Genotypic and Phenotypic Characterization of Staphylococcus aureus Isolates from the Respiratory Tract in Mechanically-Ventilated Patients

Staphylococcus aureus is a commensal and frequent colonizer of the upper respiratory tract. When mechanical ventilation disrupts natural defenses, S. aureus is frequently isolated from the lower airways, but distinguishing between colonization and infection is difficult. The objectives of this study were (1) to investigate the bacterial genome sequence in consecutive isolates in order to identify changes related to the pathological adaptation to the lower respiratory tract and (2) to explore the relationship between specific phenotypic and genotypic features with the patient's study group, persistence of the clinical isolate and clinical outcome. A set of 94 clinical isolates were selected and corresponded to 34 patients that were classified as having pneumonia (10), tracheobronchitis (11) and bronchial colonization (13). Clinical strains were phenotypically characterized by conventional identification and susceptibility testing methods. Isolates underwent whole genome sequencing using Illumina HiSeq4000. Genotypic characterization was performed with an in-house pipeline (BacterialTyper). Genomic variation arising within-host was determined by comparing mapped sequences and de novo assemblies. Virulence factors important in staphylococcal colonization and infection were characterized using previously established functional assays. (1) Toxin production was assessed using a THP-1 cytotoxicity assay, which reports on the gross cytotoxicity of individual isolates. In addition, we investigated the expression of the major virulence factor, alpha-toxin (Hla) by Western blot. (2) Adhesion to the important extracellular matrix molecule, fibronectin, was determined using a standardized microtitre plate assay. Finally, invasion experiments using THP-1 and A539 cell lines and selected clinical strains were also performed. Repeated isolation of S. aureus from endotracheal aspirate usually reflects persistence of the same strain. Within-host variation is detectable in this setting, but it shows no evidence of pathological adaptation related to virulence, resistance or niche adaptations. Cytotoxicity was variable among isolates with 14 strains showing no cytotoxicity, with these latter presenting an unaltered Fn binding capacity. No changes on cytotoxicity were reported when comparing study groups. Fn binding capacity was reported for almost all strains, with the exception of two strains that presented the lowest values. Strains isolated from patients with pneumonia presented a lower capacity of adhesion in comparison to those isolated during tracheobronchitis (p = 0.002). Hla was detected in 71 strains (75.5%), with most of the producer strains in pneumonia and bronchial colonization group (p = 0.06). In our cohort, Hla expression (presence or absence) in sequential isolates was usually preserved (70%) although in seven cases the expression varied over time. No relationship was found between low cytotoxicity and intracellular persistence in invasion experiments. In our study population, persistent S. aureus isolation from airways in ventilated patients does not reflect pathological adaptation. There is an important diversity of sequence types. Cytotoxicity is variable among strains, but no association with study groups was found, whereas isolates from patients with pneumonia had lower adhesion capability. Favorable clinical outcome correlated with increased bacterial adhesion in vitro. Most of the strains isolated from the lower airways were Hla producers and no correlation with an adverse outcome was reported. The identification of microbial factors that contribute to virulence is relevant to optimize patient management during lower respiratory tract infections

Microbead-based spoligotyping of Mycobacterium tuberculosis from Ziehl-Neelsen-stained microscopy preparations in Ethiopia

The worldwide dissemination of Mycobacterium tuberculosis strains has led to the study of their genetic diversity. One of the most used genotyping methods is spoligotyping, based on the detection of spacers in the clustered regularly interspaced short palindromic repeats (CRISPR) locus. This study assessed the performance of a microbead-based spoligotyping assay using samples extracted from Ziehl-Neelsen-stained smear-microscopy preparations and described the genetic diversity of Mycobacterium tuberculosis among new TB patients in Southern Nations, Nationalities and Peoples’ Region (SNNPR) in Ethiopia. Among the 91 samples analysed, 59 (64.8%) generated spoligotyping patterns. Fifty (84.7%) samples were classified into 12 clusters (mostly Lineage 4 or 3) comprising 2–11 samples and nine had unique spoligotyping patterns. Among the 59 spoligotyping patterns, 25 belonged to the T1 sublineage, 11 to the T3-ETH, 5 to the URAL, 4 to the H3 and 14 to other L4 sublineages. There was a remarkable variation in genetic distribution in SNNPR compared to other regions of the country. Microbead-based spoligotyping is an easy-to-perform, high-throughput assay that can generate genotyping information using material obtained from smear microscopy preparations. The method provides an opportunity to obtain data of the M. tuberculosis genetic epidemiology in settings with limited laboratory resources

