Antibiotic resistance and hospital acquired infections are on the rise worldwide. Vancomycin-resistant enterococci have been reported in clinical settings in recent decades. In this multiomics study, we provide comprehensive proteomic and transcriptomic analyses of a vancomycin-resistant Enterococcus faecalis clinical isolate from a patient with a urinary tract infection. The previous genotypic profile of the strain C2620 indicated the presence of antibiotic resistance genes characteristic of the vanB cluster. To further investigate the transcriptome of this pathogenic strain, we used whole genome sequencing and RNA-sequencing to detect and quantify the genes expressed. In parallel, we used two-dimensional gel electrophoresis followed by MALDI-TOF/MS (Matrix-assisted laser desorption/ionization-Time-of-flight/Mass spectrometry) to identify the proteins in the proteome. We studied the membrane and cytoplasm subproteomes separately. From a total of 207 analysis spots, we identified 118 proteins. The protein list was compared to the results obtained from the full transcriptome assay. Several genes and proteins related to stress and cellular response were identified, as well as some linked to antibiotic and drug responses, which is consistent with the known state of multiresistance. Even though the correlation between transcriptome and proteome data is not yet fully understood, the use of multiomics approaches has proven to be increasingly relevant to achieve deeper insights into the survival ability of pathogenic bacteria found in health care facilities.

Antibiotic resistance and hospital acquired infections are on the rise worldwide. Vancomycin-resistant enterococci have been reported in clinical settings in recent decades. In this multiomics study, we provide comprehensive proteomic and transcriptomic analyses of a vancomycin-resistant Enterococcus faecalis clinical isolate from a patient with a urinary tract infection. The previous genotypic profile of the strain C2620 indicated the presence of antibiotic resistance genes characteristic of the vanB cluster. To further investigate the transcriptome of this pathogenic strain, we used whole genome sequencing and RNA-sequencing to detect and quantify the genes expressed. In parallel, we used two-dimensional gel electrophoresis followed by MALDI-TOF/MS (Matrix-assisted laser desorption/ionization-Time-of-flight/Mass spectrometry) to identify the proteins in the proteome. We studied the membrane and cytoplasm subproteomes separately. From a total of 207 analysis spots, we identified 118 proteins. The protein list was compared to the results obtained from the full transcriptome assay. Several genes and proteins related to stress and cellular response were identified, as well as some linked to antibiotic and drug responses, which is consistent with the known state of multiresistance. Even though the correlation between transcriptome and proteome data is not yet fully understood, the use of multiomics approaches has proven to be increasingly relevant to achieve deeper insights into the survival ability of pathogenic bacteria found in health care facilities.

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli strain C999 was isolated of a Spanish patient with urinary tract infection. Previous genotyping indicated that this strain presented a multidrug-resistance phenotype and carried beta-lactamase genes encoding CTX-M-15, TEM-1, and OXA-1 enzymes. The whole-cell proteome, and the membrane, cytoplasmic, periplasmic and extracellular sub-proteomes of C999 were obtained in this work by two-dimensional gel electrophoresis (2DE) followed by fingerprint sequencing through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). A total of 602 proteins were identified in the different cell fractions, several of which are related to stress response systems, cellular responses, and antibiotic and drug responses, consistent with the multidrug-resistance phenotype. In parallel, whole genome sequencing (WGS) and RNA sequencing (RNA-Seq) was done to identify and quantify the genes present and expressing. The in silico prediction following WGS confirmed our strain as being serotype O25:H4 and sequence type ST131. The presence of proteins related to antibiotic resistance and virulence in an O25:H4-ST131 E. coli clone are serious indicators of the continued threat of antibiotic resistance spread amongst healthcare institutions. On a positive note, a multiomics approach can facilitate surveillance and more detailed characterization of virulent bacterial clones from hospital environments.

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli strain C999 was isolated of a Spanish patient with urinary tract infection. Previous genotyping indicated that this strain presented a multidrug-resistance phenotype and carried beta-lactamase genes encoding CTX-M-15, TEM-1, and OXA-1 enzymes. The whole-cell proteome, and the membrane, cytoplasmic, periplasmic and extracellular sub-proteomes of C999 were obtained in this work by two-dimensional gel electrophoresis (2DE) followed by fingerprint sequencing through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). A total of 602 proteins were identified in the different cell fractions, several of which are related to stress response systems, cellular responses, and antibiotic and drug responses, consistent with the multidrug-resistance phenotype. In parallel, whole genome sequencing (WGS) and RNA sequencing (RNA-Seq) was done to identify and quantify the genes present and expressing. The in silico prediction following WGS confirmed our strain as being serotype O25:H4 and sequence type ST131. The presence of proteins related to antibiotic resistance and virulence in an O25:H4-ST131 E. coli clone are serious indicators of the continued threat of antibiotic resistance spread amongst healthcare institutions. On a positive note, a multiomics approach can facilitate surveillance and more detailed characterization of virulent bacterial clones from hospital environments.

