Caracterización microbiológica de cepas de Cutibacterium acnes aisladas de infección protésica articular

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Medicina Preventiva y Salud Pública y Microbiología. Fecha de Lectura: 12-05-2023Este estudio se realizado dentro del proyecto sobre infección protésica causada por Cutibacterium spp. patrocinado por el ESCMID Study Group for Implant Associated Infections (ESGIAI

Microbiological Characterization of Cutibacterium acnes Strains Isolated from Prosthetic Joint Infections

Aims: This study aimed to characterize 79 Cutibacterium acnes strains isolated from prosthetic joint infections (PJIs) originated from eight European hospitals. Methods: Isolates were phylotyped according to the single-locus sequence typing (SLST) scheme. We evaluated the ability of the biofilm formation of C. acnes strains isolated from PJIs and 84 isolates recovered from healthy skin. Antibiotic susceptibility testing of planktonic and biofilm cells of PJI isolates and skin isolates was performed. Results: Most of the isolates from PJIs belonged to the SLST class H/phylotype IB (34.2%), followed by class D/phylotype IA1 (21.5%), class A/phylotype IA1 (18.9%), and class K/phylotype II (13.9%). All tested isolates were biofilm producers; no difference in biofilm formation was observed between the healthy skin group and the PJI group of strains. Planktonic and sessile cells of C. acnes remained highly susceptible to a broad spectrum of antibiotics, including beta-lactams, clindamycin, fluoroquinolones, linezolid, rifampin, and vancomycin. The minimal inhibitory concentrations (MICs) for planktonic and biofilm states coincided in most cases. However, the minimal biofilm eradication concentration (MBEC) was high for all antimicrobial drugs tested (>32 mg/L), except for rifampin (2 mg/L). Conclusions: C. acnes strains isolated from healthy skin were able to produce biofilm to the same extent as isolates recovered from PJIs. All C. acnes strains in planktonic and sessile states were susceptible to most antibiotics commonly used for PJI treatment, although rifampin was the only antimicrobial agent able to eradicate C. acnes embedded in biofilm. Keywords: Cutibacteriumacnes; antimicrobial susceptibility; biofilm; phylotyping; prosthetic joint infection

Multicenter evaluation of BioFire JI panel related to improved microbiological diagnostics on acute osteoarticular infections

Microbiological diagnosis of osteoarticular infections (OI) is crucial for a successful treatment. A prospective multicenter study including 262 synovial fluids with suspicion of acute OI was performed between July 2021 and October of 2022. BioFire Joint Infection Panel multiplex-PCR test was performed and results were compared with conventional cultures of synovial fluid specimens. In total, 136 microorganisms were detected, and fourteen samples were positive for more than one microorganism. In monomicrobial infections (n = 87) agreement with culture was 69%. In 26 samples, the multiplex PCR yield an additional positive result when culture result was negative. It helped in the detection of fastidious microorganisms as K. kingae and N. gonorrhoeae. This multiplex PCR has proven to be a useful technique that can be used for patients with high suspicion of acute OI in a rapid and automated manner

Vaccine breakthrough infections with SARS-CoV-2 Alpha mirror mutations in Delta Plus, Iota, and Omicron