Genetic characterization of Mycobacterium tuberculosis complex isolates circulating in Abuja, Nigeria

WOS:000446783800001Objective: Nigeria ranks fourth among the high tuberculosis (TB) burden countries. This study describes the prevalence of drug resistance and the genetic diversity of Mycobacterium tuberculosis in Abuja's Federal Capital Territory. Materials and methods: Two hundred and seventy-eight consecutive sputum samples were collected from adults with presumptive TB during 2013-2014. DNA was extracted from Lowenstein-Jensen cultures and analyzed for the identification of nontuberculous mycobacteria species, detection of drug resistance with line probe assays, and high-throughput spacer oligonucleotide typing (spoligotyping) using microbead-based hybridization. Results: Two hundred and two cultures were positive for M. tuberculosis complex, 24 negative, 38 contaminated, and 15 positive for nontuberculous mycobacteria. Five (2.5%)M. tuberculosis complex isolates were resistant to rifampicin (RIF) and isoniazid (multidrug resistant), nine (4.5%) to RIF alone, and 15 (7.4%) to isoniazid alone; two RIF-resistant isolates were also resistant to fluoroquinolones and ethambutol, and one multidrug resistant isolate was also resistant to ethambutol. Among the 180 isolates with spoligotyping results, 164 (91.1%) were classified as lineage 4 (Euro-American), 13 (7.2%) as lineage 5 (West African 1), two (1.1%) as lineage 2 (East Asia), and one (0.6%) as lineage 6 (West African 2). One hundred and fifty-six (86.7%) isolates were grouped in 17 clusters (2-108 isolates/cluster), of which 108 (60.0%) were grouped as L4.6.2/Cameroon (spoligotype international type 61). Conclusion: The description of drug resistance prevalence and genetic diversity of M tuberculosis in this study may be useful for improving TB control in Nigeria

Accessory gene regulator (Agr) functionality in Staphylococcus aureus derived from lower respiratory tract infections

Altres ajuts: This work has been funded by the project PI13/01418 which is part of "Plan Nacional de I+D+I" and co-funded by ISCIII-Subdirección General de Evaluacioón and "Fondo Europeo de Desarrollo Regional"(FEDER).D. Domínguez-Villanueva is funded by "Plan Nacional de I+D+I" and co-funded by ISCIII-Subdirección General de Evaluación and "Fondo Europeo de Desarrollo Regional"(FEDER). M. Gomes-Fernandes is funded by CAPES Foundation, Ministry of Education of Brazil (Brasılia, Brazil). Maisem Laabei was supported by a joint ERS/SEPAR fellowship (LTRF2015).This work also received a grant from the Spanish Society of Pneumology and Thoracic Surgery (SEPAR054/2011).Objective. Characterization of Staphylococcus aureus clinical isolates derived from lower respiratory tract infections (LRTIs), and correlation between the functionality of the accessory gene regulator (Agr) and genotypic and phenotypic characteristics, clinical variables and clinical outcome. Methods. S aureus isolates derived from LRTIs and control groups (nasal carriage and bacteraemia) were genotyped using StaphyType DNA microarray. Agr activity was evaluated using the CAMP synergistic haemolysis assay and the Vesicle Lysis Test (VLT). Discordant strains were analysed by quantitative reverse- transcriptase real-time PCR (qRT-PCR). Results. Agr was functional in 79.7% and 84.5% of strains according to the CAMP and VLT assays respectively. Higher concordance with RNAIII expression measured by qRT-PCR was observed with the VLT assay (76.2%) compared with the CAMP assay (23.8%). No statistically significant differences were observed in Agr functionality between the study groups, nor the phenotypical/genotypical bacterial characteristics. No association between increased mortality/respiratory complications and Agr function was observed. Conclusions. Agr activity was high (82.2%) in isolates from LRTIs suggesting the importance of this global regulator in lower respiratory tract colonisation and infection. However, equally high Agr activity was observed in isolates derived from nasal carriage and bacteraemia, contradictory to previous observations. Agr functionality measured by the VLT assay was superior to CAMP assay

