Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus in a Tertiary Hospital from the Comunidad Valenciana (Spain).

To reduce the high rates of morbidity and mortality caused by methicillin-resistant Staphylococcus aureus (MRSA) strains, it is essential to prevent their transmission. This can be achieved through molecular surveillance of the infecting strains, for which the detection of the entry of new strains, the analysis of antimicrobial resistance, and their containment are essential. In this study, we have analyzed 190 MRSA isolates obtained at the Consorcio Hospital General Universitario de Valencia (Spain) from 2013 to 2018 with three approaches: Multilocus Sequence Typing, spa, and SCCmec typing. Although the incidence of S. aureus infections detected in the hospital increased in the study period, the frequency of MRSA isolates decreased from 33% to 18%. One hundred seventy-two MRSA isolates were resistant to three or more classes of antimicrobials, especially to fluoroquinolones. No relevant temporal trend in the distribution of antibiotic susceptibility was observed. The combination of the three typing schemes allowed the identification of 74 different clones, of which the combination ST125-t067-IV was the most abundant in the study (27 cases). Members of three clonal complexes, CC5, CC8, and CC22, comprised 91% of the isolates, and included 32 STs and 32 spa types. The emergence of low incidence strains throughout the study period and a large number of isolates resistant to different classes of antibiotics shows the need for epidemiological surveillance of this pathogen. Our study demonstrates that epidemiological and molecular surveillance is a powerful tool to detect the emergence of clinically important MRSA clones.

Evolutionary and Phenotypic Characterization of Two Spike Mutations in European Lineage 20E of SARS-CoV-2.

We have detected two mutations in the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at amino acid positions 1163 and 1167 that appeared independently in multiple transmission clusters and different genetic backgrounds. Furthermore, both mutations appeared together in a cluster of 1,627 sequences belonging to clade 20E. This cluster is characterized by 12 additional single nucleotide polymorphisms but no deletions. The available structural information on the S protein in the pre- and postfusion conformations predicts that both mutations confer rigidity, which could potentially decrease viral fitness. Accordingly, we observed reduced infectivity of this spike genotype relative to the ancestral 20E sequence in vitro, and the levels of viral RNA in nasopharyngeal swabs were not significantly higher. Furthermore, the mutations did not impact thermal stability or antibody neutralization by sera from vaccinated individuals but moderately reduce neutralization by convalescent-phase sera from the early stages of the pandemic. Despite multiple successful appearances of the two spike mutations during the first year of SARS-CoV-2 evolution, the genotype with both mutations was displaced upon the expansion of the 20I (Alpha) variant. The midterm fate of the genotype investigated was consistent with the lack of advantage observed in the clinical and experimental data. IMPORTANCE We observed repeated, independent emergence of mutations in the SARS-CoV-2 spike involving amino acids 1163 and 1167, within the HR2 functional motif. Conclusions derived from evolutionary and genomic diversity analysis suggest that the co-occurrence of both mutations might pose an advantage for the virus and therefore a threat to effective control of the epidemic. However, biological characterization, including in vitro experiments and analysis of clinical data, indicated no clear benefit in terms of stability or infectivity. In agreement with this, continuous epidemiological surveillance conducted months after the first observations revealed that both mutations did not successfully outcompete other variants and stopped circulating 9 months after their initial detection. Additionally, we evaluated the potential of both mutations to escape neutralizing antibodies, finding that the presence of these two mutations on their own is not likely to confer antibody escape. Our results provide an example of how newly emerged spike mutations can be assessed to better understand the risk posed by new variants and indicate that some spike mutations confer no clear advantage to the virus despite independently emerging multiple times and are eventually displaced by fitter variants.

The first wave of the COVID-19 epidemic in Spain was associated with early introductions and fast spread of a dominating genetic variant.

The coronavirus disease 2019 (COVID-19) pandemic has affected the world radically since 2020. Spain was one of the European countries with the highest incidence during the first wave. As a part of a consortium to monitor and study the evolution of the epidemic, we sequenced 2,170 samples, diagnosed mostly before lockdown measures. Here, we identified at least 500 introductions from multiple international sources and documented the early rise of two dominant Spanish epidemic clades (SECs), probably amplified by superspreading events. Both SECs were related closely to the initial Asian variants of SARS-CoV-2 and spread widely across Spain. We inferred a substantial reduction in the effective reproductive number of both SECs due to public-health interventions (Re < 1), also reflected in the replacement of SECs by a new variant over the summer of 2020. In summary, we reveal a notable difference in the initial genetic makeup of SARS-CoV-2 in Spain compared with other European countries and show evidence to support the effectiveness of lockdown measures in controlling virus spread, even for the most successful genetic variants.

One is not enough: On the effects of reference genome for the mapping and subsequent analyses of short-reads.

