Molecular characterization of consecutive isolates of OXA-48-producing Klebsiella pneumoniae: changes in the virulome using next-generation sequencing (NGS).

Little is known about the clonality of consecutive OXA-48 producing-Klebsiella pneumoniae isolates from the same patient and the possibility of changes in their virulomes over time. We studied the molecular characteristics of twenty OXA-48-producing K. pneumoniae consecutive isolates from six patients using whole-genome sequencing. The genomes were screened for antimicrobial resistance and virulence factor genes and for replicon groups. MLST and SNPs analysis was performed. MLST analysis found 3 STs: ST11 (n = 13; 65.0%); ST4975 (n = 5, 25.0%); ST307 (n = 2; 10.0%). AcrAb efflux pump, siderophore enterobactin and rcsAB capsule synthesis regulator were detected in all sequenced isolates. The regulator of mucoid phenotype A (rmpA) and rmpA2 were not detected. Isolates also carried type 3 fimbriae (n = 19; 95.0%), yersiniabactin (n = 15; 75.0%) and type 1 fimbriae (7; 35.0%). Type 3 fimbriae and yersiniabactin were lost and recovered in consecutive isolates of two patients, probably acquired by horizontal gene transfer. Our findings reveal that recurrent infections are due to the same isolate, with an average of 2.69 SNPs per month, with different virulence profiles, and that the acquisition of virulence factor genes over time is possible.

Cardiovascular-related proteomic changes in ECFCs exposed to the serum of COVID-19 patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection significantly affects the cardiovascular system, causing vascular damage and thromboembolic events in critical patients. Endothelial dysfunction represents one of the first steps in response to COVID-19 that might lead to cardiovascular complications and long-term sequelae. However, despite the enormous efforts in the last two years, the molecular mechanisms involved in such processes remain poorly understood. Herein, we analyzed the protein changes taking place in endothelial colony forming cells (ECFCs) after the incubation with the serum from individuals infected with COVID-19, whether asymptomatic or critical patients, by application of a label free-quantitative proteomics approach. Specifically, ECFCs from healthy individuals were incubated ex-vivo with the serum of either COVID-19 negative donors (PCR-/IgG-, n:8), COVID-19 asymptomatic donors at different infective stages (PCR+/ IgG-, n:8and PCR-/IgG+, n:8), or hospitalized critical COVID-19 patients (n:8), followed by proteomics analysis. In total, 590 proteins were differentially expressed in ECFCs in response to all infected serums. Predictive analysis highlighted several proteins like CAPN5, SURF4, LAMP2 or MT-ND1, as highly discriminating features between the groups compared. Protein changes correlated with viral infection, RNA metabolism or autophagy, among others. Remarkably, the angiogenic potential of ECFCs in response to the infected serums was impaired, and many of the protein alterations in response to the serum of critical patients were associated with cardiovascular-related pathologies.

Detection of High Level of Co-Infection and the Emergence of Novel SARS CoV-2 Delta-Omicron and Omicron-Omicron Recombinants in the Epidemiological Surveillance of Andalusia.

Recombination is an evolutionary strategy to quickly acquire new viral properties inherited from the parental lineages. The systematic survey of the SARS-CoV-2 genome sequences of the Andalusian genomic surveillance strategy has allowed the detection of an unexpectedly high number of co-infections, which constitute the ideal scenario for the emergence of new recombinants. Whole genome sequence of SARS-CoV-2 has been carried out as part of the genomic surveillance programme. Sample sources included the main hospitals in the Andalusia region. In addition to the increase of co-infections and known recombinants, three novel SARS-CoV-2 delta-omicron and omicron-omicron recombinant variants with two break points have been detected. Our observations document an epidemiological scenario in which co-infection and recombination are detected more frequently. Finally, we describe a family case in which co-infection is followed by the detection of a recombinant made from the two co-infecting variants. This increased number of recombinants raises the risk of emergence of recombinant variants with increased transmissibility and pathogenicity.

Serum microRNAs targeting ACE2 and RAB14 genes distinguish asymptomatic from critical COVID-19 patients.