Infecciones respiratorias por Staphylococcus aureus: implicación clínica de factores de virulencia y persistencia

Staphylococcus aureus es uno de los principales patógenos comensales y oportunistas responsables de causar infecciones del tracto respiratorio inferior (ITRI). Es conocida la dificultad de distinguir entre colonización e ITRI por S. aureus tanto en pacientes crónicos como en pacientes sin neumopatía de base. La prevalencia de colonización por este microorganismo dificulta establecer el valor clínico del aislado en muestra respiratoria en determinados grupos de pacientes y establecer la verdadera indicación del tratamiento. En pacientes sometidos a ventilación mecánica (VM), la potencial gravedad del desarrollo de neumonía da lugar a frecuente prescripción antibiótica de forma empírica, favoreciendo a su vez el desarrollo de resistencias. El estudio de factores tanto del microorganismo como del hospedador, debería facilitar la distinción entre los estadios de colonización e infección. Por tanto, el objetivo de este trabajo es determinar la importancia clínica del aislamiento de S. aureus en las vías respiratorias inferiores de pacientes sometidos a VM, mediante el estudio de factores asociados con la persistencia y la adaptación del microorganismo a este nicho anatómico. Esto representaría un cambio importante en el diagnóstico y el manejo de las ITRI causadas por este microorganismo. Inicialmente se estudió qué factores del hospedador y del microorganismo estarían relacionados con la evolución clínica y el aislamiento persistente de S. aureus en muestra respiratoria. Esto se llevó a cabo en una cohorte de pacientes ingresados en UCI y sometidos a VM, que fueron clasificados en diferentes grupos en cuanto a la relación del aislado con el hospedador (infección vs. colonización). Se demostró que, de entre las diferentes variables evaluadas, el resultado clínico desfavorable es independiente de los días bajo VM y de estancia en la UCI, así como del perfil genético y el patrón de resistencia a la cloxacilina de los aislados. Además, hemos demostrado que el aislamiento persistente de S. aureus, observado en aproximadamente el 40% de los pacientes, tampoco se asoció con la evolución clínica desfavorable. Sin embargo, aunque también se observaron casos de aislados MSSA persistentes, la resistencia a la cloxacilina se correlacionó con la persistencia en el aislamiento. Por otro lado, la persistencia de S. aureus en muestra respiratoria resultó ser frecuente en los pacientes que presentaban un aislamiento consecutivo de Pseudomonas aeruginosa. Esta asociación nos llevó a investigar las interacciones entre S. aureus y P. aeruginosa, otro importante agente etiológico de ITRI frecuentemente aislado en la misma localización anatómica. El estudio de las interacciones entre estos microorganismos nos permitió ampliar el conocimiento respecto a las relaciones interespecies y su impacto sobre la virulencia, persistencia y adaptación al tracto respiratorio, y la progresión o cronicidad de la infección. A continuación, se analizó la funcionalidad del sistema quorum sensing (QS)- agr (accessory gene regulator), uno de los principales sistemas de regulación de la virulencia en S. aureus, en aislados provenientes de pacientes con ITRI. Alrededor del 83% de los aislados resultaron tener el sistema Agr funcional. Se observaron correlaciones entre la funcionalidad del Agr y la capacidad de formación de biofilm de los aislados. No obstante, la actividad de este sistema regulador de virulencia parece no tener influencia en el resultado clínico desfavorable de los pacientes, ni tampoco en el aislamiento persistente. Los hallazgos obtenidos en este estudio destacan que tanto la funcionalidad del sistema Agr, como el aislamiento persistente, observados a menudo en los aislados provenientes de pacientes sometidos a VM, corresponden más a fenotipos de adaptación del microorganismo al microbioma del tracto respiratorio, que a fenotipos de virulencia relacionados con el resultado clínico desfavorable. Por lo tanto, esto refuerza la observación de que las infecciones del tracto respiratorio a menudo se manejan de forma subóptima con respecto al diagnóstico correcto, indicación, dosis y duración del tratamiento antimicrobiano. En cuanto a las relaciones interespecíficas entre aislados de S. aureus y P. aeruginosa en pacientes con ITRI aguda, se observó que de forma similar a lo que ocurre en aislados provenientes de pacientes con enfermedades respiratorias crónicas, P. aeruginosa compite y supera a S. aureus en condiciones de cultivo estándar de laboratorio. Sin embargo, la presencia de un medio de cultivo que simula in vitro la mucosidad del tracto respiratorio, favorece la coexistencia entre ellos, e influye en la actividad de los sistemas QS y en el desarrollo de fenotipos relacionados con la virulencia como es la formación de biofilm. Finalmente, este trabajo sugiere el uso de un enfoque que combine métodos fenotípicos y genotípicos, donde además se consideren las relaciones interespecies entre los diferentes componentes del microbioma