Neisseria gonorrhoeae, the bacterium responsible for the sexually transmitted disease gonorrhoea, has shown an extraordinary ability to develop antimicrobial resistance (AMR) to multiple classes of antimicrobials. With no available vaccine, managing N. gonorrhoeae infections demands effective preventive measures, antibiotic treatment and epidemiological surveillance. The latter two are progressively being supported by the generation of whole-genome sequencing (WGS) data on behalf of national and international surveillance programmes. In this context, this study aims to perform N. gonorrhoeae clustering into genogroups based on WGS data, for enhanced prospective laboratory surveillance. Particularly, it aims to identify the major circulating WGS-genogroups in Europe and to establish a relationship between these and AMR. Ultimately, it enriches public databases by contributing with WGS data from Portuguese isolates spanning 15 years of surveillance. A total of 3791 carefully inspected N. gonorrhoeae genomes from isolates collected across Europe were analysed using a gene-by-gene approach (i.e. using cgMLST). Analysis of cluster composition and stability allowed the classification of isolates into a two-step hierarchical genogroup level determined by two allelic distance thresholds revealing cluster stability. Genogroup clustering in general agreed with available N. gonorrhoeae typing methods [i.e. MLST (multilocus sequence typing), NG-MAST (N. gonorrhoeae multi-antigen sequence typing) and PubMLST core-genome groups], highlighting the predominant genogroups circulating in Europe, and revealed that the vast majority of the genogroups present a dominant AMR profile. Additionally, a non-static gene-by-gene approach combined with a more discriminatory threshold for potential epidemiological linkage enabled us to match data with previous reports on outbreaks or transmission chains. In conclusion, this genogroup assignment allows a comprehensive analysis of N. gonorrhoeae genetic diversity and the identification of the WGS-based genogroups circulating in Europe, while facilitating the assessment (and continuous monitoring) of their frequency, geographical dispersion and potential association with specific AMR signatures. This strategy may benefit public-health actions through the prioritization of genogroups to be controlled, the identification of emerging resistance carriage, and the potential facilitation of data sharing and communication.

Neisseria gonorrhoeae, the bacterium responsible for the sexually transmitted disease gonorrhoea, has shown an extraordinary ability to develop antimicrobial resistance (AMR) to multiple classes of antimicrobials. With no available vaccine, managing N. gonorrhoeae infections demands effective preventive measures, antibiotic treatment and epidemiological surveillance. The latter two are progressively being supported by the generation of whole-genome sequencing (WGS) data on behalf of national and international surveillance programmes. In this context, this study aims to perform N. gonorrhoeae clustering into genogroups based on WGS data, for enhanced prospective laboratory surveillance. Particularly, it aims to identify the major circulating WGS-genogroups in Europe and to establish a relationship between these and AMR. Ultimately, it enriches public databases by contributing with WGS data from Portuguese isolates spanning 15 years of surveillance. A total of 3791 carefully inspected N. gonorrhoeae genomes from isolates collected across Europe were analysed using a gene-by-gene approach (i.e. using cgMLST). Analysis of cluster composition and stability allowed the classification of isolates into a two-step hierarchical genogroup level determined by two allelic distance thresholds revealing cluster stability. Genogroup clustering in general agreed with available N. gonorrhoeae typing methods [i.e. MLST (multilocus sequence typing), NG-MAST (N. gonorrhoeae multi-antigen sequence typing) and PubMLST core-genome groups], highlighting the predominant genogroups circulating in Europe, and revealed that the vast majority of the genogroups present a dominant AMR profile. Additionally, a non-static gene-by-gene approach combined with a more discriminatory threshold for potential epidemiological linkage enabled us to match data with previous reports on outbreaks or transmission chains. In conclusion, this genogroup assignment allows a comprehensive analysis of N. gonorrhoeae genetic diversity and the identification of the WGS-based genogroups circulating in Europe, while facilitating the assessment (and continuous monitoring) of their frequency, geographical dispersion and potential association with specific AMR signatures. This strategy may benefit public-health actions through the prioritization of genogroups to be controlled, the identification of emerging resistance carriage, and the potential facilitation of data sharing and communication.