Despite the extraordinary advances achieved to beat COVID-19 disease, many questions remain unsolved, including the mechanisms of action of SARS-CoV-2 and which factors determine why individuals respond so differently to the viral infection. Herein, we performed an in silico analysis to identify host microRNA targeting ACE2, TMPRSS2, and/or RAB14, all genes known to participate in viral entry and replication. Next, the levels of six microRNA candidates previously linked to viral and respiratory-related pathologies were measured in the serum of COVID-19-negative controls (n = 16), IgG-positive COVID-19 asymptomatic individuals (n = 16), and critical COVID-19 patients (n = 17). Four of the peripheral microRNAs analyzed (hsa-miR-32-5p, hsa-miR-98-3p, hsa-miR-423-3p, and hsa-miR-1246) were upregulated in COVID-19 critical patients compared with COVID-19-negative controls. Moreover, hsa-miR-32-5p and hsa-miR-1246 levels were also altered in critical versus asymptomatic individuals. Furthermore, these microRNA target genes were related to viral infection, inflammatory response, and coagulation-related processes. In conclusion, SARS-CoV-2 promotes the alteration of microRNAs targeting the expression of key proteins for viral entry and replication, and these changes are associated with disease severity. The microRNAs identified could be taken as potential biomarkers of COVID-19 progression as well as candidates for future therapeutic approaches against this disease.

Transmission of toxigenic Clostridiodes difficile between a pet dog with diarrhea and a 10-month-old infant.

We describe a case of interspecies transmission of toxigenic Clostridioides difficile involving a female and her dog, both with diarrhea without another diagnosis. Genomic analysis showed that isolates were grouped into MLST clade I, closely related to ribotype 020 and shared identical genotypes.

Synthesis of Degraded Limonoid Analogs as New Antibacterial Scaffolds against Staphylococcus aureus.

Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) have become serious infections in humans and ruminants. S. aureus strains are showing rapid changes to develop resistance in traditional antibiotic-containing systems. In the continuous fierce fight against the emergent multi-drug resistant bacterial strains, straightforward and scalable synthetic procedures to produce new active molecules are in demand. Analysis of molecular properties points to degraded limonoids as promising candidates. In this article, we report a simple synthetic approach to obtain degraded limonoid analogs as scaffolds for new antibacterial molecules. The minimum inhibitory concentrations against S. aureus were evaluated for the stereoisomer mixtures by the broth microdilution method. Analysis of results showed that the acetylated derivatives were the most active of them all.

Traqueobronquitis obstructiva por Aspergillus fumigatus en un paciente inmunocompetente / Obstructive tracheobronchitis due to Aspergillus fumigatus in an immunocompetent patient

Antecedentes: La traqueobronquitis aspergilar (TBA) es una forma clínica infrecuente de aspergilosis pulmonar invasiva donde la afectación fúngica se limita al árbol traqueobronquial. Aunque las formas más graves, como la TBA pseudomembranosa y ulcerativa, son casi exclusivas de pacientes inmunocomprometidos, la forma obstructiva, más leve, puede cursar en pacientes sin déficit inmunitario. Caso clínico: Se presenta el caso de un varón de 32 años sin antecedentes de interés que es evaluado por presentar neumonía recidivante del lóbulo inferior derecho. En los estudios microbiológicos del esputo destacaba el crecimiento de Serratia marcescens y escaso crecimiento de Aspergillus fumigatus, que se interpretó como una contaminación de la muestra. La fibrobroncoscopia reveló al nivel B10 del lóbulo inferior derecho un tapón mucoso muy denso que no se pudo extraer; no hubo otros hallazgos macroscópicos de interés. Durante la hospitalización el paciente logró expectorar el tapón mucoso y presentó una importante broncorrea posterior; en los cultivos microbiológicos se observaron numerosas colonias de A. fumigatus. Se indicó tratamiento con voriconazol, lo que llevó a la resolución del cuadro, sin nuevas recidivas. Conclusiones: La TBA obstructiva se caracteriza por la producción excesiva de moco denso cargado de hifas que puede llegar a obstruir la luz de la vía aérea y generar neumonías postobstructivas recidivantes. Es importante considerar este diagnóstico en pacientes inmunocompetentes con infecciones respiratorias recurrentes que presentan aislamiento repetido de colonias de Aspergillus en el esputo, aunque sean en escasa cuantía

Background: Aspergillus tracheobronchitis (ATB) is an uncommon type of invasive pulmonary aspergillosis in which fungal involvement is limited to the tracheobronchial tree. While the more severe forms, such as pseudomembranous and ulcerative ATB, occur almost exclusively in immunocompromised patients, the milder obstructive form may occur in patients without immune deficiency. Case report: The case of a 32 year-old man with no previous history of illness, who was evaluated for recurrent right lower lobe pneumonia, is presented. Microbiological sputum studies revealed growth of Serratia marcescens, and a limited growth of Aspergillus fumigatus, the latter interpreted as a contaminant in the specimen. Bronchoscopy revealed a dense mucous plug at level B10 of the right lower lobe, which could not be removed; no other macroscopic findings of interest were observed. During his hospital admission, the patient expectorated the mucous plug and had a significant subsequent bronchorrhoea. A substantial number of colonies of A. fumigatus grown in the sputum cultures. The patient was given voriconazole, leading to a clinical resolution, with no recurrences. Conclusions: Obstructive ATB is characterised by the excessive production of thick, hyphae-laden mucus, which can obstruct the airway lumen and generate relapsing post-obstructive pneumonias. It is important to consider this diagnosis in immunocompetent patients with recurrent respiratory infections and who show repeated isolation of Aspergillus colonies in the sputum, even in small quantities

Detection and Characterization of Extended-Spectrum Beta-Lactamases-Producing Escherichia coli in Animals.