respiratorio, a la hora de investigar con mayor precisión las posibles asociaciones entre los factores microbianos y la evolución clínica y la susceptibilidad a los antibióticos en las ITRI por S. aureus.Staphylococcus aureus is one of the most important commensal and opportunistic pathogen responsible for causing lower respiratory tract infections (LRTI). The difficulty in distinguishing between colonization and LRTI by S. aureus is well known in both chronic patients and patients without underlying lung diseases. The prevalence of colonization by this microorganism makes it difficult to determine the clinical value of an isolation in a respiratory sample in certain groups of patients and to establish an appropriate treatment. In patients undergoing mechanical ventilation (MV), the potential severity of developing pneumonia gives rise to frequent empirical antimicrobial prescription, favoring at the same time the development of resistance. The study of factors from both the microorganism and the host should facilitate the distinction between the stages of colonization and infection. Therefore, the objective of the present work is to determine the clinical importance of the isolation of S. aureus in the lower respiratory tract of patients undergoing MV, by studying factors associated with the persistence and adaptation of the microorganism to this anatomical niche. This would represent a significant change in the diagnosis and management of LRTIs caused by this microorganism. Initially, we focused on studying which factors of the host and the microorganism could be related to the clinical course and the persistent isolation of S. aureus in respiratory samples. This was carried out in a cohort of patients admitted to the ICU and undergoing MV, who were classified into different groups regarding the relationship of the isolate with the host (infection vs. colonization). We shown that among the different variables evaluated, an unfavorable clinical outcome is independent of the days under MV and stay in the ICU, as well as the genetic profile and the resistance pattern to cloxacillin of the isolates. In addition, we have shown that persistent isolation of S. aureus, observed in approximately 40% of patients, was also not associated with the clinical outcome. However, although cases of persistent MSSA isolates were also observed, resistance to cloxacillin was significantly associated with persistence. On the other hand, the persistence of S. aureus in the respiratory sample was found to be frequent in patients with consecutive isolation of Pseudomonas aeruginosa. This association led us to investigate the interactions between S. aureus and P. aeruginosa, another important etiological agent of LRTIs frequently isolated in the same anatomical location. The study of the interactions between these two microorganisms would expand the knowledge on interspecies relationships and their impact on virulence, persistence, and adaptation to the respiratory tract, as well as the progression or chronicity of the infection. Next, we analyzed the functionality of the quorum sensing system (QS) - agr (accessory gene regulator), one of the main virulence regulation systems in S. aureus, in isolates from patients with LRTI. Approximately 83% of the isolates were found to have a functional Agr system. Correlations were observed between the functionality of the Agr and the biofilm formation capacity of the clinical isolates. However, the activity of this virulence regulatory system does not seem to influence on the unfavorable clinical outcome of the patients, nor on the persistent isolation. The findings obtained in this study highlight that both the functionality of the Agr system and persistent isolation, often observed in isolates from patients undergoing MV, correspond more to adaptation phenotypes of the microorganism to the respiratory tract microbiome than to virulence phenotypes related to the unfavorable clinical result. Therefore, this reinforces the observation that respiratory tract infections are often handled suboptimally with respect to the correct diagnosis, indication, dosing and duration of antibiotic regimens. Regarding the interspecific relationships between S. aureus and P. aeruginosa isolated from patients with acute LRTI, we observed that similarly to what occurs in isolates from patients with chronic respiratory diseases, P. aeruginosa inhibits and outcompetes S. aureus under standard laboratory culture conditions. However, the presence of a mucus-like medium favors the coexistence between them and influences the activity of the QS systems and the development of phenotypes related to virulence such as biofilm formation in both microorganisms. Finally, this work proposes the use of a combined approach that includes both phenotypic and genotypic methods, and that also considers the interspecies relations among the different components of the respiratory microbiome, to investigate accurately the possible associations between the microbial factors and the clinical outcome, and antimicrobial susceptibility in LTRIs caused by S. aureus

