Genomic analysis of the initial dissemination of carbapenem-resistant Klebsiella pneumoniae clones in a tertiary hospital.

Carbapenem-resistant Klebsiella pneumoniae is a major cause of hospital-acquired infections and the fastest-growing pathogen in Europe. Carbapenem resistance was detected at the Consorcio Hospital General Universitario de Valencia (CHGUV) in early 2015, and there has been a significant increase in carbapenem-resistant isolates since then. In this study, we collected carbapenem-resistant isolates from this hospital during the period of increase (from 2015 to 2019) and studied how K. pneumoniae carbapenem-resistant isolates emerged and spread in the hospital. A total of 225 isolates were subjected to whole-genome sequencing with Illumina NextSeq. We characterized the isolates by identifying lineages and antimicrobial resistance genes and plasmids, especially those related to reduced carbapenem susceptibility. Our findings show that the initial carbapenem resistance emergence and dissemination at the CHGUV occurred during a short period of 1 year. Furthermore, it was complex, involving six different lineages of types ST307, ST11, ST101 and ST437, different resistance-determinant factors, including OXA-48, NDM-1, NDM-23 and DHA-1, and different plasmids.

Genomic Surveillance of Salmonella from the Comunitat Valenciana (Spain).

Salmonella enterica subspecies enterica is one of the most important foodborne pathogens and the causative agent of salmonellosis, which affects both humans and animals producing numerous infections every year. The study and understanding of its epidemiology are key to monitoring and controlling these bacteria. With the development of whole-genome sequencing (WGS) technologies, surveillance based on traditional serotyping and phenotypic tests of resistance is being replaced by genomic surveillance. To introduce WGS as a routine methodology for the surveillance of food-borne Salmonella in the region, we applied this technology to analyze a set of 141 S. enterica isolates obtained from various food sources between 2010 and 2017 in the Comunitat Valenciana (Spain). For this, we performed an evaluation of the most relevant Salmonella typing methods, serotyping and sequence typing, using both traditional and in silico approaches. We extended the use of WGS to detect antimicrobial resistance determinants and predicted minimum inhibitory concentrations (MICs). Finally, to understand possible contaminant sources in this region and their relationship to antimicrobial resistance (AMR), we performed cluster detection combining single-nucleotide polymorphism (SNP) pairwise distances and phylogenetic and epidemiological data. The results of in silico serotyping with WGS data were highly congruent with those of serological analyses (98.5% concordance). Multi-locus sequence typing (MLST) profiles obtained with WGS information were also highly congruent with the sequence type (ST) assignment based on Sanger sequencing (91.9% coincidence). In silico identification of antimicrobial resistance determinants and minimum inhibitory concentrations revealed a high number of resistance genes and possible resistant isolates. A combined phylogenetic and epidemiological analysis with complete genome sequences revealed relationships among isolates indicative of possible common sources for isolates with separate sampling in time and space that had not been detected from epidemiological information. As a result, we demonstrate the usefulness of WGS and in silico methods to obtain an improved characterization of S. enterica enterica isolates, allowing better surveillance of the pathogen in food products and in potential environmental and clinical samples of related interest.

Tracking the Emergence and Dissemination of a blaNDM-23 Gene in a Multidrug Resistance Plasmid of Klebsiella pneumoniae.