The detection of multidrug-resistant bacteria is a growing problem; however, the role of domesticated animals in the propagation of antimicrobial resistance has barely been studied. The aim of this study was to identify extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli strains in domestic animal feces to assess their antimicrobial resistance profile and carry out molecular characterization of the ß-lactamases. A total of 325 samples were collected from eight animal species. Of these, 34 bacterial isolates were identified as E. coli. The antibiotic resistance profile of the E. coli strains was as follows: 100% resistant to amoxicillin, aztreonam, and cephalosporins; 58.8% resistant to nalidixic acid, ciprofloxacin, and trimethoprim/sulfamethoxazole; 41.2% resistant to gentamicin and tobramycin; 11.8% resistant and 32.4% intermediate to cefoxitin; 97.1% sensible and 2.9% intermediate to amoxicillin/clavulanate; and 100% sensible to ertapenem, minocycline, imipenem, meropenem, amikacin, nitrofurantoin, fosfomycin, and colistin. All 34 E. coli strains met criteria for ESBL production. In total, 46 ß-lactamase genes were detected: 43.5% blaTEM, 30.4% blaCTX-M (23.9% blaCTX-M-1 and 6.5% blaCTX-M-9), and 26.1% blaSHV (17.4% blaSHV-5 and 8.7% blaSHV-12). All the ß-lactamases were found in dogs except for four blaSHV found in falcons. No plasmidic AmpC genes were found. The high prevalence of ESBL-producing E. coli strains in animals could become a zoonotic transmission vector.

[Obstructive tracheobronchitis due to Aspergillus fumigatus in an immunocompetent patient]. / Traqueobronquitis obstructiva por Aspergillus fumigatus en un paciente inmunocompetente.

BACKGROUND: Aspergillus tracheobronchitis (ATB) is an uncommon type of invasive pulmonary aspergillosis in which fungal involvement is limited to the tracheobronchial tree. While the more severe forms, such as pseudomembranous and ulcerative ATB, occur almost exclusively in immunocompromised patients, the milder obstructive form may occur in patients without immune deficiency. CASE REPORT: The case of a 32 year-old man with no previous history of illness, who was evaluated for recurrent right lower lobe pneumonia, is presented. Microbiological sputum studies revealed growth of Serratia marcescens, and a limited growth of Aspergillus fumigatus, the latter interpreted as a contaminant in the specimen. Bronchoscopy revealed a dense mucous plug at level B10 of the right lower lobe, which could not be removed; no other macroscopic findings of interest were observed. During his hospital admission, the patient expectorated the mucous plug and had a significant subsequent bronchorrhoea. A substantial number of colonies of A. fumigatus grown in the sputum cultures. The patient was given voriconazole, leading to a clinical resolution, with no recurrences. CONCLUSIONS: Obstructive ATB is characterised by the excessive production of thick, hyphae-laden mucus, which can obstruct the airway lumen and generate relapsing post-obstructive pneumonias. It is important to consider this diagnosis in immunocompetent patients with recurrent respiratory infections and who show repeated isolation of Aspergillus colonies in the sputum, even in small quantities.

Direct identification of microorganisms from thioglycolate broth by MALDI-TOF MS.

We developed an easy MALDI-TOF MS-based assay to identify microorganisms directly from thioglycolate broth. A total of 101 positive thioglycolate broths inoculated with 15 different kinds of samples were evaluated. In 91 samples (90.1%), direct MALDI-TOF MS identifications were the same as those obtained after conventional laboratory procedures including subcultures. In 10 samples misidentified by direct processing, yeasts or mixed cultures grew in the thioglycolate subcultures, or high cellular debris hampered a correct analysis. This rapid method can provide a fast, clinically- relevant species-level identification without disturbing the daily workflow in clinical microbiology laboratories.

A modified ESBL Nordmann/Dortet/Poirel-based protocol to optimize early sepsis management.

