Epidemiološke in klinične značilnosti hude gnilobe čebelje zalege v povezavi z genotipom Paenibacillus larvae

Žugelj, Alenka (author), Zdovc, Irena (mentor)

Paenibacillus larvae je po Gramu pozitivna sporogena bakterija, ki povzroča hudo gnilobo čebelje zalege (HGČZ). Poznanih je pet genotipov ERIC, ki se med seboj razlikujejo tudi fenotipsko. Izolate P. larvae smo izolirali ob izbruhih bolezni v Sloveniji v letih 2017 – 2019. Skupaj z izolati smo pridobili tudi pripadajoče epidemiološke podatke. Za identifikacijo izolatov P. larvae smo validirali metodo masne spektrometrije MALDI TOF in s tem pohitrili in pocenili bakteriološko preiskavo. Kolonije P. larvae so na gojiščih rasle v obliki dveh osnovnih morfotipov. Ugotovili smo, da genotip ERIC I fenotipsko večinoma izraža morfotip I – sive hrapave kolonije, genotip ERIC II pa izraža morfotip II – gladke sive ali pigmentirane kolonije. Z metodo ERIC-PCR smo prvič tipizirali in ugotovili pojavnost genotipov ERIC v Sloveniji. Prevladoval je genotip ERIC II (70,2 %). Pri nekaterih izolatih genotipa ERIC I smo ugotovili nekoliko spremenjen profil pomnožkov ERIC, zato smo 59 izbranim izolatom genotipa ERIC I sekvencirali tudi celotne genome (WGS). Kljub odstopanjem od klasičnega profila ERIC I, smo po tipizaciji z metodo WGS in silico MLST pri vseh izolatih ugotovili isti sekvenčni tip ST2. Ko smo z metodo wgMLST primerjali zaporedja lokusov v celotnih genomih z zaporedji, dostopnimi v javnih podatkovnih zbirkah, so se vsi naši izolati filogenetsko razvrstili v klad ERIC I in gručo ERIC I-ST2. Z molekularnimi in fenotipskimi metodami smo potrdili prisotnost različnih morfotipov in genotipov P. larvae v enem kužnem krogu, celo v istem čebelnjaku. Rezultati anketnega vprašalnika so statistično potrdili značilne klinične znake za genotipa ERIC I in II, kljub številnim drugim naključnim dejavnikom, ki lahko vplivajo na izražanje klinične slike HGČZ, in jih v naši raziskavi nismo spremljali. V celotnih genomih izolatov P. larvae smo ugotovili prisotnost gruč genov za sintezo inhibitornih substanc. Z disk difuzijsko metodo smo ugotovili, da posamezni sevi genotipa ERIC I izločajo protimikrobne snovi, ki inhibirajo rast nekaterih po Gramu pozitivnih bakterij in kvasovke Malassezia pachydermatis. Vpeljane diagnostične metode in rezultati raziskave predstavljajo pomemben doprinos k razumevanju genetske raznolikosti povzročitelja P. larvae in proučevanju načinov širjenja HGČZ.

• Language: Slovenian
• Keywords: Čebele, mikrobiologija, Paenibacillus larvae, diagnostika, izolacija, genotipizacija, sekvenciranje celotnega genoma, verižna reakcija s polimerazo, masna spektrometrija, ionizacija v matriksu z desorpcijo z laserjem
• Work type: Doctoral dissertation
• Organization: VF - Veterinary Faculty
• Year: 2022
• PID: 20.500.12556/RUL-137191
• Publication date in RUL: 05.06.2022

Secondary language

• Language: English
• Title: Epidemiological and clinical characteristics of American Foulbrood in connection with Paenibacillus larvae genotype

Abstract

Paenibacillus larvae is a Gram-positive sporogenic bacterium that causes American Foulbrood (AFB). Five ERIC genotypes are described, which also differ phenotypically. In 2017–2019, P. larvae isolates were obtained during AFB outbreaks in Slovenia. Together with the isolates, we also obtained the corresponding epidemiological data. For the determination of P. larvae by bacteriological examination, we validated the MALDI TOF mass spectrometry method, thus speeding up and reducing the cost of the investigation. Two morphotypes of P. larvae colonies were observed. We found that the ERIC I genotype phenotypically mostly shows one morphotype – gray rough colonies, and the ERIC II genotype another – gray smooth or pigmented colonies. For the first time, ERIC-PCR was used for P. larvae typing in Slovenia and the frequency of ERIC genotypes could be determined. ERIC II was the predominant genotype (70,2 %). Some ERIC I isolates exhibited a slightly altered ERIC I banding pattern, thus 59 isolates were selected for the whole-genome sequencing (WGS). Despite showing non-classical ERIC I pattern, WGS-typing using the in silico MLST revealed that all of the investigated isolates belonged to the sequence type ST2. When comparing the whole-genome loci using the wgMLST analysis of the investigated isolates supplemented with all the publicly available P. larvae genomes, all Slovenian isolates fell within the ERIC I clade and phylogenetically clustered with the remaining ERIC I-ST2 genomes. Using molecular and phenotypic methods, we confirmed the presence of different morphotypes and genotypes of P. larvae in the same AFB zone or even in the same apiary. Results of the questionnaire statistically confirmed the presence of clinical signs characteristic for genotypes ERIC I and II, despite many other random factors that may affect the expression of AFB clinical picture and were not monitored in the scope of our study. In the genomes of P. larvae isolates, we identified the presence of gene clusters for the synthesis of inhibitory substances. Using the diffusion method, the ability to inhibit the growth of some Gram-positive bacteria and the yeast Malassezia pachydermatis was identified for individual ERIC I strains. The implemented diagnostic methods and study results represent an important contribution to the understanding of P. larvae genetic characteristics and AFB transmission routes.

• Keywords: Bees, microbiology, Paenibacillus larvae, diagnosis, isolation, genotyping, whole genome sequencing, polymerase chain reaction, spectrometry, mass, matrix-assisted laser desorption ionization