Increasing antimicrobial resistance in nosocomial pathogens, such as Acinetobacter baumannii, is becoming a serious threat to public health. It is necessary to detect β-lactamase-producing microorganisms in clinical settings to be able to control the spread of carbapenem resistance. This study was conducted to evaluate the presence of β-lactamases in a selected clinical isolate of A. baumannii of ST2P/ST195Ox and to characterize possible enzymes, as well as its β-lactam resistome, using PCR and whole-genome sequencing analysis. PCR and sequencing confirmed that the isolate harbored five bla gene alleles, namely, blaADC-73, blaTEM-1, blaOXA-23, blaOXA-58 and blaOXA-66, as well as aminoglycosides, macrolides, sulfonamides and tetracyclines resistance determinants, which were either chromosomally and/or plasmid located. Furthermore, a gene order comparison using MAUVE alignment showed multiple changes compared with the clinical isolate of Malaysian A. baumannii AC30 genome and 76 regions with high homology. This study suggests that resistance to β-lactams in this A. baumannii isolate is mainly due to an overproduction of β-lactamases in combination with other resistance mechanism (efflux pump system).
- Publikační typ
- kazuistiky MeSH
The growing bacterial resistance to available β-lactam antibiotics is a very serious public health problem, especially due to the production of a wide range of β-lactamases. At present, clinically important bacteria are increasingly acquiring new elements of resistance to carbapenems and polymyxins, including extended-spectrum β-lactamases (ESBLs), carbapenemases and phosphoethanolamine transferases of the MCR type. These bacterial enzymes limit therapeutic options in human and veterinary medicine. It must be emphasized that there is a real risk of losing the ability to treat serious and life-threatening infections. The present study aimed to design specific oligonucleotides for rapid PCR detection of ESBL-encoding genes and in silico analysis of selected ESBL enzymes. A total of 58 primers were designed to detect 49 types of different ESBL genes. After comparing the amino acid sequences of ESBLs (CTX-M, SHV and TEM), phylogenetic trees were created based on the presence of conserved amino acids and homologous motifs. This study indicates that the proposed primers should be able to specifically detect more than 99.8% of all described ESBL enzymes. The results suggest that the in silico tested primers could be used for PCR to detect the presence of ESBL genes in various bacteria, as well as to monitor their spread.
- Publikační typ
- časopisecké články MeSH
Increasing antimicrobial resistance of nosocomial pathogens is becoming a serious threat to public health. To control the spread of this resistance, it is necessary to detect β-lactamase-producing organisms in the clinical setting. The aims of the study were to design a PCR assay for rapid detection of clinically encountered β-lactamase genes described in Enterobacteriaceae and Gram-negative non-fermenting bacteria. The functionality of proposed primers was verified using eight reference strains and 17 strains from our collection, which contained 29 different β-lactamase genes. PCR products of the test strains were confirmed by Sanger sequencing. Sequence analysis was performed using bioinformatics software Geneious. Overall, 67 pairs of primers for detecting 12 members of the class C β-lactamase family, 15 members of class A β-lactamases, six gene families of subclass B1, one member each of subclasses B2, B3 and class D β-lactamases were designed, of which 43 pairs were experimentally tested in vitro. All 29 β-lactamase genes, including 10 oxacillinase subgroups, were correctly identified by PCR. The proposed set of primers should be able to specifically detect 99.7% of analyzed β-lactamase subtypes and more than 79.8% of all described β-lactamase genes.
- MeSH
- Bacteria enzymologie genetika izolace a purifikace MeSH
- bakteriologické techniky * MeSH
- beta-laktamasy genetika metabolismus MeSH
- beta-laktamová rezistence genetika MeSH
- DNA bakterií genetika MeSH
- DNA primery MeSH
- lidé MeSH
- polymerázová řetězová reakce * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Oxacillinases (OXA) have been mostly described in Enterobacteriaceae, Acinetobacter, and Pseudomonas species. Recent years have witnessed an increased prevalence of intrinsic and/or acquired β-lactamase-producing Acinetobacter in food-producing animals. This study was conducted to assess the prevalence of OXA among selected bacterial species and to characterize these enzymes by in silico analysis. Screening of OXA was performed by PCR amplification using specific pairs of oligonucleotides. Overall, 40 pairs of primers were designed, of which 6 were experimentally tested in vitro. Among 49 bacterial isolates examined, the presence of blaOXA-1-like genes was confirmed in 20 cases (41%; 19 times in Klebsiella pneumoniae and once in Enterobacter cloacae). No OXA were found in animal isolates. The study results confirmed the specificity of the designed oligonucleotide pairs. Furthermore, the designed primers were found to possess the ability to specifically detect 90.2% of all OXA. These facts suggest that the in silico and in vitro tested primers could be used for single or multiplex PCR to screen for the presence of OXA in various bacteria, as well as to monitor their spread. At the same time, the presence of conserved characteristic amino acids and motifs was confirmed by in silico analysis of sequences of representative members of OXA.
