Odpornost bakterij rodu Bacillus proti protimikrobnim snovem v odpadnih vodah : magistrsko delo

Vuković, Sara (author), Godič Torkar, Karmen (mentor), Griessler Bulc, Tjaša (reviewer)

Uvod: Odpornost bakterij proti protimikrobnim snovem predstavlja vedno večji svetovni problem ter resno grožnjo za globalno zdravje. Proti antibiotikom odporne bakterije in geni za odpornost niso omejeni samo na klinično okolje, ampak se širijo tudi v zunanja okolja. V magistrskem delu smo preučevali odpornost bakterij rodu Bacillus iz odpadne vode proti različnim vrstam antibiotikov, predvsem proti sulfonamidom in tetraciklinom. Namen: Namen magistrskega dela je bil preučiti prisotnost proti antibiotikom odpornih sevov rodu Bacillus na različnih stopnjah čiščenja v dveh centralnih čistilnih napravah za čiščenje komunalne odpadne vode z različno sestavo in izvorom vhodne vode. Pri sevih smo podrobneje preučili odpornost proti sulfonamidom in tetraciklinom. Metode dela: Iz vzorcev odpadne vode, ki smo jih odvzeli na različnih stopnjah čiščenja v obeh čistilnih napravah, smo osamili bakterijske seve in jih identificirali z uporabo osnovnih biokemijskih testov. Za določitev fenotipske odpornosti bakterij proti antibiotikom smo uporabili difuzijski metodi z diski in z gradientom. Molekularno genetske metode so vključevale ekstrakcijo nukleinske kisline, multipleksno verižno reakcijo s polimerazo in elektroforezo. Poleg tega smo izvedli kinetični test baktericidnega delovanja, s katerim smo spremljali dinamiko razmnoževanja bakterijskih celic pod vplivom antibiotikov v različnih časovnih intervalih. Rezultati: Rezultati so pokazali, da je povprečna koncentracija bakterij rodu Bacillus v vtoku odpadne vode v obeh čistilnih napravah od log 3 CFU/mL do log 4 CFU/mL in da se po čiščenju v iztoku zniža za povprečno manj kot log 1 CFU/mL. Ugotovili smo znatne razlike v odpornosti proti posameznim antibiotikom med sevi iz obeh čistilnih naprav. Sevi iz ene čistilne naprave so bili najpogosteje odporni proti kloramfenikolu (78,9 %) in klindamicinu (60,5 %). V drugi čistilni napravi pa je bilo največ sevov odpornih proti linezolidu in trimetoprimu v kombinaciji s sulfametoksazolom (87,5 %). Gene za odpornost proti sulfonamidom sulII in sulIII smo potrdili pri 50 % testiranih sevov, gene za odpornost proti tetraciklinom pa pri 17,9 % primerov. Geni tetM in tetL so bili najpogostejši pri testiranih sevih. Število odpornih bakterijskih sevov se je zmanjšalo po dodatni obdelavi z visoko-pretočnim algnim bazenom. Razprava in zaključek: Čistilne naprave so pomemben rezervoar bakterij, odpornih proti protimikrobnim snovem, in genov za odpornost, kar predstavlja tveganje za njihovo širjenje v naravna okolja. Pri sevih Bacillus je bila pogosta tudi večkratna odpornost proti trem ali več skupinam antibiotikov. Velik delež proti protimikrobnim snovem odpornih mikroorganizmov in genov za odpornost se v okolje širi z odpadnimi vodami, zato je nujno v čistilnih napravah uvesti ustrezne postopke čiščenja, s katerimi bi to preprečili. Med najučinkovitejše sodijo napredne oksidacijske metode (npr. ozonacija), membranska filtracija (npr. reverzna osmoza), ultravijolična razgradnja, aktivno oglje ter biološki sistemi z mikroalgami in bakterijami.

• Language: Slovenian
• Keywords: magistrska dela, sanitarno inženirstvo, odpornost proti protimikrobnim snovem, Bacillus spp., odpadna voda, sulfonamidi, tetraciklini
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: ZF - Faculty of Health Sciences
• Place of publishing: Ljubljana
• Publisher: [S. Vuković]
• Year: 2025
• Number of pages: 89 str., [16] str. pril.
• PID: 20.500.12556/RUL-169167
• UDC: 614
• COBISS.SI-ID: 236101379
• Publication date in RUL: 16.05.2025

Secondary language

• Language: English
• Title: Resistance of Bacillus species to antimicrobial agents in wastewater : master thesis

Abstract

Introduction: Bacterial resistance to antimicrobials is a growing global problem and presents a serious threat to global health. Antimicrobial-resistant bacteria and resistance genes are not only limited to the clinical setting, but are also spreading in the natural environment. In this study, the resistance of bacteria of the genus Bacillus isolated from wastewater to different types of antibiotics, mainly sulfonamides and tetracyclines, was investigated. Purpose: The purpose of the Master's thesis was to investigate the presence of resistant strains Bacillus spp. in different treatment stages in two central wastewater treatment plants for the treatment of municipal wastewater with different composition and origin of the input water. The resistance of the strains to sulfonamides and tetracyclines was investigated in more detail. Methods: Bacterial strains were isolated from wastewater samples taken at different treatment stages in the two wastewater treatment plants and identified using basic biochemical tests. Disk diffusion and gradient diffusion methods were used to determine the phenotypic resistance of the bacteria to antibiotics. Molecular genetic methods included nucleic acid extraction, multiplex polimerase chain reaction and electrophoresis. In addition, we performed a kinetic assay of bactericidal activity to observe the dynamics of bacterial cell multiplication under the influence of antibiotics at different time intervals. Results: The results showed that the average concentration of bacteria of the genus Bacillus in the wastewater influent in both wastewater treatment plants ranged between log 3 CFU/mL and log 4 CFU/mL and decreased on average by less than log 1 CFU/mL after treatment in the effluent. The significant differences in resistance to individual antibiotics between strains of both wastewater treatment plants were confirmed. Strains from one wastewater treatment plant were most frequently resistant to chloramphenicol (78.9 %) and clindamycin (60.5 %), while the highest percentage of strains from another wastewater treatment plant were resistant to linezolid and trimethoprim in combination with sulfamethoxazole (87.5 %). The sulfonamide resistance genes sulII and sulIII were detected in 50 % of the strains tested and the tetracycline resistance genes were confirmed in 17.9 % of cases. The most frequently occurring genes in tested strains were tetM and tetL. The number of resistant bacterial strains decreased after additional treatment with the High Rate Algae Pond. Discussion and conclusion: Wastewater treatment plants are an important reservoir for antimicrobial resistant bacteria and resistance genes, which poses a risk for their spread in the natural environment. Multiple resistance to three or more groups of antibiotics was also common among the Bacillus strains tested. A large proportion of antimicrobial-resistant microorganisms and resistance genes are spread into the environment with wastewater, so it is crucial to implement appropriate treatment processes in wastewater treatment plants to prevent this. The most effective methods include advanced oxidation methods (e.g. ozonation), membrane filtration (e.g. reverse osmosis), ultraviolet degradation, activated carbon, and biological systems with microalgae and bacteria.

• Keywords: master's theses, sanitary engineering, antimicrobial resistance, Bacillus spp., wastewater, sulfonamides, tetracyclines