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Klebsiella pneumoniae and Colistin Susceptibility Testing: Performance Evaluation for Broth Microdilution, Agar Dilution and Minimum Inhibitory Concentration Test Strips and Impact of the Skipped Well Phenomenon

Publication . Elias, Rita; Melo Cristino, José; Lito, Luís; Pinto, Margarida; Gonçalves, Luísa; Campino, Susana; Clark, Taane; Duarte, Aida; Perdigão, João

The emergence of multidrug resistant Gram-negative pathogens, particularly carbapenemase producers, has forced clinicians to use last line antibiotics, such as colistin. Since colistin susceptibility testing presents several challenges, this study aimed at evaluating the performance of two alternative susceptibility methods for Klebsiella pneumoniae, namely, agar dilution (AD) and MIC test strips (MTS). These approaches were compared with the reference method, broth microdilution (BMD), and provide a quantitative description for the “skipped well” (SW) phenomenon. Colistin susceptibility was evaluated by BMD and AD in parallel and triplicate, using 141 K. pneumoniae clinical isolates while MTS performance was evaluated only for a subset (n = 121). Minimum inhibitory concentration analysis revealed that a substantial part (n = 26/141; 18.4%) of the initial isolates was deemed undetermined by BMD due to the following: discordance between replicates (1.4%); presence of multiple SWs (7.8%); and the combination of both events (9.2%). Both AD and MTS revealed a high number of false-susceptible strains (“very major errors”), 37.5% and 68.8%, respectively. However, AD agreement indices were reasonably high (EA = 71.3% and CA = 94.8%). For MTS these indices were lower, in particular EA (EA = 41.7% and CA = 89.6), but the approach enabled the detection of distinct sub-populations for four isolates. In conclusion, this study provides the most comprehensive study on the performance of AD and MTS for colistin susceptibility testing in K. pneumoniae, highlighting its limitations, and stressing the importance of sample size and composition. Further, this study highlights the impact of the SW phenomenon associated with the BMD method for K. pneumoniae.

2021-12-14Journal article

Open access

Genomic-based surveillance reveals high ongoing transmission of multi-drug-resistant Mycobacterium tuberculosis in Southern Brazil

Publication . Salvato, Richard Steiner; Reis, Ana Júlia; Schiefelbein, Sun Hee; Gómez, Michael Andrés Abril; Salvato, Stéphanie Steiner; Silva, Larissa Vitória da; Costa, Elis Regina Dalla; Unis, Gisela; Dias, Claudia Fontoura; Viveiros, Miguel; Portugal, Isabel; Groll, Andrea von; Silva, Pedro Eduardo Almeida da; Kritski, Afrânio Lineu; Perdigão, João; Rossetti, Maria Lucia Rosa

Genomic-based surveillance on the occurrence of drug resistance and its transmission dynamics has emerged as a powerful tool for the control of tuberculosis (TB). A whole-genome sequencing approach, phenotypic testing and clinical-epidemiological investigation were used to undertake a retrospective population-based study on drug-resistant (DR)-TB in Rio Grande do Sul, the largest state in Southern Brazil. The analysis included 305 resistant Mycobacterium tuberculosis strains sampled statewide from 2011 to 2014, and covered 75.7% of all DR-TB cases identified in this period. Lineage 4 was found to be predominant (99.3%), with high sublineage-level diversity composed mainly of 4.3.4.2 [Latin American and Mediterranean (LAM)/RD174], 4.3.3 (LAM/RD115) and 4.1.2.1 (Haarlem/RD182) sublineages. Genomic diversity was also reflected in resistance of the variants to first-line drugs. A large number of distinct resistance-conferring mutations, including variants that have not been reported previously in any other setting worldwide, and 22 isoniazid-monoresistant strains with mutations described as disputed in the rpoB gene but causing rifampicin resistance generally missed by automated phenotypic tests as BACTEC MGIT. Using a cut-off of five single nucleotide polymorphisms, the estimated recent transmission rate was 55.1%, with 168 strains grouped into 28 genomic clusters. The most worrying fact concerns multi-drug-resistant (MDR) strains, of which 73.4% were clustered. Different resistance profiles and acquisition of novel mutations intraclusters revealed important amplification of resistance in the region. This study described the diversity of M. tuberculosis strains, the basis of drug resistance, and ongoing transmission dynamics across the largest state in Southern Brazil, stressing the urgent need for MDR-TB transmission control state-wide.

