Dejavniki virulence pri vrstah rodu Aeromonas, izoliranih s kože človeške ribice in njenega vodnega okolja

Becner, Lea (author), Turk, Martina (mentor)

Proteus anguinus ali človeška ribica sodi med dvoživke in velja za največjo obligatno jamsko žival ter za edinega jamskega vretenčarja v Evropi. Njen obstoj je zaradi onesnaževanja okolja kot posledice človeške dejavnosti in neposrednega vpliva na njen habitat ogrožen. Raziskave kože človeških ribic in kraške ter akvarijske vode so potrdile prisotnost za dvoživke potencialno nevarnih sevov bakterij iz rodu Aeromonas, povzročiteljev t.i. sindroma rdečih nog. Sevom rodu Aeromonas smo določili virulentni potencial in ocenili grožnjo, ki jo predstavljajo človeškim ribicam. Seve rodu Aeromonas smo do vrste natančno identificirali s filogenetsko analizo na podlagi konkateniranih nukleotidnih zaporedij hišnih genov dnaJ, dnaX, gyrB, recA in rpoD ter gena za 16S rRNA v programu MEGA-X. Na podlagi gena za 16S rRNA prepoznane izolate najbolj patogene vrste A. hydrophila smo identificirali kot A. media, potrdili pa smo prisotnost za ribe in druge vodne živali potencialno patogeni vrsti A. salmonicida in A. veronii. Analize so nakazale na večjo vrstno raznolikost aeromonasov v kožni mikrobioti zdravih človeški ribic napram bolnim ter potrdili dejstvo, da kožna mikrobiota promovira rast v okolju manj zastopanih bakterijskih vrst. Z metodo PCR smo ugotavljali prisotnosti genov za virulentne dejavnike act, alt, ast, aerA, ahpA, epr, hlyA in GCAT in s povprečno 2,13 geni na sev rodu Aeromonas potrdili nizek virulentni potencial. Kljub temu smo odkrili 2 potencialno citotoksična seva z genotipom act+/alt+/aerA+/hlyA+, 19 potencialno citotoksičnih sevov genotipa act+/alt+/aerA+ in 4 potencialno zelo virulentne seve genotipa alt+/aerA+/hlyA+. Kar 93 % sevov je bilo odpornih proti vsaj enemu testiranemu antibiotiku, proti gentamicinu (93,49 % sevov), imipenemu (49,11 % sevov), eritromicinu (34,91 % sevov), ceftriaksonu (34, 32 % sevov), aztreonamu (2,96 % sevov) in enrofloksacinu (2,96 % sevov), ki se uporabljajo v veterini ali humani medicini, kar je zaskrbljujoče. Seve z največjim povprečnim številom antibiotikov, proti katerim so bili odporni, smo izolirali iz lokacije, kjer je v preteklosti prišlo do onesnaženja s težkimi kovinami, kar se sklada s konceptom navskrižne odpornosti. Ugotovitve nakazujejo na verjetno onesnaženje kraških vod z odpadnimi vodami, živinorejo in človeškimi fekalijami ter posledično širjenje genskega materiala, ki vsebuje zapise za mehanizme odpornosti in virulence med bakterijami.

• Language: Slovenian
• Keywords: Aeromonas, človeška ribica, Proteus anguinus, dejavniki virulence, odpornost proti antibiotikom
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Place of publishing: Ljubljana
• Publisher: [L. Becner]
• Year: 2022
• PID: 20.500.12556/RUL-143464
• UDC: 579.25/.26:579.84:597.92
• COBISS.SI-ID: 135003395
• Publication date in RUL: 22.12.2022

Secondary language

• Language: English
• Title: Virulence factors of Aeromonas spp. isolated from from the skin of the olm and its aquatic environment

Abstract

The amphibian Proteus anguinus, also known as the olm, is considered the largest obligate cave animal and the only cave-dwelling vertebrate in Europe. Its existence is threatened by the direct impact on its habitat caused by environmental pollution due to human activities. Microbiome studies of its skin and aquatic enviroment led to the discovery of potentially dangerous and red-leg syndrome causing bacteria of the genus Aeromonas. We determined the virulence potential and assessed the threat Aeromonas spp. pose to the olm. We identified the isolated strains of Aeromonas spp. to species by phylogenetic analyzes based on the concatenated sequences of the housekeeping genes dnaJ, dnaX, gyrB, recA and rpoD and the 16S rRNA gene using the MEGA-X program. We were able to identify strains previously recognized as A. hydrophila as A. media and confirm the presence of the potential fish pathogen species A. salmonicida and A. veronii. The results indicated a greater diversity of Aeromonas species isolated from the skin microbiome of healthy olms compared to unhealthy animals, and confirmed the fact that the skin microbiome promotes the growth of the less represented Aeromonas species from the aquatic enviroment. Using the PCR method, we determined the presence of the virulence factor genes act, alt, ast, aerA, ahpA, epr, hlyA and GCAT and confirmed an average of 2.13 genes per Aeromonas spp. strain, indicating a low virulence potential of the isolated strains. However, we succeeded in detecting 2 potentially cytotoxic act+/alt+/aerA+/hlyA+ strains, 19 potentially cytotoxic act+/alt+/aerA+ strains, and 4 potentially highly virulent alt+/aerA+/hlyA+ strains. No less than 93 % of the strains were resistant to at least one of the antibiotics tested: Gentamicin (93.49 % of all strains), Imipenem (49.11 % of all strains), Erythromycin (34.91 % of all strains), Ceftriaxone (34.32 % of all strains), Aztreonam (2.96 % of all strains), and Enrofloxacin (2.96 % of all strains), which are used in veterinary or human medical practice, raising concern. The strains with the highest average number of antibiotics to which they were resistant were isolated from a site with a history of heavy metal contamination, consistent with the concept of cross-resistance in bacteria. The results of this study suggest that karst waters are likely contaminated by sewage, livestock and human faeces, leading to the spread of resistance and virulence mechanisms among environmental bacteria.

• Keywords: Aeromonas, olm, Proteus anguinus, virulence factors, antibiotic resistance