Occurence of colistin resistance and molecular characterisation of colistin resistant gram-negative bacilli in Central Slovenian region

Ozadje: Kolistin učinkuje proti številnim po Gramu negativnim bacilom (GNB). Desetletja se v humani medicini skorajda ni uporabljal, uporabljali pa so ga v veterini. Zaradi naraščanja odpornosti GNB proti antibiotikom, kar je porajajoči se javnozdravstveni problem, postaja znova zanimiv za zdravljenje, podatki o pojavnosti proti kolistinu odpornih izolatov (ColR) pa so omejeni. Namen doktorske naloge je bil razjasniti razsežnost problema ColR, ugotoviti delež s ColR-GNB koloniziranih bolnikov na enotah intenzivnega zdravljenja (IE), oceniti uporabnost metod za hitro določanje ColR-izolatov ter s sekvenciranjem celotnega genoma na modelu Escherichia coli pridobiti poglobljene informacije o izolatih ColR v osrednjeslovenskem prostoru. Metode: S selektivnim gojiščem smo na ColR presejalno testirali izolate GNB. Pri 1176 prospektivno zbranih izolatih GNB smo določili minimalno inhibitorno koncentracijo za kolistin. Nadzorne kužnine bolnikov na IE smo analizirali z dvema protokoloma. Rezultate treh metod za hitro določanje ColR smo primerjali z rezultati mikrodilucije. Sekvencirali smo celotni genom 21 izolatov ColR E. coli. Rezultati: Deleži ColR med izolati E. coli, Klebsiella pneumoniae, Enterobacter spp., Citrobacter spp., Pseudomonas aeruginosa in Acinetobacter baumannii so bili 3,4 %, 6,3 %, 24,7 %, 5,1 %, 11,7 % in 23,7 %. Uvedli smo dvostopenjsko epidemiološko spremljanje ColR s specifičnostjo 99,5 % in občutljivostjo 87,5 %. S ColR je bilo koloniziranih 9,4 % bolnikov. Za najustreznejši test za hitro določanje ColR sta se izkazala Rapid Polymyxin NP (Enterobacterales) in Rapid ResaPolymyxin NP (A. baumannii). Pri enem ColR-izolatu E. coli smo našli gen mcr-1, pri ostalih ColR-izolatih smo našli le kromosomske mutacije genov povezanih z odpornostjo proti kolistinu. Zaključki: Ugotovili smo višji delež ColR izolatov od pričakovanega. Za ugotavljanje kolonizacije s ColR je optimalen protokol s predinkubacijo. Nekaj metod za hitro določanje ColR-izolatov je obetavnih, rezultat je potrebno potrditi z mikrodilucijo. Odpornost posredovana z genom mcr-1 pri E. coli je redka, prevladujejo kromosomske mutacije, izolati se pojavljajo sporadično.Background: The antibiotic colistin is effective against many Gram-negative bacilli (GNB). For decades, it was almost no longer used in human medicine, but it was used in veterinary medicine. Due to the increasing resistance of GNB to antibiotics, it is again interesting for the treatment of patients, though data of colistin-resistant isolates (ColR) are scarce. The purpose of the thesis was to assess the extent of the ColR problem, to determine the proportion of ColR GNB colonisied patients in intensive care units (ICU), to evaluate the usefulness of methods for the rapid determination of ColR isolates, and to obtain in-depth information about ColR isolates in the central Slovenian region. Methods: The minimal inhibitory concentration for colistin for 1176 prospectively collected GNB isolates was determined. Control samples from ICU patients were processed using two protocols. The results of three methods for rapid determination of ColR were compared with the results of the reference method. The whole genome of 21 colR Escherichia coli isolates was sequenced and analyzed. Results: Colistin resistance rates were 3.4%, 6.3%, 24.7%, 5.1%, 11.7% and 23.7% among E. coli, Klebsiella pneumoniae, Enterobacter spp., Citrobacter spp., Pseudomonas aeruginosa and Acinetobacter baumannii isolates, respectively. A two-step epidemiologocial screening with specificity of 99.5% and sensitivity of 87.5% was introduced. 9.4% of ICU patientes were colonised with ColR isolates. Rapid Polymyxin NP (Enterobacterales) and Rapid ResaPolymyxin NP (A. baumannii) proved to be the most suitable tests for the rapid determination of ColR. A single mcr-1 gene was found in an E. coli isolate, in other isolates chromosomal adaptation were found. Conclusions: ColR rates among GNB isolates were higher than expected. Based on our findings, protocol with pre-enrichment step is mandatory to identify ColR carriers. Two methods for rapid detection of ColR isolates were promising, but the final result must be confirmed with the reference method. Sequencing of ColR E. coli isolates revealed sporadic cases of ColR isolates with complex resistance mechanisms

Intrahost norovirus evolution in chronic infection over 5 years of shedding in a kidney transplant recipient