We evaluated a modification of a colorimetric test recently described by Dortet et al. (2015) for the rapid detection of ESBL-producing Enterobacteriaceae directly from positive blood cultures that requires less manipulation, materials and hands-on time. The simplified protocol showed a sensitivity and specificity of 100% and 95.7% respectively.

HTLV infection in HCV-antibody positive patients in Spain.

Since hepatitis C virus (HCV) and human T-lymphotropic virus (HTLV) share transmission routes, dual infection could be frequent. In Spain, HTLV underdiagnosis is highlighted by the high proportion of patients presenting either with tropical spastic paraparesis (TSP) or adult T-cell leukemia (ATL) at first diagnosis. We examined whether the renewed efforts for expanding HCV testing may provide a sentinel population that might selectively be targeted to unveil asymptomatic HTLV carriers. The presence of anti-HTLV antibodies was examined in 3,838 consecutive individuals with reactive HCV serology attended during the last three years at 13 hospitals distributed across the Spanish geography. Overall 71% were male and the median age was 41-years old. Foreigners represented 9% of the study population. A total of 50 individuals (1.3%) were seroreactive for HTLV, being 30 confirmed as HTLV-2 and two as HTLV-1 (0.12%). The remaining 18 had indeterminate Western blot patterns. Most individuals with HTLV-2 and HTLV indeterminate serology were HIV-positive, former injection drug users and native Spaniards. In contrast, the two HTLV-1 infections were found in men coming from Brazil and the Dominican Republic, respectively. In summary, the overall prevalence of HTLV infection in individuals living in Spain seropositive for HCV is 1.3%, more than 10-fold greater than in general outclinics in Spain. However, immigrants from HTLV-1 endemic regions and former injection drug users with HTLV-2 infection are by far the major contributory groups in HCV patients. Therefore, testing for HTLV in newly diagnosed HCV individuals would not contribute much to improve late HTLV diagnosis in Spain.

Fungemia por Trichosporon asahii en un paciente con neoplasia hematológica / Fungemia due to Trichosporon asahii in a patient with hematological malignancy

Antecedentes. La tricosporonosis es una infección oportunista debida a hongos levaduriformes del género Trichosporon. La mayoría de los casos de tricosporonosis invasiva acontecen en individuos inmunodeficientes. Caso clínico. Describimos un caso de infección diseminada por Trichosporon asahii en un paciente hematológico. Se trata de un varón de 52 años diagnosticado de leucemia linfoblástica aguda que desarrolla un cuadro febril durante el tercer ciclo de quimioterapia de inducción. A las 24 h de incubación se observó positividad en los hemocultivos extraídos, visualizándose en la tinción de Gram estructuras alargadas compatibles con elementos fúngicos. La identificación del hongo como Trichosporon asahii se llevó a cabo mediante la asimilación de compuestos de carbono y la amplificación y secuenciación de los dominios D1/D2 y la región espaciadora interna transcrita del ADN ribosómico. El hongo se aisló además de unas lesiones pustulosas que presentaba el paciente en la región pectoral. Tras tratamiento con anfotericina B, el paciente evolucionó favorablemente de las lesiones y del proceso febril. Conclusiones. Trichosporon asahii es un patógeno emergente en pacientes inmunodeprimidos y su presencia no debe ser considerada como colonización, pues existe riesgo de infección invasiva (AU)

Background. Trichosporonosis is an opportunistic infection caused by the genus Trichosporon. The majority of cases of invasive trichosporonosis occurs in immunocompromised individuals. Case report. We describe a case of disseminated infection by Trichosporon asahii in a hematology patient. A 52-year-old man diagnosed with acute lymphoblastic leukemia developed a febrile episode during the third cycle of the induction chemotherapy. The blood cultures were positive after 24 h incubation, showing elongated structures compatible with fungal elements in the Gram stain. The identification of the fungus as Trichosporon asahii was carried out by the assimilation of compounds of carbon and the amplification and sequencing of the D1/D2 domain and the internal transcribed spacer of the ribosomal DNA. The fungus was also isolated from the pustular lesions that the patient had in the chest. After treatment with amphotericin B, the patient progressed satisfactorily. Conclusions. Trichosporon asahii is an emergent pathogen in immunosupressed patients and its presence should not be considered as colonization, as there is risk of invasive infection (AU)

[Fungemia due to Trichosporon asahii in a patient with hematological malignancy]. / Fungemia por Trichosporon asahii en un paciente con neoplasia hematológica.