Replication of SARS-CoV-2 in the human population is defined by distributions of mutants that are present at different frequencies within the infected host and can be detected by ultra-deep sequencing techniques. In this study, we examined the SARS-CoV-2 mutant spectra of amplicons from the spike-coding (S-coding) region of 5 nasopharyngeal isolates derived from patients with vaccine breakthrough. Interestingly, all patients became infected with the Alpha variant, but amino acid substitutions that correspond to the Delta Plus, Iota, and Omicron variants were present in the mutant spectra of the resident virus. Deep sequencing analysis of SARS-CoV-2 from patients with vaccine breakthrough revealed a rich reservoir of mutant types and may also identify tolerated substitutions that can be represented in epidemiologically dominant variants.This work was supported by the Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation (COVID-19 Research Call COV20/00181) and co-financed by the European Development Regional Fund “A way to achieve Europe.” The work was also supported by grants CSIC-COV19-014 from the CSIC, project 525/C/2021 from the Fundació La Marató de TV3; PID2020-113888RB-I00 from the Ministerio de Ciencia e Innovación; BFU2017-91384-EXP from the Ministerio de Ciencia, Innovación y Universidades (MCIU);PI18/00210 and PI21/00139 from the Instituto de Salud Carlos III; and S2018/BAA-4370 (PLATESA2) from the Comunidad de Madrid/ FEDER. This research work was also funded by the European Commission – NextGenerationEU (regulation EU 2020/2094), through the CSIC’s Global Health Platform (PTI Salud Global). CP and PM are supported by the Miguel Servet programme of the Instituto de Salud Carlos III (CPII19/00001 and CP16/00116, respectively), cofinanced by the European Regional Development Fund (ERDF). CIBERehd is funded by the Instituto de Salud Carlos III. Institutional grants from the Fundación Ramón Areces and Banco Santander to the CBMSO are also acknowledged. The team at CBMSO belongs to the Global Virus Network (GVN). BMG is supported by predoctoral contract PFIS FI19/00119 from the Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo), cofinanced by the Fondo Social Europeo (FSE). CGC is supported by predoctoral contract PRE2018- 083422 from the MCIU. BS was supported by a predoctoral research fellowship (Doctorados Industriales, DI-17-09134) from the Spanish Ministry of Economy and Competitiveness (MINECO).Peer reviewe

SARS-CoV-2 mutant spectra reveal differences between COVID-19 severity categories

Trabajo presentado en el XVI Congreso Nacional de Virología, celebrado en Málaga (España) del 06 al 09 de septiembre de 2022.RNA virus populations are composed of complex mixtures of genomes that are termed mutant spectra. SARS-CoV-2 replicates as a viral quasispecies, and mutations that are detected at low frequencies in a host can be dominant in subsequent variants. We have studied mutant spectrum complexities of SARS-CoV-2 populations derived from thirty nasopharyngeal swabs of patients infected during the first wave (April 2020) in the Hospital Universitario Fundación Jiménez Díaz. The patients were classified according to the COVID-19 severity in mild (non-hospitalized), moderate (hospitalized) and exitus (hospitalized with ICU admission and who passed away due to COVID-19). Using ultra-deep sequencing technologies (MiSeq, Illumina), we have examined four amplicons of the nsp12 (polymerase)-coding region and two amplicons of the spike-coding region. Ultra-deep sequencing data were analyzed with different cut-off frequency for mutation detection. Average number of different point mutations, mutations per haplotype and several diversity indices were significantly higher in SARS-CoV-2 isolated from patients who developed mild disease. A feature that we noted in the SARS-CoV-2 mutant spectra from diagnostic samples is the remarkable absence of mutations at intermediate frequencies, and an overwhelming abundance of mutations at frequencies lower than 10%. Thus, the decrease of the cut-off frequency for mutation detection from 0.5% to 0.1% revealed an increasement (50- to 100 fold) in the number of different mutations. The significantly higher frequency of mutations in virus from patients displaying mild than moderate or severe disease was maintained with the 0.1% cut- off frequency. To evaluate whether the frequency repertoire of amino acid substitutions differed between SARS-CoV-2 and the well characterized hepatitis C virus (HCV), we performed a comparative study of mutant spectra from infected patients using the same bioinformatics pipelines. HCV did not show the deficit of intermediate frequency substitutions that was observed with SARS-CoV-2. This difference was maintained when two functionally equivalent proteins, the corresponding viral polymerases, were compared. In conclusion, SARS-CoV-2 mutant spectra are rich reservoirs of mutants, whose complexity is not uniform among clinical isolates. Virus from patients who developed mild disease may be a source of new variants that may acquire epidemiological relevance.This work was supported by Instituto de Salud Carlos III, Spanish Ministry of Science and In-novation (COVID-19 Research Call COV20/00181), and co-financed by European Development Regional Fund ‘A way to achieve Europe’. The work was also supported by grants CSIC-COV19-014 from Consejo Superior de Investigaciones Científicas (CSIC), project 525/C/2021 from Fundació La Marató de TV3, PID2020-113888RB-I00 from Ministerio de Ciencia e Innovación, BFU2017-91384-EXP from Ministerio de Ciencia, Innovación y Universidades (MCIU), PI18/00210 and PI21/00139 from Instituto de Salud Carlos III, and S2018/BAA-4370 (PLATESA2 from Comunidad de Madrid/FEDER). C.P., M.C., and P.M. are supported by the Miguel Servet programme of the Instituto de Salud Carlos III (CPII19/00001, CPII17/00006, and CP16/00116, respectively) co-financed by the European Regional Development Fund (ERDF). CIBERehd (Centro de Investi-gación en Red de Enfermedades Hepáticas y Digestivas) is funded by Instituto de Salud Carlos III. Institutional grants from the Fundación Ramón Areces and Banco Santander to the CBMSO are also acknowledged. The team at CBMSO belongs to the Global Virus Network (GVN). B.M.-G. is supported by predoctoral contract PFIS FI19/00119 from Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo) cofinanced by Fondo Social Europeo (FSE). R.L.-V. is supported by predoctoral contract PEJD-2019-PRE/BMD-16414 from Comunidad de Madrid. C.G.-C. is sup-ported by predoctoral contract PRE2018-083422 from MCIU. BS was supported by a predoctoral research fellowship (Doctorados Industriales, DI-17-09134) from Spanish MINECO

