins, rapid and accurate detection is requested in common clinical practice. Conclusions. Currently, the detection of extended-spectrum ß-lactamases is primarily based on the determination of bacterial phenotypes rather than genotypes. This is because therapeutic decisions are based on assessing the susceptibility rather than presence of resistance genes. One of the main disadvantages of genetic methods is high costs, including those of laboratory equipment. On the other hand, if these modern methods are introduced into diagnostics, they often help in rapid and accurate detection of certain microorganisms or their resistance and pathogenic determinants.
K dôležitým bakteriálnym b-laktamázam so stúpajúcim klinickým významom patria v súčasnej dobe AmpC enzýmy. Cieľom štúdie bolo stanovenie ich výskytu u kmeňov Klebsiella pneumoniaeizolovaných od hemato-onkologických pacientov na základe prospektívnej štúdie. Materiál a metódy: Z klinického materiálu pacientov hospitalizovaných na Hemato-onkologickej klinike Fakultnej nemocnice Olomouc boli v priebehu dvoch mesiacov izolované kmene daného species. K identifikácii boli použité štandardné mikrobiologické postupy a automatizovaný systém Vitek 2. Produkcia AmpC b-laktamáz bola orientačne stanovená fenotypovými testami a následne konfirmovaná PCR dôkazom génov kódujúcich tieto enzýmy. Výsledky: V uvedenom období bolo získaných celkovo 35 izolátov K. pneumoniae. U 7 z nich bola fenotypovo predbežne preukázaná produkcia AmpC b-laktamáz. S využitím metódy multiplex PCR sa potvrdilo toto určenie a stanovil sa DHA-typ u všetkých izolátov. Záver: Všetky AmpC-pozitívne izoláty vykazovali falošnú citlivosť aspoň na jeden z testovaných cefalosporínov III. generácie. Klinicky zjavná infekcia bola dokumentovaná u jedného pacienta. Z uvedených výsledkov vyplýva možnosť výskytu AmpC b-laktamáz u klinicky významných izolátov K. pneumoniae.
Currently, important bacterial b-lactamases of increasing clinical significance include AmpC enzymes. The aim was to assess their occurrence in Klebsiella pneumoniae strains isolated from patients with haematological malignancies in a prospective study. Material and methods: Over a 2-month period, strains of the species were isolated from clinical material obtained from patients hospitalized at the Department of Haemato-Oncology of the University Hospital Olomouc. The strains were identified using standard microbiological techniques and the Vitek 2 automated system. Production of AmpC b-lactamases was roughly determined by phenotypic tests and subsequently confirmed by PCR detection of genes encoding these enzymes. Results: During the above-mentioned period, a total of 35 Klebsiella pneumoniae isolates were collected. In 7 of them, production of AmpC b-lactamases was preliminarily detected by phenotypic test. The multiplex PCR method confirmed phenotyping and determined DHA types in all the isolates. Conclusions: All AmpC-positive isolates were false-susceptible to at least one of the tested third-generation cephalosporins. In one patient, clinically apparent infection caused by this strain was documented. The reported results suggest the possibility of occurrence of AmpC- b-lactamases in Klebsiella pneumoniae strains with clinical significance.
Cieľ práce: Cieľom štúdie bolo stanovenie prevalencie ESBL-pozitívnych izolátov Klebsielh pneumoniae u pacientov v intenzívnej starostlivosti a ich molekulárno-biologická analýza. Materiál a metódy: V období 5 mesiacov boli od pacientov hospitalizovaných na Klinike anestéziológie a resuscitácie Fakultnej nemocnice Olomouc izolované kmene Klehsiella pneumoniae. U každého izolátu bol určený antibiogram štandardnou dilučnou mikrometódou a produkcia ESBL bola stanovená modifikovaným double disk synergy testom. Na dôkaz prítomnosti génu blaTEM a blaSHV bola použitá PCR. Izoláty produkujúce SHV a TEM typy β-laktamáz boh ďalej typizované použitím metódy polymorfizmu dĺžky restrikčných fragmentov (RFLP) na identifikáciu najbežnejšie sa vyskytujúcich mutácií zodpovedných za vznik ESBL fenotypu. Posúdenie podobnosti, resp. identity izolátov, bolo uskutočnené pulznou gelovou elektroforézou (PFGE) fragmentov DNA, naštiepených pomocou reštrikčnej endonukleázy Xbal. Výsledky: Celkovo bolo získaných 67 izolátov Klebsiella pneumoniae. U 13 z nich bola stanovená produkcia ESBL a pomocou PCR dokázaná pritomnost WasHv génu. Reštrikčné štiepenie pomocou Nhel odhalilo výskyt mutácie v pozícii 238 u všetkých SHV-pozitívnych PCR produktov. Prítomnosť génu kódujúceho širokospektrú β-laktamázu TEM typu však nebola potvrdená. Molekulárno-biologická typizácia pomocou PFGE zistila prítomnosť 11 rôznych kmeňov. Záver. Prevalencia ESBL-pozitívnych kmeňov KlebsieJJa pneumoniae dosiahla u sledovanej skupiny pacientov v intenzívnej starostlivosti hodnoty 19,4 %. Analýza SHV a TEM produktov PCR metódou RFLP preukázala výskyt ESBL typu SHV. Celkovo 84,6 % kmenov malo jedinečný reštrikčný profil. Výsledky dokladujú nielen dobrú úroveň hygienicko-epidemiologických režimov na sledovanej klinike, ale aj racionálnu antibiotickú politiku.
Objectives: The study aimed at the assessment of the prevalence of ESBL-positive isolates of Klebsiella pneumoniae in intensive care patients and their molecular biology analysis. Material and methods: Over a 5-month period, Klebsiella pneumoniae strains were isolated from patients hospitalized at the Department of Anaesthesiology and Resuscitation of the University Hospital in Olomouc. For each isolate, an antibiogram was performed by the standard microdilution method and the production of ESBL was determined by the modified double-disk synergy test. PCR was used to demonstrate the presence of the blaTEM and blaSHV genes. The isolates producing SHV- and TEM-types of β-lactamases were typed using the restriction fragment length polymorphism (RFLP) method to identify the most common mutations responsible for the development of an ESBL phenotype. Similar or identical isolates were determined by pulsed-field gel electrophoresis (PFGE) of DNA fragments cleaved by the XbaI restriction endonuclease. Results: A total of 67 isolates of KJebsieJJa pneumoniae were obtained. In 13 of them, the production of ESBL was detected and the presence of the blaSHV gene was confirmed by PCR. Restriction cleavage by Nhei revealed mutations at position 238 in all SHV-positive PCR products. The restriction analysis did not confirm the presence of the gene encoding TEM-type extended-spectrum β-lactamase. Molecular biology typing by PFGE detected the presence of 11 different strains. Conclusions: In the observed group of intensive care patients, the prevalence of ESBL-positive strains of Klebsiella pneumoniae reached 19.4 %. The analysis of SHV and TEM products of PCR by the RFLPP method showed the prevalence of SHV-type ESBL. Overail, 84.6 % of the strains had unique restriction profiles. The results suggest both high levels of hygienic and epidemiological measures at the monitored department and rational antibiotic policy.