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální proteiny genetika metabolismus MeSH
- beta-laktamasy genetika metabolismus MeSH
- DNA primery chemická syntéza metabolismus MeSH
- Enterobacter cloacae klasifikace účinky léků enzymologie genetika MeSH
- Escherichia coli klasifikace účinky léků enzymologie genetika MeSH
- exprese genu MeSH
- fylogeneze MeSH
- gramnegativní bakteriální infekce diagnóza epidemiologie mikrobiologie veterinární MeSH
- Klebsiella pneumoniae klasifikace účinky léků enzymologie genetika MeSH
- kur domácí mikrobiologie MeSH
- lidé MeSH
- maso mikrobiologie MeSH
- mikrobiální testy citlivosti MeSH
- multiplexová polymerázová řetězová reakce metody MeSH
- peniciliny farmakologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
Antibiotic resistance is an ever-increasing global problem. Major commercial antibiotics often fail to fight common bacteria, and some pathogens have become multi-resistant. Polymyxins are potent bactericidal antibiotics against gram-negative bacteria. Known resistance to polymyxin includes intrinsic, mutational and adaptive mechanisms, with the recently described horizontally acquired resistance mechanisms. In this review, we present several strategies for bacteria to develop enhanced resistance to polymyxins, focusing on changes in the outer membrane, efflux and other resistance determinants. Better understanding of the genes involved in polymyxin resistance may pave the way for the development of new and effective antimicrobial agents. We also report novel in silico tested primers for PCR assay that may be able distinguish colistin-resistant isolates carrying the plasmid-encoded mcr genes and will assist in combating the spread of colistin resistance in bacteria.
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální léková rezistence účinky léků genetika MeSH
- bakteriální proteiny účinky léků genetika MeSH
- gramnegativní bakteriální infekce farmakoterapie genetika MeSH
- kolistin farmakologie MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- polymyxiny farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
BACKGROUND: Various food-producing animals have been recognized in recent years as a potential reservoir for the spread of antibiotic resistant bacteria that may pose a risk to human health and therefore their dissemination in the food production chain needs to be assessed. METHODS: In this study, 450 boot swabs from chicken farms were analyzed for the presence of antimicrobial resistance with a focus on β-lactams resistance in Acinetobacter species. RESULTS: Two β-lactamase-encoding genes were first time identified in Acinetobacter lwoffii and Acinetobacter schindleri isolates. The deduced amino acid sequence of OXA-496 shared 93.8% identity with OXA-363. The second OXA-134-like enzyme, OXA-537, had the highest sequence identity (97.8%) with OXA-235 and OXA-237. CONCLUSIONS: The results of this study illustrate the occurrence of new OXA-134-like β-lactamases, called OXA-496 and OXA-537, carrying strains of Acinetobacter lwoffii and Acinetobacter schindleri in chicken farm litter, and highlight the possible role of Acinetobacter as a reservoir of resistance genes.
- MeSH
- Acinetobacter genetika MeSH
- bakteriální léková rezistence genetika MeSH
- beta-laktamasy genetika MeSH
- beta-laktamová rezistence genetika MeSH
- farmy MeSH
- kur domácí MeSH
- mikrobiální testy citlivosti MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
BACKGROUND: Planktonic stationary and exponential cultures of Pseudomonas aeruginosa are highly resistant to killing by bactericidal antimicrobials because of the presence of persisters, cells that are multidrug tolerant and play a key role in the recalcitrance of biofilm infections. AIM: The aim of this study was to investigate the formation of persister cells in P. aeruginosa stationary vs. exponential cultures using different class antimicrobials. METHODS: The susceptibilities of P. aeruginosa PAO1 wild-type and mutant strains to antimicrobials were determined by standard microtiter broth dilution method. In order to determine persister formation, dose- and time-dependent killing experiments were performed with antibiotics. RESULTS: Ceftazidime (Cephalosporin) showed little efficacy against either culture. Stationary-phase cells were more tolerant to imipenem (Carbapenem) than exponential cells, leaving a small fraction of persisters at high imipenem concentration in both populations. Polymyxin B (Polymyxin) appeared to be ineffective at low concentrations against both cell populations. Very high polymyxin B concentration completely eradicated exponential cells and regrowth was seen in a stationary population. Stationary cells were more tolerant to tobramycin (Aminoglycoside) than exponential cells but a higher concentration of tobramycin completely eliminated survivors. Ciprofloxacin (Fluoroquinolone) at a low concentration resulted in killing of both cultures of P. aeruginosa, producing persisters that were invulnerable to killing. CONCLUSIONS: Stationary cells appear to be somewhat more tolerant than exponential cells in all of these assays. We also showed that nutrient deprivation (serine starvation) regulated by stringent and general stress response, contribute to the increased tolerance of P. aeruginosa exponential and stationary planktonic cells via production of persisters.
Antimikrobiálna rezistencia nozokomiálnych patogénov sa ukázala v novom tisícročí ako jeden z najvýznamnejších problémov v zdravotnej starostlivosti. V tomto prehľade uvádzame nové metódy na testovanie citlivosti baktérií na antibiotiká založené na detekcii markerov antibiotikami sprostredkovanej bunkovej smrti, ktoré by sa mohli stať v blízkej budúcnosti cennými alternatívami k už existujúcim fenotypovým metódam.