Mapping of high-throughput sequencing (HTS) reads to a single arbitrary reference genome is a frequently used approach in microbial genomics. However, the choice of a reference may represent a source of errors that may affect subsequent analyses such as the detection of single nucleotide polymorphisms (SNPs) and phylogenetic inference. In this work, we evaluated the effect of reference choice on short-read sequence data from five clinically and epidemiologically relevant bacteria (Klebsiella pneumoniae, Legionella pneumophila, Neisseria gonorrhoeae, Pseudomonas aeruginosa and Serratia marcescens). Publicly available whole-genome assemblies encompassing the genomic diversity of these species were selected as reference sequences, and read alignment statistics, SNP calling, recombination rates, dN/dS ratios, and phylogenetic trees were evaluated depending on the mapping reference. The choice of different reference genomes proved to have an impact on almost all the parameters considered in the five species. In addition, these biases had potential epidemiological implications such as including/excluding isolates of particular clades and the estimation of genetic distances. These findings suggest that the single reference approach might introduce systematic errors during mapping that affect subsequent analyses, particularly for data sets with isolates from genetically diverse backgrounds. In any case, exploring the effects of different references on the final conclusions is highly recommended.

Use of next generation sequencing technologies for the diagnosis and epidemiology of infectious diseases. / Uso de las tecnologías de secuenciación masiva para el diagnóstico y epidemiología de enfermedades infecciosas.

For the first time, next generation sequencing technologies provide access to genomic information at a price and scale that allow their implementation in routine clinical practice and epidemiology. While there are still many obstacles to their implementation, there are also multiple examples of their major advantages compared with previous methods. Their main advantage is that a single determination allows epidemiological information on the causative microorganism to be obtained simultaneously, as well as its resistance profile, although these advantages vary according to the pathogen under study. This review discusses several examples of the clinical and epidemiological use of next generation sequencing applied to complete genomes and microbiomes and reflects on its future in clinical practice.

Uso de las tecnologías de secuenciación masiva para el diagnóstico y epidemiología de enfermedades infecciosas / Use of next generation sequencing technologies for the diagnosis and epidemiology of infectious diseases

Por primera vez, la tecnología de secuenciación masiva permite acceder a la información genómica a un precio y a una escala tales, que se está implementado en la práctica clínica y epidemiológica rutinaria. Los obstáculos para dicha implementación son todavía muchos. Sin embargo, ya existen muchos ejemplos de las grandes ventajas que supone en comparación con métodos anteriores. Esto es, sobre todo, porque con una sola determinación podemos obtener simultáneamente información epidemiológica del microorganismo causante, así como de su perfil de resistencias, si bien estas ventajas están más o menos desarrolladas según el patógeno considerado. En esta revisión se repasan varios ejemplos del uso clínico y epidemiológico de la secuenciación masiva aplicada a genomas completos y microbiomas, y se reflexiona sobre su futuro en la práctica clínica

For the first time, next generation sequencing technologies provide access to genomic information at a price and scale that allow their implementation in routine clinical practice and epidemiology. While there are still many obstacles to their implementation, there are also multiple examples of their major advantages compared with previous methods. Their main advantage is that a single determination allows epidemiological information on the causative microorganism to be obtained simultaneously, as well as its resistance profile, although these advantages vary according to the pathogen under study. This review discusses several examples of the clinical and epidemiological use of next generation sequencing applied to complete genomes and microbiomes and reflects on its future in clinical practice

Emergence and dissemination of colistin-resistant Klebsiella pneumoniae isolates expressing OXA-48 plus CTX-M-15 in patients not previously treated with colistin in a Spanish university hospital.

Dissemination of multidrug-resistant Klebsiella pneumoniae in the hospital environment represents a primary target of resistance containment and stewardship programs. At present, polymyxins, mostly in combination, exemplify a last-resort alternative. Colistin-resistant K. pneumoniae isolates harboring OXA-48 plus CTX-M-15 (n = 21) with the simultaneous colistin-susceptible counterparts (n = 9) were recovered from 14 hospitalized patients (January 2014-January 2015) admitted in different wards. In most cases, patients had not previously received colistin. Genetic relatedness experiments demonstrated that 93% (28/30) of isolates belonged to the ST11 high-risk clone. Heteroresistance and the fitness cost of colistin resistance were addressed in susceptible and resistant isolates as well as in in vitro-obtained stable mutants, and results appeared to be strain dependent. Whole genome sequencing demonstrated molecular changes in pmrA, pmrB, and mgrB genes. Plasmid-mediated colistin resistance genes were not found. Colistin resistance in multidrug-resistant K. pneumoniae isolates should be continuously monitored to detect its potential emergence, even in patients not previously exposed to colistin.