SARS-CoV-2 Point Mutation and Deletion Spectra, and Their Association with Different Disease Outcome

Mutant spectra of RNA viruses are important to understand viral pathogenesis, and response to selective pressures. There is a need to characterize the complexity of mutant spectra in coronaviruses sampled from infected patients. In particular, the possible relationship between SARS-CoV-2 mutant spectrum complexity and disease associations has not been established. In the present study, we report an ultra-deep sequencing (UDS) analysis of the mutant spectrum of amplicons from the nsp12 (polymerase)- and spike (S)-coding regions of thirty nasopharyngeal isolates (diagnostic samples) of SARS-CoV-2 of the first COVID-19 pandemic wave (Madrid, Spain, April 2020) classified according to the severity of ensuing COVID-19. Low frequency mutations and deletions, counted relative to the consensus sequence of the corresponding isolate, were overwhelmingly abundant. We show that the average number of different point mutations, mutations per haplotype and several diversity indices was significantly higher in SARS-CoV-2 isolated from patients who developed mild disease than in those associated with moderate or severe disease (exitus). No such bias was observed with RNA deletions. Location of amino acid substitutions in the three dimensional structures of nsp12 (polymerase) and S suggest significant structural or functional effects. Thus, patients who develop mild symptoms may be a richer source of genetic variants of SARS-CoV-2 than patients with moderate or severe COVID-19.This work was supported by Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation (COVID-19 Research Call COV20/00181), and co‐financed by European Development Regional Fund ‘A way to achieve Europe’. The work was also supported by grants CSIC-COV19-014 from Consejo Superior de Investigaciones Científicas (CSIC), project 525/C/2021 from Fundació La Marató de TV3, PID2020-113888RB-I00 from Ministerio de Ciencia e Innovación, BFU2017-91384-EXP from Ministerio de Ciencia, Innovación y Universidades (MCIU), PI18/00210 and PI21/00139 from Instituto de Salud Carlos III and S2018/BAA-4370 (PLATESA2 from Comunidad de Madrid/FEDER). C.P., M.C. and P.M. are supported by the Miguel Servet programme of the Instituto de Salud Carlos III (CPII19/00001, CPII17/00006 and CP16/00116, respectively) cofinanced by the European Regional Development Fund (ERDF). CIBERehd (Centro de Investigación en Red de Enfermedades Hepáticas y Digestivas) is funded by Instituto de Salud Carlos III. Institutional grants from the Fundación Ramón Areces and Banco Santander to the CBMSO are also acknowledged. The team at CBMSO belongs to the Global Virus Network (GVN). B.M.-G. is supported by predoctoral contract PFIS FI19/00119 from Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo) cofinanced by Fondo Social Europeo (FSE). R.L.- V. is supported by predoctoral contract PEJD-2019-PRE/BMD-16414 from Comunidad de Madrid. C.G.-C. is supported by predoctoral contract PRE2018-083422 from MCIU. BS was supported by a predoctoral research fellowship (Doctorados Industriales, DI-17- 09134) from Spanish MINECON