Antimicrobial resistance among nosocomial pathogens has emerged as one of the most important health care problems in the new millennium. In this review, we present new methods for bacterial antimicrobial susceptibility testing, based on the detection of antibiotic-mediated cell death markers that could provide valuable alternatives to existing phenotypic approaches in the very near future.
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální léková rezistence MeSH
- buněčná smrt * účinky léků MeSH
- kvantitativní polymerázová řetězová reakce * metody MeSH
- lidé MeSH
- mikrobiální testy citlivosti * MeSH
- Pseudomonas aeruginosa MeSH
- reaktivní formy kyslíku MeSH
- volné radikály MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
BACKGROUND: During the last decade, the prevalence of carbapenem-resistant Enterobacteriaceae in human patients has increased. Carbapenemase-producing bacteria are usually multidrug resistant. Therefore, early recognition of carbapenemase producers is critical to prevent their spread. OBJECTIVES: The objective of this study was to develop the primers for single and/or multiplex PCR amplification assays for simultaneous identification of class A, class B, and class D carbapenem hydrolyzing β-lactamases in Enterobacteriaceae and then to evaluate their efficiency. MATERIALS AND METHODS: The reference sequences of all genes encoding carbapenemases were downloaded from GenBank. Primers were designed to amplify the following 11 genes: bla KPC, bla OXA, bla VIM, bla NDM, bla IMP, bla SME, bla IMI, bla GES , bla GIM, bla DIM and bla CMY . PCR conditions were tested to amplify fragments of different sizes. Two multiplex PCR sets were created for the detection of clinically important carbapenemases. The third set of primers was included for detection of all known carbapenemases in Enterobacteriaceae. They were evaluated using six reference strains and nine clinical isolates. RESULTS: Using optimized conditions, all carbapenemase-positive controls yielded predicted amplicon sizes and confirmed the specificity of the primers in single and multiplex PCR. CONCLUSIONS: We have reported here a reliable method, composed of single and multiplex PCR assays, for screening all clinically known carbapenemases. Primers tested in silico and in vitro may distinguish carbapenem-resistant Enterobacteriaceae and could assist in combating the spread of carbapenem resistance in Enterobacteriaceae.
- Publikační typ
- časopisecké články MeSH
Za posledné dve desaťročia pozorujeme rýchly nárast infekcií spôsobených mikroorganizmami s mnohonásobnou rezistenciou. Naše možnosti antibiotickej terapie sú vystavené obrovskému nebezpečenstvu v dôsledku šírenia rezistentných patogénov. Mnoho štúdií dokazuje, že včasné poskytnutie primeranej antibiotickej liečby je nesmierne dôležité pri záchrane života. Momentálne sa lekári spoliehaju na klinické, epidemiologické a demografické údaje, ktoré im napomáhajú pri výbere empirickej terapie, nakoľko výsledky kultivácie a testovania citlivosti na antibiotiká môžu trvať až 48 hod. a dlhšie. A preto je nevyhnutný záujem o skorú a citlivejšiu detekciu rezistentných baktérií. V tomto prehľade poskytujeme zhrnutie najpokročilejších metód (techniky založené na PCR, prietoková cytometria, hmotnostná spektrometria, mikročipy a iné) určených na rýchlu detekciu antibiotickej rezistencie u baktérií, ktoré by sa mohli stať vo veľmi blízkej budúcnosti cennými alternatívami k už existujúcim metódam (fenotypové metódy).
The past two decades have witnessed increasing infections due to multidrug-resistant bacteria. Therefore, transmission of these pathogens could limit the antibiotic therapy options. Many reports suggest that initiation of appropriate antimicrobial therapy can be lifesaving. Physicians rely on combination of clinical, epidemiological and demographic data to guide empirical therapy because results of culture and antimicrobial susceptibility testing may require 48 hours or longer. Therefore, an ongoing effort for the development of earUer and more sensitive detection of resistant bacteria is inevitable. This review presents a summary of the most advanced methods (e.g. PCR-based techniques, flow cytometry, mass spectrometry, microarrays and others) that are able to rapidly detect antibiotic resistance in bacterial pathogens which have the potential to become valuable alternatives to the existing methods (standard phenotypic resistance testing) in the very near future.
- Klíčová slova
- MALDI-TOF, vážení mikrobiálních buněk pomocí vibrujících konzol, izotermická mikrokalorimetrie, magnetická rotace kuliček,
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální léková rezistence MeSH
- časové faktory MeSH
- kalorimetrie metody MeSH
- lidé MeSH
- magnety MeSH
- mikrobiální testy citlivosti * metody trendy MeSH
- mikrofluidní analytické techniky metody MeSH
- mikrotechnologie MeSH
- nanotechnologie MeSH
- průtoková cytometrie MeSH
- RNA genetika MeSH
- rotace MeSH
- sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