Since the discovery of blaNDM-1, NDM ß-lactamases have become one of the most widespread carbapenemases worldwide. To date, 43 different NDM variants have been reported but some, such as blaNDM-23, have not been characterized in detail yet. Here, we describe the emergence of a novel blaNDM-23 allele from a blaNDM-1 ancestor and the multidrug resistance plasmid that has disseminated it through a Klebsiella pneumoniae ST437 clone in several Spanish hospitals. Between 2016 and 2019, 1,972 isolates were collected in an epidemiological survey for extended-spectrum-ß-lactamase (ESBL)-producing Klebsiella pneumoniae in the Comunitat Valenciana (Spain). Three carbapenem-resistant strains failed to be detected by carbapenemase-producing Enterobacteriaceae (CPE) screening tests. These isolates carried a blaNDM-23 gene. To characterize this gene, its emergence, and its dissemination, we performed antimicrobial susceptibility tests, hybrid sequencing with Illumina and Nanopore technologies, and phylogenetic analyses. The MICs of the blaNDM-23 allele were identical to those of the blaNDM-1 allele. The blaNDM-23 allele was found in 14 isolates on a 97-kb nonmobilizable, multidrug-resistant plasmid carrying 19 resistance genes for 9 different antimicrobial families. In this plasmid, the blaNDM-23 gene is in the variable region of a complex class 1 integron with a singular genetic environment. The small genetic distance between blaNDM-23-producing isolates reflects a 5-year-long clonal dispersion involving several hospitals and interregional spread. We have characterized the genomic and epidemiological contexts in the emergence and community spread of a new blaNDM-23 allele in a multidrug resistance (MDR) plasmid of Klebsiella pneumoniae. IMPORTANCE At a time when antimicrobial resistance has become one of the biggest concerns worldwide, the emergence of novel alleles and extremely drug-resistant plasmids is a threat to public health worldwide, especially when they produce carbapenem resistance in one of the most problematic pathogens, such as Klebsiella pneumoniae. We used genomic epidemiology to describe the emergence of a novel NDM-23 allele and identify it in a MDR plasmid that has disseminated through a K. pneumoniae ST437 clone in several hospitals in Spain. Using bioinformatic and phylogenetic analyses, we have traced the evolutionary and epidemiological route of the new allele, the hosting plasmid, and the strain that carried both of them from Pakistan to Spain. A better understanding of the NDM-producing K. pneumoniae populations and plasmids has made evident the spread of this clone through the region, enhancing the importance of genomic surveillance in the control of antimicrobial resistance.

Genetic determinants of host tropism in Klebsiella phages.

Bacteriophages play key roles in bacterial ecology and evolution and are potential antimicrobials. However, the determinants of phage-host specificity remain elusive. Here, we isolate 46 phages to challenge 138 representative clinical isolates of Klebsiella pneumoniae, a widespread opportunistic pathogen. Spot tests show a narrow host range for most phages, with <2% of 6,319 phage-host combinations tested yielding detectable interactions. Bacterial capsule diversity is the main factor restricting phage host range. Consequently, phage-encoded depolymerases are key determinants of host tropism, and depolymerase sequence types are associated with the ability to infect specific capsular types across phage families. However, all phages with a broader host range found do not encode canonical depolymerases, suggesting alternative modes of entry. These findings expand our knowledge of the complex interactions between bacteria and their viruses and point out the feasibility of predicting the first steps of phage infection using bacterial and phage genome sequences.

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.

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.

The widespread presence of a family of fish virulence plasmids in Vibrio vulnificus stresses its relevance as a zoonotic pathogen linked to fish farms.

Vibrio vulnificus is a pathogen of public health concern that causes either primary septicemia after ingestion of raw shellfish or secondary septicemia after wound exposure to seawater. In consequence, shellfish and seawater are considered its main reservoirs. However, there is one aspect of its biology that is systematically overlooked: its association with fish in its natural environment. This association led in 1975 to the emergence of a zoonotic clade within phylogenetic lineage 2 following successive outbreaks of vibriosis in farmed eels. Although this clade is now worldwide distributed, no new zoonotic clades were subsequently reported. In this work, we have performed phylogenetic, genomic and functional studies to show that other zoonotic clades are in fact present in 4 of the 5 lineages of the species. Further, we associate these clades, most of them previously but incompletely described, with the acquisition of a family of fish virulence plasmids containing genes essential for resistance to the immune system of certain teleosts of interest in aquaculture. Consequently, our results provide several pieces of evidence about the importance of this species as a zoonotic agent linked to fish farms, as well as on the relevance of these artificial environments acting as drivers that accelerate the evolution of the species.

Complete Analysis of the Epidemiological Scenario around a SARS-CoV-2 Reinfection: Previous Infection Events and Subsequent Transmission.