Insights into the genomic adaptive traits of Treponema pallidum, the causative bacterium of syphilis, have long been hampered due to the absence of in vitro culture models and the constraints associated with its propagation in rabbits. Here, we have bypassed the culture bottleneck by means of a targeted strategy never applied to uncultivable bacterial human pathogens to directly capture whole-genome T. pallidum data in the context of human infection. This strategy has unveiled a scenario of discreet T. pallidum interstrain single-nucleotide-polymorphism-based microevolution, contrasting with a rampant within-patient genetic heterogeneity mainly targeting multiple phase-variable loci and a major antigen-coding gene (tprK). TprK demonstrated remarkable variability and redundancy, intra- and interpatient, suggesting ongoing parallel adaptive diversification during human infection. Some bacterial functions (for example, flagella- and chemotaxis-associated) were systematically targeted by both inter- and intrastrain single nucleotide polymorphisms, as well as by ongoing within-patient phase variation events. Finally, patient-derived genomes possess mutations targeting a penicillin-binding protein coding gene (mrcA) that had never been reported, unveiling it as a candidate target to investigate the impact on the susceptibility to penicillin. Our findings decode the major genetic mechanisms by which T. pallidum promotes immune evasion and survival, and demonstrate the exceptional power of characterizing evolving pathogen subpopulations during human infection.

A sífilis é uma doença sexualmente transmissível causada pela bactéria Treponema pallidum e constitui um problema de saúde pública mundial, em parte devido à ausência de uma vacina para prevenção da sua transmissão. A investigação desta doença tem sido atrasada pela incapacidade histórica de cultivar T. pallidum in vitro, dificultando por exemplo o desenvolvimento de estudos genómicos. De facto, há uma grande lacuna no conhecimento da epidemiologia molecular deste agente patogénico, assim como da base molecular que medeia a patogénese da sífilis. No estudo aqui apresentado, foi possível implementar uma abordagem inovadora para capturar o genoma de T. pallidum no contexto de infeção humana, evitando-se, assim, a necessidade da cultura da bactéria em modelo animal. Esta estratégia permitiu estudar, pela primeira vez, como é que este agente patogénico vai alterando o seu genoma para se adaptar e sobreviver como agente infecioso humano. Nomeadamente permitiu descodificar os principais mecanismos genéticos pelos quais a bactéria T. pallidum evade o sistema imunitário e se adapta ao Homem nesta complexa e multifásica doença. A aplicação desta estratégia inovadora de monitorização da interação Homem-bactéria poderá ser importante para o desenvolvimento de novas medidas profiláticas e/ou terapêuticas. Acresce que esta abordagem constitui também um ponto de viragem para o aperfeiçoamento de metodologias de diagnóstico e de epidemiologia molecular, o que permitirá aumentar o conhecimento da distribuição geográfica, das vias de transmissão e das propriedades de virulência deste agente patogénico para bem da saúde pública.

We show that the SARS-CoV-2 B.1.1.7 lineage is highly disseminated in Portugal, with the odds of B.1.1.7 proportion increasing at an estimated 89% (95% confidence interval: 83-95%) per week until week 3 2021. RT-PCR spike gene target late detection (SGTL) can constitute a useful surrogate to track B.1.1.7 spread, besides the spike gene target failure (SGTF) proxy. SGTL/SGTF samples were associated with statistically significant higher viral loads, but not with substantial shift in age distribution compared to non-SGTF/SGTL cases.

A tuberculose multirresistente continua a ser um dos principais desafios no controlo da Tuberculose em Portugal. Os métodos de diagnóstico e vigilância clássicos são trabalhosos, muito demorados e nem sempre fáceis de interpretar. A introdução de metodologias moleculares permitiu ultrapassar alguns destes obstáculos, mas, sendo limitada no número de alvos genéticos que analisa, não permite uma análise completa das características das estirpes de M. tuberculosis isoladas. Para ultrapassar esta questão, tivemos como objectivo a implementação de uma metodologia baseada na sequenciação total do genoma que, para além de permitir um screening de todas as mutações conhecidas associadas a resistência, permite fazer uma vigilância molecular das estirpes com uma sensibilidade muito elevada, possibilitando a inter venção das Autoridades de Saúde de forma atempada e otimizada. O Laboratório Nacional de Referência de Micobactérias/Tuberculose do Instituto Nacional de Doutor Ricardo Jorge está, neste momento, capacitado para responder eficazmente a estas necessidades garantindo um dignóstico e vigilância em “tempo real” de todas as estirpes de M. tuberculosis multirresistentes.