Infecciones respiratorias por Staphylococcus aureus : implicación clínica de factores de virulencia y persistencia /

Staphylococcus aureus es uno de los principales patógenos comensales y oportunistas responsables de causar infecciones del tracto respiratorio inferior (ITRI). Es conocida la dificultad de distinguir entre colonización e ITRI por S. aureus tanto en pacientes crónicos como en pacientes sin neumopatía de base. La prevalencia de colonización por este microorganismo dificulta establecer el valor clínico del aislado en muestra respiratoria en determinados grupos de pacientes y establecer la verdadera indicación del tratamiento. En pacientes sometidos a ventilación mecánica (VM), la potencial gravedad del desarrollo de neumonía da lugar a frecuente prescripción antibiótica de forma empírica, favoreciendo a su vez el desarrollo de resistencias. El estudio de factores tanto del microorganismo como del hospedador, debería facilitar la distinción entre los estadios de colonización e infección. Por tanto, el objetivo de este trabajo es determinar la importancia clínica del aislamiento de S. aureus en las vías respiratorias inferiores de pacientes sometidos a VM, mediante el estudio de factores asociados con la persistencia y la adaptación del microorganismo a este nicho anatómico. Esto representaría un cambio importante en el diagnóstico y el manejo de las ITRI causadas por este microorganismo. Inicialmente se estudió qué factores del hospedador y del microorganismo estarían relacionados con la evolución clínica y el aislamiento persistente de S. aureus en muestra respiratoria. Esto se llevó a cabo en una cohorte de pacientes ingresados en UCI y sometidos a VM, que fueron clasificados en diferentes grupos en cuanto a la relación del aislado con el hospedador (infección vs. colonización). Se demostró que, de entre las diferentes variables evaluadas, el resultado clínico desfavorable es independiente de los días bajo VM y de estancia en la UCI, así como del perfil genético y el patrón de resistencia a la cloxacilina de los aislados. Además, hemos demostrado que el aislamiento persistente de S. aureus, observado en aproximadamente el 40% de los pacientes, tampoco se asoció con la evolución clínica desfavorable. Sin embargo, aunque también se observaron casos de aislados MSSA persistentes, la resistencia a la cloxacilina se correlacionó con la persistencia en el aislamiento. Por otro lado, la persistencia de S. aureus en muestra respiratoria resultó ser frecuente en los pacientes que presentaban un aislamiento consecutivo de Pseudomonas aeruginosa. Esta asociación nos llevó a investigar las interacciones entre S. aureus y P. aeruginosa, otro importante agente etiológico de ITRI frecuentemente aislado en la misma localización anatómica. El estudio de las interacciones entre estos microorganismos nos permitió ampliar el conocimiento respecto a las relaciones interespecies y su impacto sobre la virulencia, persistencia y adaptación al tracto respiratorio, y la progresión o cronicidad de la infección. A continuación, se analizó la funcionalidad del sistema quorum sensing (QS)- agr (accessory gene regulator), uno de los principales sistemas de regulación de la virulencia en S. aureus, en aislados provenientes de pacientes con ITRI. Alrededor del 83% de los aislados resultaron tener el sistema Agr funcional. Se observaron correlaciones entre la funcionalidad del Agr y la capacidad de formación de biofilm de los aislados. No obstante, la actividad de este sistema regulador de virulencia parece no tener influencia en el resultado clínico desfavorable de los pacientes, ni tampoco en el aislamiento persistente. Los hallazgos obtenidos en este estudio destacan que tanto la funcionalidad del sistema Agr, como el aislamiento persistente, observados a menudo en los aislados provenientes de pacientes sometidos a VM, corresponden más a fenotipos de adaptación del microorganismo al microbioma del tracto respiratorio, que a fenotipos de virulencia relacionados con el resultado clínico desfavorable. Por lo tanto, esto refuerza la observación de que las infecciones del tracto respiratorio a menudo se manejan de forma subóptima con respecto al diagnóstico correcto, indicación, dosis y duración del tratamiento antimicrobiano. En cuanto a las relaciones interespecíficas entre aislados de S. aureus y P. aeruginosa en pacientes con ITRI aguda, se observó que de forma similar a lo que ocurre en aislados provenientes de pacientes con enfermedades respiratorias crónicas, P. aeruginosa compite y supera a S. aureus en condiciones de cultivo estándar de laboratorio. Sin embargo, la presencia de un medio de cultivo que simula in vitro la mucosidad del tracto respiratorio, favorece la coexistencia entre ellos, e influye en la actividad de los sistemas QS y en el desarrollo de fenotipos relacionados con la virulencia como es la formación de biofilm. Finalmente, este trabajo sugiere el uso de un enfoque que combine métodos fenotípicos y genotípicos, donde además se consideren las relaciones interespecies entre los diferentes componentes del microbioma