BACKGROUND: Trichosporonosis is an opportunistic infection caused by the genus Trichosporon. The majority of cases of invasive trichosporonosis occurs in immunocompromised individuals. CASE REPORT: We describe a case of disseminated infection by Trichosporon asahii in a hematology patient. A 52-year-old man diagnosed with acute lymphoblastic leukemia developed a febrile episode during the third cycle of the induction chemotherapy. The blood cultures were positive after 24h incubation, showing elongated structures compatible with fungal elements in the Gram stain. The identification of the fungus as Trichosporon asahii was carried out by the assimilation of compounds of carbon and the amplification and sequencing of the D1/D2 domain and the internal transcribed spacer of the ribosomal DNA. The fungus was also isolated from the pustular lesions that the patient had in the chest. After treatment with amphotericin B, the patient progressed satisfactorily. CONCLUSIONS: Trichosporon asahii is an emergent pathogen in immunosupressed patients and its presence should not be considered as colonization, as there is risk of invasive infection.

Use of Cervista HPV HR assay for detection of human papillomavirus in samples with hybrid capture borderline negative results.

We have evaluated Cervista HPV HR for papillomavirus detection in 65 samples previously borderline negative by the hybrid capture method (Digene), using InnoLipa and sequencing as confirmatory techniques. Nine samples were found to be positive by Cervista HPV HR, of which five (7.6%) were confirmed by InnoLipa. Four samples (6.1%) were false positive, of which three samples were reactive for A9 probes. The Cervista HPV HR assay can detect HPV-positive samples from those with hybrid capture borderline results but can produce false-positive results when tested for reactivity with A9 probes.

Comparison of human papillomavirus genotyping using commercial assays based on PCR and reverse hybridization methods.

Different tests for human papillomavirus (HPV) screening are commercially available, detecting high-risk oncogenic HPV types with a pool of genotype-specific probes. However, it is necessary to establish reliable methods for the identification of individual genotypes. The purpose of this study was to compare three different commercial methods for HPV genotyping: INNO-LiPA HPV Genotyping v2 (LiPA), Linear Arrays HPV Genotyping Test (LA) and Clinical Arrays Human Papillomavirus (CA). A total of 83 HPV DNA-positive samples by hybrid capture method were genotyped (82, 78 and 81 by LiPA, LA and CA, respectively). Comparison analysis was limited to the HPV genotypes common to the three assays. There were concordant results (absolute agreement between assays) in 31 samples (39.7%) and compatible results (correspondence for some but not all genotypes) were found in 44 samples (56.4%). Only three samples (3.8%) were considered as discordant (did not show any similarity between the tests). Analyzing kappa values we have a very good agreement (>0.8) for HPV16 and HPV31 and good agreement (0.6-0.8) for HPV types 6, 18, 53 and 66 when all methods are compared. We conclude that all genotyping methods tested are highly comparable and suitable for clinical and epidemiological studies.

Sesquiterpenes as immunosuppressants.

BACKGROUND: The search for new immunosuppressive drugs is a high priority. Sesquiterpenes constitute a family of compounds with a great variety of biological activities due to their reactive moieties. METHODS: Human tumor cell lines and murine primary cells and human peripheral blood mononuclear cells or primary CD4+ cells from healthy individuals were stimulated in the presence of sesquiterpenes. Cell division was analyzed by 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester, cell cycle progression by Hoecht, and cell death by Anexin-V and propidium iodine staining. Cytokine secretion was analyzed by means of a bioplex assay. RESULTS: Two sesquiterpene derivatives of the 18 previously shown to inhibit vegetal cell growth are shown to block cell division and cell cycle progression in human and murine cell lines and primary cells. Cytokine secretion is also impaired on stimulation in the presence of sesquiterpenes. CONCLUSIONS: Here, we show that sesquiterpenes heliannuols constitute a novel family of molecules with potential use as immunosuppressants. Moreover, we show that an assay based on the allelopathic effect of plant leads can be used as a cost-effective screening previous to studies in mammalian cells.

Prevalence and genetic characterization of hepatitis E virus in paired samples of feces and serum from naturally infected pigs.

This study describes the distribution of Hepatitis E virus (HEV) in a naturally infected swine population and the genetic relatedness of HEV strains on swine farms in Spain. Of fecal and serum samples collected from 131 pigs and manure-ditch samples collected from 17 farms, HEV was detected in 16%, 14%, and 59%, respectively, for an overall prevalence rate of 23%. The maximum prevalence rates for feces and serum were in pigs 5 to 12 wk old. A high prevalence of the virus in feces (18%) was observed in sows. Gene sequencing was performed on 6 strains from feces, serum, and manure ditch: the nucleotide identities varied from 81.5% to 99% when compared with those of other strains of genotype 3 isolated from swine. This is the first study in Europe to show the variation in virus distribution by age in feces and serum in a naturally infected swine population.