Identification of polysaccharide capsules among extensively drug-resistant genitourinary Haemophilus parainfluenzae isolates

The human commensal Haemophilus parainfluenzae is emerging as an opportunistic multidrug-resistant pathogen. The objectives of this work were to characterise a new capsular operon of extensively drug-resistant (XDR) H. parainfluenzae clinical isolates and study their resistance mechanisms using whole-genome sequencing. All strains were resistant to: ß-lactams, via amino acid changes in PBP3 (S385T, I442F, V511A, N526K and V562I); quinolones, by alterations in GyrA (S84F and D88Y) and ParC (S84F and S138T); chloramphenicol, through the presence of catS; macrolides, via the presence of mel and mef(E)-carrying MEGA element; and tetracycline, through the presence of tet(M) and/or tet(B). Phylogenetic analysis revealed high genomic diversity when compared to the H. parainfluenzae genomes available on the NCBI, the isolates from this study being closely related to the Swiss XDR AE-2096513. A full capsular operon showing homology to that of H. influenzae was identified, in accordance with the observation of a capsular structure by TEM. This study describes for the first time a capsular operon in H. parainfluenzae, a major determinant of pathogenicity that may contribute to increased virulence in XDR clinical isolates. Moreover, phylogenetic analysis suggests the possible spread of an XDR-encapsulated strain in Europe

Assessment of trimethoprim-sulfamethoxazole susceptibility testing methods for fastidious Haemophilus spp.

Objectives: To compare the determinants of trimethoprim-sulfamethoxazole resistance with established susceptibility values for fastidious Haemophilus spp., to provide recommendations for optimal trimethoprim-sulfamethoxazole measurement. Methods: We collected 50 strains each of Haemophilus influenzae and Haemophilus parainfluenzae at Bellvitge University Hospital. Trimethoprim-sulfamethoxazole susceptibility was tested by microdilution, E-test and disc diffusion using both Mueller-Hinton fastidious (MH-F) medium and Haemophilus test medium (HTM) following EUCAST and CLSI criteria, respectively. Mutations in folA, folP and additional determinants of resistance were identified in whole-genome-sequenced isolates. Results: Strains presented generally higher rates of trimethoprim-sulfamethoxazole resistance when grown on HTM than on MH-F, independent of the methodology used (average MIC 2.6-fold higher in H. influenzae and 1.2-fold higher in H. parainfluenzae). The main resistance-related determinants were as follows: I95L and F154S/V in folA; 3- and 15-bp insertions and substitutions in folP; acquisition of sul genes; and FolA overproduction potentially linked to mutations in -35 and -10 promoter motifs. Of note, 2 of 19 H. influenzae strains (10.5%) and 9 of 33 H. parainfluenzae strains (27.3%) with mutations and assigned as resistant by microdilution were inaccurately considered susceptible by disc diffusion. This misinterpretation was resolved by raising the clinical resistance breakpoint of the EUCAST guidelines to ≤30 mm. Conclusions: Given the routine use of disc diffusion, a significant number of strains could potentially be miscategorized as susceptible to trimethoprim-sulfamethoxazole despite having resistance-related mutations. A simple modification to the current clinical resistance breakpoint given by the EUCAST guideline for MH-F ensures correct interpretation and correlation with the reference standard method of microdilution

Dinamics of invasive disease caused by streptococcus pneumoniae clones related to the PCV13 serotypes not included in the PCV7

Treballs Finals de Grau de Farmàcia, Facultat de Farmàcia, Universitat de Barcelona, 2017. Tutora: M. Carme Fusté Munné[en] Streptococcus pneumoniae is an important cause of some infectious diseases that affect the population such as community-acquired pneumonia or meningitis. The diagnosis of Pneumococcal disease is based on a variety of laboratory tests that confirm the presence of the bacteria as the causal agent, starting with a Gram staining for the previous microscopic identification and the bacterial growth in a culture media. Then, it is followed by the MALDI-TOF identification once the bacterial strain is isolated; in the case of urine samples, it proceeds with an lateral flow immunoassay test. Finally the antimicrobial susceptibility profile is required to establish an appropriate treatment as well as other useful complementary properties in clinical studies. After completing this process, the monitoring of the disease is established by serotyping tests, genotyping tests, and characterization of resistance mechanisms, especially transferable by Tn916 transposon in ermB and mef genes associated with macrolide resistance and the tetM gene associated with tetracycline resistance. This whole process is needed to confirm the existence of different multidrug resistant clones emerged and spread throughout the world during the early use of antibiotics decades ago and generated by selective adaptation while naturally residing within the human upper respiratory pathways, also inhabited by other species of nosocomial streptococcus able to generate and transmit antibiotic resistances. Nowadays, the prevention of Pneumococcal disease is based on vaccination; the capsular polysaccharide is the major determinant of virulence of S. pneumoniae and it is considered the basis of the current vaccine development. The introduction of the 7-valent Pneumococcal conjugate vaccine or PCV7 has changed the epidemiology of Pneumococcal disease worldwide (2001), and later the PCV13 in 2010. However, some serotypes of PCV13 not included in the PCV7 remained as a cause of invasive disease.[sp] Streptococcus pneumoniae es un causante importante de algunas de las enfermedades infecciosas que afectan a la población como la neumonía adquirida en la comunidad o la meningitis. El diagnóstico de la enfermedad neumocócica se basa en una variedad de pruebas de laboratorio confirmatorias de la presencia de la bacteria como agente causal, empezando con una tinción de Gram para la previa identificación microscópica junto con la siembra de la muestra en medios de cultivo enriquecido, y seguido posteriormente de la identificación por MALDI-TOF una vez aislada la cepa bacteriana; en el caso de las muestras urinarias, se procede con una prueba inmunocromatográfica en tira reactiva. Finalmente se requieren las pruebas de sensibilidad a antimicrobianos para establecer un perfil adecuado de tratamiento, así como otras propiedades complementarias útiles en estudios clínicos. Una vez completado este procedimiento, se establece la vigilancia de la enfermedad mediante pruebas de identificación del serotipo, del genotipo, y la caracterización de los mecanismos de resistencia, especialmente los transferibles a través de transposones Tn916 relacionados con los genes ermB y mef asociados a resistencias a macrólidos, y al gen tetM asociado a resistencias a la tetraciclina. Todo este proceso es necesario para confirmar la existencia de diferentes clones multirresistentes que surgieron y se difundieron por todo el mundo durante los primeros usos de antibióticos décadas atrás y los cuales se generan a través de la adaptación selectiva mientras residen de forma natural dentro de las vías respiratorias superiores humanas, habitadas también por otras especies de estreptococos nosocomiales capaces de generar resistencias y transmitirlas. Actualmente, la prevención de la enfermedad neumocócica se basa en la vacunación; el polisacárido capsular es el principal determinante de virulencia de S. pneumoniae y también es la base del desarrollo de la vacuna actual. La introducción de la vacuna neumocócica conjugada heptavalente o PCV7 ha cambiado la epidemiología de las enfermedades neumocócicas en todo el mundo (2001) y posteriormente la PCV13 en el año 2010. Sin embargo, algunos serotipos de la PCV13 no incluidos en la vacuna PCV7 se mantuvieron como causa de la enfermedad invasiva