respiratorio, a la hora de investigar con mayor precisión las posibles asociaciones entre los factores microbianos y la evolución clínica y la susceptibilidad a los antibióticos en las ITRI por S. aureus.Staphylococcus aureus is one of the most important commensal and opportunistic pathogen responsible for causing lower respiratory tract infections (LRTI). The difficulty in distinguishing between colonization and LRTI by S. aureus is well known in both chronic patients and patients without underlying lung diseases. The prevalence of colonization by this microorganism makes it difficult to determine the clinical value of an isolation in a respiratory sample in certain groups of patients and to establish an appropriate treatment. In patients undergoing mechanical ventilation (MV), the potential severity of developing pneumonia gives rise to frequent empirical antimicrobial prescription, favoring at the same time the development of resistance. The study of factors from both the microorganism and the host should facilitate the distinction between the stages of colonization and infection. Therefore, the objective of the present work is to determine the clinical importance of the isolation of S. aureus in the lower respiratory tract of patients undergoing MV, by studying factors associated with the persistence and adaptation of the microorganism to this anatomical niche. This would represent a significant change in the diagnosis and management of LRTIs caused by this microorganism. Initially, we focused on studying which factors of the host and the microorganism could be related to the clinical course and the persistent isolation of S. aureus in respiratory samples. This was carried out in a cohort of patients admitted to the ICU and undergoing MV, who were classified into different groups regarding the relationship of the isolate with the host (infection vs. colonization). We shown that among the different variables evaluated, an unfavorable clinical outcome is independent of the days under MV and stay in the ICU, as well as the genetic profile and the resistance pattern to cloxacillin of the isolates. In addition, we have shown that persistent isolation of S. aureus, observed in approximately 40% of patients, was also not associated with the clinical outcome. However, although cases of persistent MSSA isolates were also observed, resistance to cloxacillin was significantly associated with persistence. On the other hand, the persistence of S. aureus in the respiratory sample was found to be frequent in patients with consecutive isolation of Pseudomonas aeruginosa. This association led us to investigate the interactions between S. aureus and P. aeruginosa, another important etiological agent of LRTIs frequently isolated in the same anatomical location. The study of the interactions between these two microorganisms would expand the knowledge on interspecies relationships and their impact on virulence, persistence, and adaptation to the respiratory tract, as well as the progression or chronicity of the infection. Next, we analyzed the functionality of the quorum sensing system (QS) - agr (accessory gene regulator), one of the main virulence regulation systems in S. aureus, in isolates from patients with LRTI. Approximately 83% of the isolates were found to have a functional Agr system. Correlations were observed between the functionality of the Agr and the biofilm formation capacity of the clinical isolates. However, the activity of this virulence regulatory system does not seem to influence on the unfavorable clinical outcome of the patients, nor on the persistent isolation. The findings obtained in this study highlight that both the functionality of the Agr system and persistent isolation, often observed in isolates from patients undergoing MV, correspond more to adaptation phenotypes of the microorganism to the respiratory tract microbiome than to virulence phenotypes related to the unfavorable clinical result. Therefore, this reinforces the observation that respiratory tract infections are often handled suboptimally with respect to the correct diagnosis, indication, dosing and duration of antibiotic regimens. Regarding the interspecific relationships between S. aureus and P. aeruginosa isolated from patients with acute LRTI, we observed that similarly to what occurs in isolates from patients with chronic respiratory diseases, P. aeruginosa inhibits and outcompetes S. aureus under standard laboratory culture conditions. However, the presence of a mucus-like medium favors the coexistence between them and influences the activity of the QS systems and the development of phenotypes related to virulence such as biofilm formation in both microorganisms. Finally, this work proposes the use of a combined approach that includes both phenotypic and genotypic methods, and that also considers the interspecies relations among the different components of the respiratory microbiome, to investigate accurately the possible associations between the microbial factors and the clinical outcome, and antimicrobial susceptibility in LTRIs caused by S. aureus