2021-10Journal article

Restricted access

A phylogenomic approach for the analysis of colistin resistance-associated genes in Klebsiella pneumoniae, its mutational diversity and implications for phenotypic resistance

Publication . Elias, Rita; Spadar, Anton; Phelan, Jody; Melo-Cristino, José; Lito, Luís; Pinto, Margarida; Gonçalves, Luísa; Campino, Susana; Clark, Taane; Duarte, Aida; Perdigão, João

The emergence of carbapenemase-producing Klebsiella pneumoniae strains has triggered the use of old antibiotics such as colistin. This is driving the emergence of colistin resistance in multidrug-resistant strains that underlie life-threatening infections. This study analyses the mutational diversity of 22 genes associated with colistin resistance in 140 K. pneumoniae clinical isolates integrated in a high-resolution phylogenetic scenario. Colistin susceptibility was accessed by broth microdilution. A total of 98 isolates were susceptible and 16 were resistant, 10 of which were carbapenemase producers. Across the 22 genes examined, 171 non-synonymous mutations and 9 mutations associated with promoter regions were found. Eighty-five isolates had a truncation and/or deletion in at least one of the 22 genes. However, only seven mutations, the complete deletion of mgrB or insertion sequence (IS)-mediated disruption, were exclusively observed in resistant isolates. Four of these (mgrB Ile13fs, pmrB Gly207Asp, phoQ His339Asp and ramA Ile28Met) comprised novel mutations that are potentially involved in colistin resistance. One strain bore a ISEcp1-blaCTX-M-15::mgrB disruption, underlying co-resistance to third-generation cephalosporins and colistin. Moreover, the high-resolution phylogenetic context shows that most of the mutational diversity spans multiple phylogenetic clades, and most of the mutations previously associated with colistin resistance are clade-associated and present in susceptible isolates, showing no correlation with colistin resistance. In conclusion, the present study provides relevant data on the genetic background of genes involved with colistin resistance deeply rooted across monophyletic groups and provides a better understanding of the genes and mutations involved in colistin resistance.

2022-06Journal article

Open access

Expanded tracking of a Beijing Mycobacterium tuberculosis strain involved in an outbreak in France

Publication . Genestet, Charlotte; Perdigão, João; Herranz, Marta; Maus, Sandra R.; Berland, Jean-Luc; Chiner-Oms, Álvaro; Comas, Iñaki; Muñoz, Patricia; Portugal, Isabel; Dumitrescu, Oana; Pérez-Lago, Laura; García De Viedma, Darío

2021-11Journal article

Restricted access

The burden of bacterial antimicrobial resistance in the WHO European region in 2019: a cross-country systematic analysis

Publication . Mestrovic, Tomislav; Robles Aguilar, Gisela; Swetschinski, Lucien R; Ikuta, Kevin S; Gray, Authia P; Davis Weaver, Nicole; Han, Chieh; Wool, Eve E; Hayoon, Anna Gershberg; Hay, Simon I; Dolecek, Christiane; Sartorius, Benn; Murray, Christopher J L; Addo, Isaac Yeboah; Ahinkorah, Bright Opoku; Ahmed, Ayman; Aldeyab, Mamoon A; Allel, Kasim; Ancuceanu, Robert; Anyasodor, Anayochukwu Edward; Ausloos, Marcel; Barra, Fabio; Bhagavathula, Akshaya Srikanth; Bhandari, Dinesh; Bhaskar, Sonu; Cruz-Martins, Natália; Dastiridou, Anna; Dokova, Klara; Dubljanin, Eleonora; Durojaiye, Oyewole Christopher; Fagbamigbe, Adeniyi Francis; Ferrero, Simone; Gaal, Peter Andras; Gupta, Veer Bala; Gupta, Vijai Kumar; Gupta, Vivek Kumar; Herteliu, Claudiu; Hussain, Salman; Ilic, Irena M; Ilic, Milena D; Jamshidi, Elham; Joo, Tamas; Karch, André; Kisa, Adnan; Kisa, Sezer; Kostyanev, Tomislav; Kyu, Hmwe Hmwe; Lám, Judit; Lopes, Graciliana; Mathioudakis, Alexander G; Mentis, Alexios-Fotios A; Michalek, Irmina Maria; Moni, Mohammad Ali; Moore, Catrin E; Mulita, Francesk; Negoi, Ionut; Negoi, Ruxandra Irina; Palicz, Tamás; Pana, Adrian; Perdigão, João; Petcu, Ionela-Roxana; Rabiee, Navid; Rawaf, David Laith; Rawaf, Salman; Shakhmardanov, Murad Ziyaudinovich; Sheikh, Aziz; Silva, Luís Manuel Lopes Rodrigues; Skryabin, Valentin Yurievich; Skryabina, Anna Aleksandrovna; Socea, Bogdan; Stergachis, Andy; Stoeva, Temenuga Zhekova; Sumi, Chandra Datta; Thiyagarajan, Arulmani; Tovani-Palone, Marcos Roberto; Yesiltepe, Metin; Zaman, Sojib Bin; Naghavi, Mohsen