SARS-CoV-2 Point Mutation and Deletion Spectra and Their Association with Different Disease Outcomes

Mutant spectra of RNA viruses are important to understand viral pathogenesis and response to selective pressures. There is a need to characterize the complexity of mutant spectra in coronaviruses sampled from infected patients. In particular, the possible relationship between SARS-CoV-2 mutant spectrum complexity and disease associations has not been established. In the present study, we report an ultradeep sequencing (UDS) analysis of the mutant spectrum of amplicons from the nsp12 (polymerase)- and spike (S)-coding regions of 30 nasopharyngeal isolates (diagnostic samples) of SARS-CoV-2 of the first COVID-19 pandemic wave (Madrid, Spain, April 2020) classified according to the severity of ensuing COVID-19. Low-frequency mutations and deletions, counted relative to the consensus sequence of the corresponding isolate, were overwhelmingly abundant. We show that the average number of different point mutations, mutations per haplotype, and several diversity indices was significantly higher in SARS-CoV-2 isolated from patients who developed mild disease than in those associated with moderate or severe disease (exitus). No such bias was observed with RNA deletions. Location of amino acid substitutions in the three-dimensional structures of nsp12 (polymerase) and S suggest significant structural or functional effects. Thus, patients who develop mild symptoms may be a richer source of genetic variants of SARS-CoV-2 than patients with moderate or severe COVID-19.Peer reviewe

SARS-CoV-2 Mutant Spectra at Different Depth Levels Reveal an Overwhelming Abundance of Low Frequency Mutations

Populations of RNA viruses are composed of complex and dynamic mixtures of variant genomes that are termed mutant spectra or mutant clouds. This applies also to SARS-CoV-2, and mutations that are detected at low frequency in an infected individual can be dominant (represented in the consensus sequence) in subsequent variants of interest or variants of concern. Here we briefly review the main conclusions of our work on mutant spectrum characterization of hepatitis C virus (HCV) and SARS-CoV-2 at the nucleotide and amino acid levels and address the following two new questions derived from previous results: (i) how is the SARS-CoV-2 mutant and deletion spectrum composition in diagnostic samples, when examined at progressively lower cut-off mutant frequency values in ultra-deep sequencing; (ii) how the frequency distribution of minority amino acid substitutions in SARS-CoV-2 compares with that of HCV sampled also from infected patients. The main conclusions are the following: (i) the number of different mutations found at low frequency in SARS-CoV-2 mutant spectra increases dramatically (50- to 100-fold) as the cut-off frequency for mutation detection is lowered from 0.5% to 0.1%, and (ii) that, contrary to HCV, SARS-CoV-2 mutant spectra exhibit a deficit of intermediate frequency amino acid substitutions. The possible origin and implications of mutant spectrum differences among RNA viruses are discussed.This work was supported by Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation (COVID-19 Research Call COV20/00181), and co-financed by European Development Regional Fund ‘A way to achieve Europe’. The work was also supported by grants CSIC-COV19-014 from Consejo Superior de Investigaciones Científicas (CSIC), project 525/C/2021 from Fundació La Marató de TV3, PID2020-113888RB-I00 from Ministerio de Ciencia e Innovación, BFU2017-91384-EXP from Ministerio de Ciencia, Innovación y Universidades (MCIU), PI18/00210 and PI21/00139 from Instituto de Salud Carlos III, and S2018/BAA-4370 (PLATESA2 from Comunidad de Madrid/FEDER). C.P., M.C., and P.M. are supported by the Miguel Servet programme of the Instituto de Salud Carlos III (CPII19/00001, CPII17/00006, and CP16/00116, respectively) cofinanced by the European Regional Development Fund (ERDF). CIBERehd (Centro de Investigación en Red de Enfermedades Hepáticas y Digestivas) is funded by Instituto de Salud Carlos III. Institutional grants from the Fundación Ramón Areces and Banco Santander to the CBMSO are also acknowledged. The team at CBMSO belongs to the Global Virus Network (GVN). B.M.-G. is supported by predoctoral contract PFIS FI19/00119 from Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo) cofinanced by Fondo Social Europeo (FSE). R.L.-V. is supported by predoctoral contract PEJD-2019-PRE/BMD-16414 from Comunidad de Madrid. C.G.-C. is supported by predoctoral contract PRE2018-083422 from MCIU. P.S. is supported by postdoctoral contract “Margarita Salas” CA1/RSUE/2021 from MCIU. B.S. was supported by a predoctoral research fellowship (Doctorados Industriales, DI-17-09134) from Spanish MINECO.Peer reviewe

