REZUMAT ETAPA III
A III-a etapă a proiectului își propune să investigheze mecanismele de transfer orizontal al CP si BLSE între mediul spitalicesc și cel acvatic prin studiul contextului genetic al secvențelor de inserție, integronilor, transpozonilor și sǎ evalueze impactului stației de epurare asupra gradului de diseminare a tulpinilor producătoare de CP si BLSE în efluent și în râul receptor. În acest scop au fost secvențiate (pair-end whole genome sequencing (WGS) – MiSeq System Sequencer, Illumina) un numǎr de 54 de tulpini de purtǎtoare de GRC, apartinand speciilor A. baumannii (Ab=34) si P. aeruginosa (Pa=20) izolate pe o perioadǎ de aproximativ un an (sept. 2018 – august 2019) din opt locatii diferite ale Romaniei atat din infectii intra-spitalicesti cat si reteaua de apǎ uzatǎ in aceeasi secventǎ temporalǎ. Sase tulpini de Ab purtǎtoare de GRC si analizate anterior prin WGS au fost selectate pentru secventiere de generatie a treia [MinION (ONT) technologies] iar analiza bioinformatică a datelor secvențiere obținute a fost realizată cu software MinKNOW prin traducerea acestora în secvențe de nucleotide, urmată de asamblarea acestora. Pentru un numǎr de 40 de tulpini de Ab purtǎtoare de GRC a fost investigate diseminarea genelor blaOXA-23 si blaOXA-24 prin conjugare pe mediu lichid si pe mediu solid. Evaluarea impactului stației de epurare asupra gradului de diseminare a tulpinilor multirezistente producătoare de carbapenemaze în efluent și în râul receptor s-a realizat analiza prin comparativǎ a numǎrului de unitǎti formatoare de colonii (UFC/100 L) apartinand bacililor Gram negativi non-fermentativi rezistenti la carbapeneme / producǎtori de beta-lactamaze de spectru extins, tulpini izolate din probele de apǎ din efluentul SEAU analizate si in raul receptor (regiunea aval a SEAU). In cazul tulpinilor apartinand speciei Ab s-au identificat asocieri ale GRC cu elemente genetice mobile de tipul secventelor de insertie de tip ISAba1, transpozonilor (Tn3) si integronilor de clasǎ 1 (qacE∆1) iar analiza filogeneticǎ a tulpinilor clinice si din reteaua de apǎ uzatǎ a demonstrat apartenenta tulpinilor la sase grupe filogenetice. In cazul tulpinilor de Pa s-a demonstrat diseminarea GRC (blaIMP-13, blaVIM-2, blaGES-1, blaVEB-9 si blaTEM-40) din mediul spitalicesc in cel acvatic prin intermediul integronilor de clasǎ 1 (qacE∆1). Secventierea de generatie a treia a permis localizarea GRC (blaOXA-72) fie în structura plasmidelor (p-MAL), fie integrate în cromosomul bacterian, putand fi identificatǎ o nouă structură genetică de tipul unui transpozon. Experimentele de conjugare au demonstrat transferul conjugativ al GRC de tip blaOXA-23 si blaOXA-24 între mediul spitalicesc și cel acvatic. Analiza comparativǎ a incǎrcǎturii microbiene in efluent si in raul receptor al SEAU analizate a demonstrat o reducere de 1 log pentru fenotipurile investigate.
SUMMARY THIRD STAGE
The third stage of the project aim to investigate the horizontal transfer mechanisms of CP and BLSE between the hospital and aquatic environment by studying the genetic context of the insertion sequences, integrons, and transposons and to assess the impact of the wastewater treatment plant (WWTP) on the dissemination of carbapenemase (CP) and extended spectrum β-lactamase producing isolates (ESBL) in the effluent and receiving river. For this, a total number of 54 carbapenemase producing strains (GRC) belonging to A. baumannii (Ab = 34) and P. aeruginosa (Pa = 20) isolated for a period of approximately one year (Sept. 2018 - August 2019) from eight different locations of Romania, from intra-hospital infections and from the wastewater network in the same temporal sequence were sequenced (pair-end whole genome sequencing (WGS) - MiSeq System Sequencer, Illumina). Six GRC Ab strains and previously analyzed by WGS were selected for third generation sequencing [MinION (ONT) technologies] and the bioinformatics analysis of the obtained sequencing data was performed with MinKNOW software by translating them into nucleotide sequences, followed of their assembly. For a total number of 40 GRC Ab strains was investigated the dissemination of blaOXA-23 and blaOXA-24 genes by conjugation on liquid and solid culture medium. Evaluation of the impact WWTP on the dissemination of multidrug-resistant CP producing strains in the effluent and in the receiving river was performed by comparative analysis of the of colony forming units (CFU / 100 L) belonging to carbapenem-resistant/ ESBL producers non-fermenting Gram-negative bacilli, strains isolated from the wastewater samples from the analyzed WWTPs effluent and from the receiving river (downstream region of the WWTPs). In Ab strains there have been identified the associations of GRC with mobile genetic elements such as ISAba1 insertion sequences, transposons (Tn3) and class 1 integrons (qacE∆1) and the phylogenetic analysis of the clinical and wastewater has demonstrated that the strains belonged to six phylogenetic groups. In the case of Pa strains, the dissemination of GRC from the hospital to the aquatic environment (blaIMP-13, blaVIM-2, blaGES-1, blaVEB-9 and blaTEM-40) was demonstrated by class 1 integrons (qacE∆1). Third-generation sequencing allowed the localization of GRC (blaOXA-72) either plasmid (p-MAL) or chromosomal located, being able to identify a new genetic structure (transposon). Conjugation experiments have demonstrated the conjugative transfer of blaOXA-23 and blaOXA-24 genes between hospital and aquatic environments. The comparative analysis of the microbial load in the effluent and in the receiving river of the analyzed WWTPs showed a reduction of 1 log for the investigated phenotypes.