Background Antimicrobial resistance (AMR) represents one of the most crucial threats to public health and modern health care. Previous studies have identified challenges with estimating the magnitude of the problem and its downstream effect on human health and mortality. To our knowledge, this study presents the most comprehensive set of regional and country-level estimates of AMR burden in the WHO European region to date. Methods We estimated deaths and disability-adjusted life-years attributable to and associated with AMR for 23 bacterial pathogens and 88 pathogen–drug combinations for the WHO European region and its countries in 2019. Our methodological approach consisted of five broad components: the number of deaths in which infection had a role, the proportion of infectious deaths attributable to a given infectious syndrome, the proportion of infectious syndrome deaths attributable to a given pathogen, the percentage of a given pathogen resistant to an antimicrobial drug of interest, and the excess risk of mortality (or duration of an infection) associated with this resistance. These components were then used to estimate the disease burden by using two counterfactual scenarios: deaths attributable to AMR (considering an alternative scenario where infections with resistant pathogens are replaced with susceptible ones) and deaths associated with AMR (considering an alternative scenario where drug-resistant infections would not occur at all). Data were solicited from a wide array of international stakeholders; these included research hospitals, surveillance networks, and infection databases maintained by private laboratories and medical technology companies. We generated 95% uncertainty intervals (UIs) for final estimates as the 25th and 975th ordered values across 1000 posterior draws, and models were cross-validated for out-of-sample predictive validity. Findings We estimated 541 000 deaths (95% UI 370 000–763 000) associated with bacterial AMR and 133 000 deaths (90 100–188 000) attributable to bacterial AMR in the whole WHO European region in 2019. The largest fatal burden of AMR in the region came from bloodstream infections, with 195 000 deaths (104 000–333 000) associated with resistance, followed by intra-abdominal infections (127 000 deaths [81 900–185 000]) and respiratory infections (120 000 deaths [94 500–154 000]). Seven leading pathogens were responsible for about 457 000 deaths associated with resistance in 53 countries of this region; these pathogens were, in descending order of mortality, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus faecium, Streptococcus pneumoniae, and Acinetobacter baumannii. Methicillin-resistant S aureus was shown to be the leading pathogen–drug combination in 27 countries for deaths attributable to AMR, while aminopenicillin-resistant E coli predominated in 47 countries for deaths associated with AMR. Interpretation The high levels of resistance for several important bacterial pathogens and pathogen–drug combinations, together with the high mortality rates associated with these pathogens, show that AMR is a serious threat to public health in the WHO European region. Our regional and cross-country analyses open the door for strategies that can be tailored to leading pathogen–drug combinations and the available resources in a specific location. These results underscore that the most effective way to tackle AMR in this region will require targeted efforts and investments in conjunction with continuous outcome-based research endeavours. Funding Bill & Melinda Gates Foundation, Wellcome Trust, and Department of Health and Social Care using UK aid funding managed by the Fleming Fund.

2022-11Journal article

Open access