The first descriptions of reinfection by SARS-CoV-2 have been recently reported. However, these studies focus exclusively on the reinfected case, without considering the epidemiological context of the event. Our objectives were to perform a complete analysis of the sequential infections and community transmission events around a SARS-CoV-2 reinfection, including the infection events preceding it, the exposure, and subsequent transmissions. Our analysis was supported by host genetics, viral whole-genome sequencing, phylogenomic viral population analysis, and refined epidemiological data obtained from interviews with the involved subjects. The reinfection involved a 53-year-old woman with asthma (Case A), with a first COVID-19 episode in April 2020 and a much more severe second episode 4-1/2 months later, with SARS-CoV-2 seroconversion in August, that required hospital admission. An extended genomic analysis allowed us to demonstrate that the strain involved in Case A's reinfection was circulating in the epidemiological context of Case A and was also transmitted subsequently from Case A to her family context. The reinfection was also supported by a phylogenetic analysis, including 348 strains from Madrid, which revealed that the strain involved in the reinfection was circulating by the time Case A suffered the second episode, August-September 2020, but absent at the time range corresponding to Case A's first episode. IMPORTANCE We present the first complete analysis of the epidemiological scenario around a reinfection by SARS-CoV-2, more severe than the first episode, including three cases preceding the reinfection, the reinfected case per se, and the subsequent transmission to another seven cases.

Draft Genome Sequences of Vibrio vulnificus Strains Recovered from Moribund Tilapia.

Potentially zoonotic Vibrio vulnificus strains were isolated from vibriosis outbreaks occurring on eastern Mediterranean tilapia farms between 2016 and 2019. In this work, the draft genome sequences of three representative isolates are presented.

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.

Molecular epidemiology and drug-resistance mechanisms in carbapenem-resistant Klebsiella pneumoniae isolated in patients from a tertiary hospital in Valencia, Spain.

OBJECTIVES: The aim of this study has been to characterize carbapenem-resistant Klebsiella pneumoniae isolates and to determine the resistance mechanisms involved, the clonal relationship between strains and clinical and demographical data of the infected patients. METHODS: Clinical and demographical data from patients were collected and statistically analysed. Antimicrobial susceptibility testing was performed and resistance genes were detected both phenotypically and genotypically. Conjugation assays were performed to show horizontal transferability of resistance genes. Clonal relationship was also studied. Next-generation sequencing (NGS) was performed to obtain information regarding resistance genes, sequence types, virulence factors and plasmid types. RESULTS: Statistical significance was shown by the presence of an infection if there had been a previous hospital stay; urinary catheter carriage and chronic renal disease also indicated higher probabilities of being infected. More than 95% of the isolates were non-susceptible to third-generation cephalosporins, and more than 90% were non-susceptible to quinolones. Phenotypic and genotypic methods for resistance detection were concordant and later confirmed by NGS. This is the first detection of OXA-48, NDM-1 and CTX-M-15 co-production in the area. No plasmid-mediated colistin resistance was found. Tetracycline, sulfonamides and aminoglycoside resistance genes were found in almost all the isolates studied. No virulence factors were detected. Multilocus sequence typing showed more than 15 different sequence types, with ST101, ST307 and ST11 being the most prevalent. CONCLUSIONS: This study is the first to report such a large group of OXA-48 carbapenemases with clonal dissemination among carbapenem-resistant K. pneumoniae in Valencia. This is also the first detection of OXA-48, NDM-1 and CTX-M-15 co-production in the area.

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.

High-throughput sequencing (HTS) for the analysis of viral populations.

The development of High-Throughput Sequencing (HTS) technologies is having a major impact on the genomic analysis of viral populations. Current HTS platforms can capture nucleic acid variation across millions of genes for both selected amplicons and full viral genomes. HTS has already facilitated the discovery of new viruses, hinted new taxonomic classifications and provided a deeper and broader understanding of their diversity, population and genetic structure. Hence, HTS has already replaced standard Sanger sequencing in basic and applied research fields, but the next step is its implementation as a routine technology for the analysis of viruses in clinical settings. The most likely application of this implementation will be the analysis of viral genomics, because the huge population sizes, high mutation rates and very fast replacement of viral populations have demonstrated the limited information obtained with Sanger technology. In this review, we describe new technologies and provide guidelines for the high-throughput sequencing and genetic and evolutionary analyses of viral populations and metaviromes, including software applications. With the development of new HTS technologies, new and refurbished molecular and bioinformatic tools are also constantly being developed to process and integrate HTS data. These allow assembling viral genomes and inferring viral population diversity and dynamics. Finally, we also present several applications of these approaches to the analysis of viral clinical samples including transmission clusters and outbreak characterization.