Potential value of a rapid syndromic multiplex PCR for the diagnosis of native and prosthetic joint infections: a real-world evidence study

Introduction: The BIOFIRE Joint Infection (JI) Panel is a diagnostic tool that uses multiplex-PCR testing to detect microorganisms in synovial fluid specimens from patients suspected of having septic arthritis (SA) on native joints or prosthetic joint infections (PJIs). Methods: A study was conducted across 34 clinical sites in 19 European and Middle Eastern countries from March 2021 to June 2022 to assess the effectiveness of the BIOFIRE JI Panel. Results: A total of 1527 samples were collected from patients suspected of SA or PJI, with an overall agreement of 88.4 % and 85 % respectively between the JI Panel and synovial fluid cultures (SFCs). The JI Panel detected more positive samples and microorganisms than SFC, with a notable difference on Staphylococcus aureus, Streptococcus species, Enterococcus faecalis, Kingella kingae, Neisseria gonorrhoeae, and anaerobic bacteria. The study found that the BIOFIRE JI Panel has a high utility in the real-world clinical setting for suspected SA and PJI, providing diagnostic results in approximately 1 h. The user experience was positive, implying a potential benefit of rapidity of results' turnover in optimising patient management strategies. Conclusion: The study suggests that the BIOFIRE JI Panel could potentially optimise patient management and antimicrobial therapy, thus highlighting its importance in the clinical setting

Molecular Diagnosis of Osteoarticular Implant-Associated Infection: Available Techniques and How We Can Use Them

Despite recent advances during the last few years, microbiological diagnosis of prosthetic joint infections remains a challenge. Molecular biology techniques have been developed to try to overcome this problem, and recently, many of them have become available for many laboratories. Some of them, especially commercial multiplex PCR-based assays and universal 16S rDNA homemade PCR assays, are now available in many laboratories. Moreover, new technologies have appeared, especially metagenomics and next-generation sequencing. These techniques have demonstrated their potential in many studies but appear to be experimental at present. A few studies have evaluated the possible use of these methods in the clinical routine, and a review of the critical aspects for the selection of a molecular method (accuracy, complexity, cost) was performed. Finally, a proposal for a protocol that includes molecular biology techniques was made according to the literature published in this field. In conclusion, molecular biology techniques are ready to be used in the clinical routine of a microbiology laboratory, but their use must be carried out in accordance with the many special characteristics of each laboratory. In all cases, the interpretation of the results must be conducted by a multidisciplinary team with experience in the management of these patients