Strain-specific interspecies interactions between co-isolated pairs of Staphylococcus aureus and Pseudomonas aeruginosa from patients with tracheobronchitis or bronchial colonization

Dual species interactions in co-isolated pairs of Staphylococcus aureus and Pseudomonas aeruginosa from patients with tracheobronchitis or bronchial colonization were examined. The genetic and phenotypic diversity between the isolates was high making the interactions detected strain-specific. Despite this, and the clinical origin of the strains, some interactions were common between some co-isolated pairs. For most pairs, P. aeruginosa exoproducts affected biofilm formation and reduced growth in vitro in its S. aureus counterpart. Conversely, S. aureus did not impair biofilm formation and stimulated swarming motility in P. aeruginosa. Co-culture in a medium that mimics respiratory mucus promoted coexistence and favored mixed microcolony formation within biofilms. Under these conditions, key genes controlled by quorum sensing were differentially regulated in both species in an isolate-dependent manner. Finally, co-infection in the acute infection model in Galleria mellonella larvae showed an additive effect only in the co-isolated pair in which P. aeruginosa affected less S. aureus growth. This work contributes to understanding the complex interspecies interactions between P. aeruginosa and S. aureus by studying strains isolated during acute infection

Genotypic and Phenotypic Characterization of Staphylococcus aureus Isolates from the Respiratory Tract in Mechanically-Ventilated Patients

Staphylococcus aureus is a commensal and frequent colonizer of the upper respiratory tract. When mechanical ventilation disrupts natural defenses, S. aureus is frequently isolated from the lower airways, but distinguishing between colonization and infection is difficult. The objectives of this study were (1) to investigate the bacterial genome sequence in consecutive isolates in order to identify changes related to the pathological adaptation to the lower respiratory tract and (2) to explore the relationship between specific phenotypic and genotypic features with the patient's study group, persistence of the clinical isolate and clinical outcome. A set of 94 clinical isolates were selected and corresponded to 34 patients that were classified as having pneumonia (10), tracheobronchitis (11) and bronchial colonization (13). Clinical strains were phenotypically characterized by conventional identification and susceptibility testing methods. Isolates underwent whole genome sequencing using Illumina HiSeq4000. Genotypic characterization was performed with an in-house pipeline (BacterialTyper). Genomic variation arising within-host was determined by comparing mapped sequences and de novo assemblies. Virulence factors important in staphylococcal colonization and infection were characterized using previously established functional assays. (1) Toxin production was assessed using a THP-1 cytotoxicity assay, which reports on the gross cytotoxicity of individual isolates. In addition, we investigated the expression of the major virulence factor, alpha-toxin (Hla) by Western blot. (2) Adhesion to the important extracellular matrix molecule, fibronectin, was determined using a standardized microtitre plate assay. Finally, invasion experiments using THP-1 and A539 cell lines and selected clinical strains were also performed. Repeated isolation of S. aureus from endotracheal aspirate usually reflects persistence of the same strain. Within-host variation is detectable in this setting, but it shows no evidence of pathological adaptation related to virulence, resistance or niche adaptations. Cytotoxicity was variable among isolates with 14 strains showing no cytotoxicity, with these latter presenting an unaltered Fn binding capacity. No changes on cytotoxicity were reported when comparing study groups. Fn binding capacity was reported for almost all strains, with the exception of two strains that presented the lowest values. Strains isolated from patients with pneumonia presented a lower capacity of adhesion in comparison to those isolated during tracheobronchitis (p = 0.002). Hla was detected in 71 strains (75.5%), with most of the producer strains in pneumonia and bronchial colonization group (p = 0.06). In our cohort, Hla expression (presence or absence) in sequential isolates was usually preserved (70%) although in seven cases the expression varied over time. No relationship was found between low cytotoxicity and intracellular persistence in invasion experiments. In our study population, persistent S. aureus isolation from airways in ventilated patients does not reflect pathological adaptation. There is an important diversity of sequence types. Cytotoxicity is variable among strains, but no association with study groups was found, whereas isolates from patients with pneumonia had lower adhesion capability. Favorable clinical outcome correlated with increased bacterial adhesion in vitro. Most of the strains isolated from the lower airways were Hla producers and no correlation with an adverse outcome was reported. The identification of microbial factors that contribute to virulence is relevant to optimize patient management during lower respiratory tract infections