Noroviruses are the leading cause of acute gastroenteritis, and they can affect humans of all age groups. In immunocompromised patients, norovirus infections can develop into chronic diarrhea or show prolonged asymptomatic virus shedding. Chronic norovirus infections are frequently reported for solid organ transplant recipients, with rapid intrahost norovirus evolution seen. In this report, we describe a case of chronic norovirus infection in an immunocompromised patient who was followed up for over 5 years. The purpose of the study was to specify the norovirus evolution in a chronically infected immunocompromised host and identify possible selection sites in norovirus capsid protein. During the follow-up period, 25 sequential stool samples were collected and nine of them were selected to generate amplicons covering viral RNA-dependent RNA polymerase (RdRp) and viral capsid protein (VP1) genes. Amplicons were sequenced using next-generation sequencing. Single nucleotide polymorphisms were defined, which demonstrated a nearly 3-fold greater mutation rate in the VP1 genome region compared to the RdRp genome region (7.9 vs. 2.8 variable sites/100 nucleotides, respectively). This indicates that mutations in the virus genome were not accumulated randomly, but are rather the result of mutant selection during the infection cycle. Using ShoRAH software we were able to reconstruct haplotypes occurring in each of the nine selected samples. The deduced amino-acid haplotype sequences were aligned and the positions were analyzed for selective pressure using the Datamonkey program. Only 12 out of 25 positive selection sites were within the commonly described epitopes A, B, C, and D of the VP1 protein. New positive selection sites were determined that have not been described before and might reflect adaptation of the norovirus toward optimal histo-blood-group antigen binding, or modification of the norovirus antigenic properties. These data provide new insights into norovirus evolutionary dynamics and indicate new putative epitope "hot-spots" of modified and optimized norovirus-host interactions

Intrahost Norovirus Evolution in Chronic Infection Over 5 Years of Shedding in a Kidney Transplant Recipient

Noroviruses are the leading cause of acute gastroenteritis, and they can affect humans of all age groups. In immunocompromised patients, norovirus infections can develop into chronic diarrhea or show prolonged asymptomatic virus shedding. Chronic norovirus infections are frequently reported for solid organ transplant recipients, with rapid intrahost norovirus evolution seen. In this report, we describe a case of chronic norovirus infection in an immunocompromised patient who was followed up for over 5 years. The purpose of the study was to specify the norovirus evolution in a chronically infected immunocompromised host and identify possible selection sites in norovirus capsid protein. During the follow-up period, 25 sequential stool samples were collected and nine of them were selected to generate amplicons covering viral RNA-dependent RNA polymerase (RdRp) and viral capsid protein (VP1) genes. Amplicons were sequenced using next-generation sequencing. Single nucleotide polymorphisms were defined, which demonstrated a nearly 3-fold greater mutation rate in the VP1 genome region compared to the RdRp genome region (7.9 vs. 2.8 variable sites/100 nucleotides, respectively). This indicates that mutations in the virus genome were not accumulated randomly, but are rather the result of mutant selection during the infection cycle. Using ShoRAH software we were able to reconstruct haplotypes occurring in each of the nine selected samples. The deduced amino-acid haplotype sequences were aligned and the positions were analyzed for selective pressure using the Datamonkey program. Only 12 out of 25 positive selection sites were within the commonly described epitopes A, B, C, and D of the VP1 protein. New positive selection sites were determined that have not been described before and might reflect adaptation of the norovirus toward optimal histo-blood-group antigen binding, or modification of the norovirus antigenic properties. These data provide new insights into norovirus evolutionary dynamics and indicate new putative epitope “hot-spots” of modified and optimized norovirus–host interactions

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<p>Noroviruses are the leading cause of acute gastroenteritis, and they can affect humans of all age groups. In immunocompromised patients, norovirus infections can develop into chronic diarrhea or show prolonged asymptomatic virus shedding. Chronic norovirus infections are frequently reported for solid organ transplant recipients, with rapid intrahost norovirus evolution seen. In this report, we describe a case of chronic norovirus infection in an immunocompromised patient who was followed up for over 5 years. The purpose of the study was to specify the norovirus evolution in a chronically infected immunocompromised host and identify possible selection sites in norovirus capsid protein. During the follow-up period, 25 sequential stool samples were collected and nine of them were selected to generate amplicons covering viral RNA-dependent RNA polymerase (RdRp) and viral capsid protein (VP1) genes. Amplicons were sequenced using next-generation sequencing. Single nucleotide polymorphisms were defined, which demonstrated a nearly 3-fold greater mutation rate in the VP1 genome region compared to the RdRp genome region (7.9 vs. 2.8 variable sites/100 nucleotides, respectively). This indicates that mutations in the virus genome were not accumulated randomly, but are rather the result of mutant selection during the infection cycle. Using ShoRAH software we were able to reconstruct haplotypes occurring in each of the nine selected samples. The deduced amino-acid haplotype sequences were aligned and the positions were analyzed for selective pressure using the Datamonkey program. Only 12 out of 25 positive selection sites were within the commonly described epitopes A, B, C, and D of the VP1 protein. New positive selection sites were determined that have not been described before and might reflect adaptation of the norovirus toward optimal histo-blood-group antigen binding, or modification of the norovirus antigenic properties. These data provide new insights into norovirus evolutionary dynamics and indicate new putative epitope “hot-spots” of modified and optimized norovirus–host interactions.</p