Proučevanje razvoja odpornosti različnih sevov bakterij vrste Staphylococcus epidermidis proti didecildimetilamonijevemu kloridu

Ribič, Urška (author), Jeršek, Barbka (mentor), Klančnik, Anja (comentor)

V raziskavah smo okarakterizirali seve S. epidermidis izolirane iz čistih prostorov in humane seve S. epidermidis iz kliničnega okolja ter raziskali njihovo sposobnost prilagoditve na didecildimetilamonijev klorid (DDAC) in razkužilo, ki vsebuje DDAC. Občutljivost za razkužilo je bila pri 57 sevih S. epidermidis iz čistih prostorov določena z minimalno inhibitorno koncentracijo (MIK) od 1,3 do 40,8 mg/L in z MIK za DDAC od 0,1 do 4,5 mg/L, ter pri 15 humanih sevih od 10,2 do 20,4 mg/L za razkužilo in za DDAC 0,1 mg/L. Večina sevov iz čistih prostorov (96,5 %) je imela gene qacA/B in qacC, 47,4 % sevov je bilo odpornih proti gentamicinu in 3,5 % proti cefoksitinu, 40,4 % sevov iz čistih prostorov je tvorilo biofilm. 7 sevov smo izpostavili naraščajočim koncentracijam razkužila in DDAC. Sevi se na razkužilo niso prilagodili, medtem ko so se 4 sevi prilagodili na DDAC in 3 sevi so postali odporni proti DDAC. Pri tem se je MIK povečala za od 2- do 180-krat. Prilagojeni/odporni sevi so bili navzkrižno odporni proti benzalkonijevemu kloridu (3/7) in antibiotikom (4/7), imeli zmanjšano velikost celic (6/7), spremenjen maščobnokislinski profil (7/7), močnejše delovanje izlivnih črpalk (5/7) in bili močnejši tvorci biofilma (3/7) v primerjavi z neprilagojenimi sevi. Analiza diferencialno izraženih genov, RNA-seq, pri kriteriju več kot 5-kratna sprememba izražanja (FC) > 5 in statistični značilnosti (p) < 0,05 prilagojenega seva (Se11Ad) in odpornega seva (Se18To) je pokazala vključenost več različnih mehanizmov. Pri obeh sevih je bilo po prilagoditvi močno povišano delovanje izlivnih črpalk (npr. izlivna črpalka za arzenove spojine) in transportnih sistemov (npr. za aminokisline, peptide, fosfatne ione, nukleotide). Pri Se11Ad je bil pomemben mehanizem znižana aktivnost sistema Agr, kar je vodilo k povečani tvorbi biofilma, pri Se18To pa različni geni, ki sodelujejo pri sintezi celične stene, kar je vodilo do zadebelitve celične stene. Študij sevov bakterij vrste S. epidermidis in prepoznavanje mehanizmov prilagoditve in razvoja odpornosti proti razkužilom so ključnega pomena pri iskanju uspešnih strategij razkuževanja.

• Language: Slovenian
• Keywords: mikrobiološka kontrola prostorov, čisti prostori, Staphylococcus epidermidis, razkuževanje, kvartarne amonijeve spojine, didecildimetilamonijev klorid, DDAC, sekvenciranje RNA, biofilmi, izlivne črpalke, mehanizmi odpornosti
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: BF - Biotechnical Faculty
• Publisher: [U. Ribič]
• Year: 2020
• PID: 20.500.12556/RUL-121692
• UDC: 579.24+579.26:615.281:648.6
• COBISS.SI-ID: 36022787
• Publication date in RUL: 23.10.2020

Secondary language

• Language: English
• Title: Examining the development of resistance in different strains of Staphylococcus epidermidis to didecyldimethylammonium chloride

Abstract

The main objectives of our research were the characterization of Staphylococcus epidermidis strains isolated from cleanrooms and human S. epidermidis strains, and study of their adaptation to didecyldimethylammonium chloride (DDAC)-based disinfectant and to DDAC itself. The susceptibilities to the disinfectant of 57 S. epidermidis strains isolated from cleanrooms were determined according to their minimum inhibitory concentrations (MICs), which ranged from 1.3 to 40.8 mg/L for the disinfectant, and from 0.1 to 4.5 mg/L for DDAC. In 15 clinical strains, the MICs were 10.2 mg/L for the disinfectant and 0.1 mg/L for DDAC. The qacA/B and qacC genes were present in the majority of cleanroom strains (96.5%), with almost half showing resistance to gentamicin (47.4%), but with low cefoxitin resistance (3.5%); 40.4% of these cleanroom strains were biofilm formers. Seven S. epidermidis strains were exposed to gradually increased concentrations of disinfectant or DDAC. Adaptation to the disinfectant was not seen, but for DDAC, 4 strains became adapted and 3 strains became resistant. Their corresponding MICs increased from 2- to 180-fold. Compared to their non-adapted counterparts, these adapted/resistant strains became cross-resistant to benzalconium chloride (3/7) and antibiotics (4/7), showed smaller cell size (6/7), altered their fatty-acid profiles (7/7), increased their efflux pump activities (5/7), and became stronger biofilm formers (3/7). Analysis for differentially expressed genes of an adapted (Se11Ad) and a resistant (Se18To) strain using RNA-seq analysis with criterium of fold change (FC) > 5 and statistical significance (p) < 0.05, revealed several adaptation mechanisms. Both strains had up-regulated genes for efflux pumps (e.g., arsenic efflux pump) and transport systems (e.g., amino acids, peptides, phosphate ions, nucleotides transport). For Se11Ad, down-regulation of the Agr system was seen, which results in stronger biofilm formation, as an important adaptation mechanism. For Se18To, several genes involved in cell-wall synthesis were down-regulated, which resulted in thickening of the cell wall. S. epidermidis studies and knowledge of their adaptation and resistance development mechanisms are crucial for the definition of effective disinfection strategies.

• Keywords: microbiological monitoring, cleanrooms, Staphylococcus epidermidis, disinfection, quaternary ammonium compounds, didecyldimethylammonium chloride, DDAC, RNA sequencing, biofilms, efflux pumps, resistance mechanisms