Accessory gene regulator (Agr) functionality in <i>Staphylococcus aureus</i> derived from lower respiratory tract infections

<div><p>Objective</p><p>Characterization of <i>Staphylococcus aureus</i> clinical isolates derived from lower respiratory tract infections (LRTIs), and correlation between the functionality of the accessory gene regulator (Agr) and genotypic and phenotypic characteristics, clinical variables and clinical outcome.</p><p>Methods</p><p><i>S aureus</i> isolates derived from LRTIs and control groups (nasal carriage and bacteraemia) were genotyped using StaphyType DNA microarray. Agr activity was evaluated using the CAMP synergistic haemolysis assay and the Vesicle Lysis Test (VLT). Discordant strains were analysed by quantitative reverse-transcriptase real-time PCR (qRT-PCR).</p><p>Results</p><p>Agr was functional in 79.7% and 84.5% of strains according to the CAMP and VLT assays respectively. Higher concordance with RNAIII expression measured by qRT-PCR was observed with the VLT assay (76.2%) compared with the CAMP assay (23.8%). No statistically significant differences were observed in Agr functionality between the study groups, nor the phenotypical/genotypical bacterial characteristics. No association between increased mortality/respiratory complications and Agr function was observed.</p><p>Conclusions</p><p>Agr activity was high (82.2%) in isolates from LRTIs suggesting the importance of this global regulator in lower respiratory tract colonisation and infection. However, equally high Agr activity was observed in isolates derived from nasal carriage and bacteraemia, contradictory to previous observations. Agr functionality measured by the VLT assay was superior to CAMP assay.</p></div

Accessory gene regulator (Agr) functionality in Staphylococcus aureus derived from lower respiratory tract infections

Altres ajuts: This work has been funded by the project PI13/01418 which is part of "Plan Nacional de I+D+I" and co-funded by ISCIII-Subdirección General de Evaluacioón and "Fondo Europeo de Desarrollo Regional"(FEDER).D. Domínguez-Villanueva is funded by "Plan Nacional de I+D+I" and co-funded by ISCIII-Subdirección General de Evaluación and "Fondo Europeo de Desarrollo Regional"(FEDER). M. Gomes-Fernandes is funded by CAPES Foundation, Ministry of Education of Brazil (Brasılia, Brazil). Maisem Laabei was supported by a joint ERS/SEPAR fellowship (LTRF2015).This work also received a grant from the Spanish Society of Pneumology and Thoracic Surgery (SEPAR054/2011).Objective. Characterization of Staphylococcus aureus clinical isolates derived from lower respiratory tract infections (LRTIs), and correlation between the functionality of the accessory gene regulator (Agr) and genotypic and phenotypic characteristics, clinical variables and clinical outcome. Methods. S aureus isolates derived from LRTIs and control groups (nasal carriage and bacteraemia) were genotyped using StaphyType DNA microarray. Agr activity was evaluated using the CAMP synergistic haemolysis assay and the Vesicle Lysis Test (VLT). Discordant strains were analysed by quantitative reverse- transcriptase real-time PCR (qRT-PCR). Results. Agr was functional in 79.7% and 84.5% of strains according to the CAMP and VLT assays respectively. Higher concordance with RNAIII expression measured by qRT-PCR was observed with the VLT assay (76.2%) compared with the CAMP assay (23.8%). No statistically significant differences were observed in Agr functionality between the study groups, nor the phenotypical/genotypical bacterial characteristics. No association between increased mortality/respiratory complications and Agr function was observed. Conclusions. Agr activity was high (82.2%) in isolates from LRTIs suggesting the importance of this global regulator in lower respiratory tract colonisation and infection. However, equally high Agr activity was observed in isolates derived from nasal carriage and bacteraemia, contradictory to previous observations. Agr functionality measured by the VLT assay was superior to CAMP assay