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

Hepatitis C virus intrinsic molecular determinants may contribute to the development of cholestatic hepatitis after liver transplantation.

Cholestatic hepatitis C (CHC) is a severe form of hepatitis C virus (HCV) infection recurrence that leads to high graft loss rates early after liver transplantation (LT). To investigate the pathogenic mechanisms of CHC, we analysed HCV quasispecies in CHC patients compared to a control group (mild hepatitis C recurrence) by deep pyrosequencing. At the time of LT, NS5B quasispecies complexity was similar between the two groups but, after LT, it decreased more sharply in CHC patients than in the control group. Interestingly, the major variant before LT propagated efficiently and remained as the dominant sequence after LT in 62 % of CHC patients versus 11 % of controls (P=0.031). Sequence analysis of the complete non-structural region in a limited number of patients revealed a potential 12 aa signature specific to the CHC group. These data suggest that intrinsic molecular determinants in the circulating HCV quasispecies may provide a fitness advantage, contributing to the development of CHC.

Hepatitis C virus early kinetics and resistance-associated substitution dynamics during antiviral therapy with direct-acting antivirals.

The emergence of resistance-associated substitutions (RASs) can compromise the high efficacy of direct-acting antivirals (DAAs). Little is known about RASs selection at very early time points during DAA treatment. Therefore, we analyzed the potential emergence of RASs immediately after therapy initiation. Samples of 71 patients treated with different DAAs were collected at baseline, during therapy (hours 4 and 8; days 1-7; weeks 2-4) or until target not detected. HCV-RNA levels were determined by qPCR, and RASs were detected by deep sequencing. Sixty-three (89%) patients achieved a sustained virological response (SVR), 7 (10%) relapsed, and 1 (1%) experienced a breakthrough. Almost all non-SVR (7/8, 88%) showed RASs either at baseline or relapse. High-frequency RASs detected at baseline (Y93H and L159F+C316N) remained detectable at early time points during therapy and reappeared as most prevalent substitutions at relapse. Conversely, emergent RASs at relapse (Q80R, D168E/V, R155K and L31V) were not observed during the first hours-days, before HCV-RNA became undetectable. HCV-RNA decay and genetic evolution of the quasispecies followed a similar pattern during the first hours of therapy in SVR and non-SVR patients. In conclusion, the absence of early RASs selection and the similar dynamics of HCV kinetics and quasispecies in SVR and non-SVR patients after therapy initiation suggest that RASs selection may occur at later stages in the remaining reservoir, where viral populations persist hidden at very low replication levels. Nevertheless, we cannot completely exclude very early selection, when RASs are present below the sensitivity limit of deep sequencing.

Phylogenetic analysis of an epidemic outbreak of acute hepatitis C in HIV-infected patients by ultra-deep pyrosequencing.

BACKGROUND: The incidence of acute hepatitis C (AHC) among HIV-infected men who have sex with men (MSM) has increased significantly in the last 10 years. Several studies point to a social and sexual network of HIV-positive MSM that extends internationally. OBJECTIVES: The aim of our study was to investigate the dynamics of HCV transmission in an outbreak of AHC in HIV-infected MSM in Barcelona by ultra-deep pyrosequencing. STUDY DESIGN: Between 2008 and 2013, 113 cases of AHC in HIV-infected MSM were diagnosed in the Infectious Diseases Unit, Hospital Clínic, Barcelona. Massive sequencing was performed using the Roche 454 GS Junior platform. To define possible transmission networks, maximum likelihood phylogenetic trees were constructed, and levels of genetic diversity within and among patients were compared. RESULTS: Among the 70 cases analyzed, we have identified 16 potential clusters of transmission: 8 for genotype 1a (23 cases involved), 1 for genotype 1b (3 cases) and 7 for genotype 4d (27 cases). Although the initial phylogenetic reconstruction suggested a local transmission cluster of HCV gt4d, our approach based on low genetic differentiation did not corroborate it. Indeed, gt4d strains formed 4 independent groups related to patients from other countries. CONCLUSIONS: Frequent clustering of HIV-positive MSM shows that HCV infection has spread through a local network in Barcelona. This outbreak is related to a large international HCV transmission network among MSM. Public health efforts are needed to reduce HCV transmission among this high-risk group.