Publications:
ISI papers:
1. Corneliu Ovidiu Vrancianu; Irina Gheorghe*; Elena-Georgiana Dobre; Ilda Barbu Czobor;
Roxana Elena Cristian; Marcela Popa; Sang Hee Lee; Carmen Limban; Ilinca Margareta Vlad; Mariana Carmen Chifiriuc. Emerging strategies to combat β-lactamase producing ESKAPE pathogens. Accepted in Int. J. Mol. Sci.(ISSN 1422-0067) on 10 November 2020.
2. Corneliu Ovidiu Vrancianu, Irina Gheorghe*, Ilda Barbu Czobor, Mariana Carmen Chifiriuc. Antibiotic Resistance Profiles, Molecular Mechanisms and Innovative Treatment Strategies of Acinetobacter baumannii. Microorganisms. 2020. 8 (6), 935 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7355832/).
3. Al Shaikhli Nawfal Haitham, Al Owaidi Ali, Mariana Carmen Chifiriuc, Grigore Mihaescu, Rana Mujahid Abdullah Alshwaikh, Sajjad Mohsin, Hamzah Basil Mohammed, Gratiela Gradisteanu Pircalabioru, Irina Gheorghe. Antibiotic resistance determinants of Acinetobacter baumannii strains isolated from nosocomial infections. Romanian Biotechnological Letter. 2020. 25 (3), 1658-1665 ( https://www.e-repository.org/rbl/vol.25/iss.3/20.pdf).
4. Marius Surleac, Ilda Czobor Barbu, Simona Paraschiv, Laura Ioana Popa, Irina Gheorghe, Luminita Marutescu, Marcela Popa, Ionela Sarbu, Daniela Talapan, Mihai Nita, Alina Viorica Iancu, Manuela Arbune, Alina Manole, Stefan Nicolescu, Oana Sandulescu, Adrian Streinu-Cercel, Dan Otelea, Mariana Carmen Chifiriuc, Whole genome sequencing snapshot of multi-drug resistant Klebsiella pneumoniae strains from hospitals and receiving wastewater treatment plants in Southern Romania. Plos One, 15(1): e0228079. (https://doi.org/10.1371/journal.pone.0228079 )(https://pubmed.ncbi.nlm.nih.gov/31999747/)
International conferences:
ECCMID 21-24 APRIL 2020 (on-line)
1. Abstract 8317 Detection of blaKPC-2 in a conjugative IncP-6 plasmid in Escherichia coli isolated from wastewater in Romania. Laura Ioana Popa, Alina Cristina Neguț, Ilda Czobor, Irina Gheorghe, Sajjad Mohsin, Mihaela Magdalena Mitache, Marcela Popa, Luminita Marutescu, Mariana Carmen Chifiriuc
2. Abstract 9452 KPC-producing and colistin-resistant Klebsiella pneumoniae ST258 persistence during wastewater treatment plant processes. Ilda Czobor, Irina Gheorghe, Laura Ioana Popa, Marius Surleac, Simona Paraschiv, Dan Otelea, Luminita Marutescu, Marcela Popa, Sajjad Mohsin, Mariana Carmen Chifiriuc.
3. Abstract 5374 Insights in the resistome of multidrug-resistant Pseudomonas aeruginosa strains isolated in Romania from nosocomial infections and wastewater. Irina Gheorghe*, Ilda Czobor, Laura Ioana Popa, Ionela Avram, Al Shaikhli Nawfal Haitham, Sajjad Mohsin, Simona Paraschiv, Marius Surleac, Violeta-Corina Cristea, Marcela Popa, Luminita Marutescu, Dan Otelea, Veronica Lazar, Mariana Carmen Chifiriuc.
FEMS 28-31 OCT. 2020 (on-line)
1. Genetic characterization of multidrug-resistant Acinetobacter baumannii strains isolated in the same temporal sequence from hospital infections and the wastewater network in South Romania. Corneliu Ovidiu Vrancianu, Irina Gheorghe*, Luminita Marutescu, Marcela Popa, Ilda Barbu, Mihai Nita, Alex Muntean, Cerasela Dragomirescu, Oana Sandulescu, Daniela Talapan, Mircea Ioan Popa, Mariana Carmen Chifiriuc