Rapid diagnostic of multidrug-resistant sepsis pathogens directly from blood culture bottles using MALDI-TOF and the EUCAST RAST.

In this study, rapid diagnostic of multidrug-resistant (MDR) sepsis pathogens, directly from positive blood culture (BC) bottles, was evaluated by combining MALDI-TOF and the EUCAST Rapid Antimicrobial Susceptibility Testing (RAST). Carbapenemase production in Escherichia coli and Klebsiella pneumoniae isolates was also evaluated by RAST. From 171 positive BC bottles analyzed, 79 (46 %) MDR species, including E. coli (4/34, 12 %), K. pneumoniae (33/48, 69 %), Pseudomonas aeruginosa (12/12, 100 %), Acinetobacter baumannii (15/15, 100 %), and Staphylococcus aureus (14/37, 38 %) displaying resistance to beta-lactams, fluoroquinolones, aminoglycosides, and/or trimethoprim/sulphamethoxazole, were identified. In this regard, turnaround time of direct MALDI-TOF identification and RAST was < 7 h, which was significantly (p< 0.05) lower than our routine method. Carbapenemase detection by RAST displayed 100% sensitivity and 88.7 % specificity at 8 h. This protocol could offer advantages for the treatment and clinical outcomes of septic patients, improving the rapid diagnostic of sepsis by MDR pathogens.

Genomic analysis of a Kpi (pilus system)-positive and CTX-M-15-producing Klebsiella pneumoniae belonging to the high-risk clone ST15 isolated from an impacted river in Brazil.

Convergence of resistance and virulence in Klebsiella pneumoniae is a critical public health issue worldwide. A multidrug-resistant CTX-M-15-producing K. pneumoniae (TIES-4900 strain) was isolated from a highly impacted urban river, in Brazil. The genome was sequenced by MiSeq Illumina platform and de novo assembled using Unicycler. In silico prediction was accomplished by bioinformatics tools. The size of the genome is 5.4 Mb with 5145 protein-coding genes. TIES-4900 strain belonged to the sequence type ST15, yersiniabactin sequence type YbST10, ICEKp4, KL24 (wzi-24) and O1v1 locus. Phylogenomics confirmed genomic relatedness with ST15 clones from human and animal hosts. Convergence of broad resistome (antibiotics, heavy-metals and biocides) and virulome, including the Kpi pilus system involved in host-pathogen interaction and persistence of ST15 clone to hospital environments, were predicted. Virulent behavior was confirmed in the Galleria mellonella infection model. This study may give genomic insights on the spread of critical-priority WHO pathogens beyond hospital settings.

Clinical outcomes and molecular characterization of drug-resistant tuberculosis in pre- and extensively drug-resistant disease based on line probe assays.

Multidrug-resistant tuberculosis (MDR-TB) represents a significant impact in transmission, outcome, and health costs. The World Health Organization recommends implementation of rapid diagnostic methods for multidrug-resistance detection. This study was performed to evaluate the frequency of pre- and extensively drug resistant tuberculosis (pre-XDR-TB and XDR-TB) among MDR-TB patients, the pattern of resistance mutations for fluoroquinolones and the clinical outcome. Adult patients followed at a Brazilian regional reference center for TB, from January 2013 to June 2019 were included. Stored Mycobacterium tuberculosis (Mtb) cultures were recovered, the DNA was extracted, and the susceptibility test was performed using the line probe assay for second line antimycobacterial drugs, Genotype MTBDRsl version 2.0 (Hain Lifescience, CmbH, Germany). Among 33 MDR-TB included patients, we diagnosed XDR-TB or pre-XDR in five (15%) cases. Of these, mutations related to fluoroquinolones resistance were observed in four Mtb isolates, including one who had no phenotypic resistance profile. In two other patients with phenotypic resistance to ofloxacin, genotypic resistance was not found. Case fatality rate was 60% in pre/XDR-TB group, compared to 3.6% in the remaining of patients. This study observed few cases of pre-XDR and XDR-TB among a MDR-TB cohort. Phenotypic and genotypic assays presented good agreement. Clinical outcome was more favorable for patients with susceptibility to fluoroquinolones and injectable drugs.

FONA-7, a Novel Extended-Spectrum ß-Lactamase Variant of the FONA Family Identified in Serratia fonticola.

Serratia fonticola is a human pathogen widely found in the environment, with birds being reported as possible natural hosts. During an epidemiological and genomic surveillance study conducted to monitor the occurrence of extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales in South American wild birds, we identified an ESBL-positive S. fonticola in a fecal sample collected from a Hudsonian Whimbrel, during its non-breeding range on the Pacific Coast of Chile. Whole genome sequencing analysis and "in silico" modeling revealed a novel variant of the class A ESBLs FONA family, designated FONA-7, which shows 96.28% amino acid identity with FONA-6; with amino acid substitutions occurring in the signal peptide sequence (Thr22→Ser), and in the mature protein (Ser39→Asn and Thr227→Ile). This finding denotes that migratory birds can be potential vectors for the transboundary spread of ESBL-producing bacteria, creating a further theoretical risk for the origin of novel plasmid-encoded ß-lactamases.

Genome Sequences of Clinical Isolates of NDM-1-Producing Klebsiella quasipneumoniae subsp. similipneumoniae and KPC-2-Producing Klebsiella quasipneumoniae subsp. quasipneumoniae from Brazil.

Klebsiella quasipneumoniae is an emerging pathogen in human medicine. We report draft genome sequences of NDM-1- and KPC-2-producing K. quasipneumoniae strains from inpatients in Brazil. K. quasipneumoniae subsp. quasipneumoniae and K. quasipneumoniae subsp. similipneumoniae harbored broad resistomes. These data could contribute to a better understanding of acquired resistance in K. quasipneumoniae.

Lymphocyte responses to Mycobacterium tuberculosis and Mycobacterium bovis are similar between BCG-vaccinated patients with cystic fibrosis and healthy controls.

BACKGROUND: The low rate of nontuberculous mycobacteria (NTM) among Brazilian patients with cystic fibrosis (CF) may be due to cross-reactive Bacille Calmette-Guérin (BCG) vaccination. In the present pilot study, we aimed to compare the lymphocyte responses against Mycobacterium tuberculosis(Mtb) and Mycobacterium bovis (BCG) in BCG-vaccinated CF patients and healthy controls. METHODS: The lymphocyte responses of CF patients (n = 10) and healthy controls (n = 10) were assessed in terms of lymphocyte proliferation index (LPI), using flow cytometry. Median rates of each cell subtype - CD4, CD8, γδ T cells and CD19 (B) cells - were also determined. RESULTS: Median LPIs (CF vs. controls) were 22.9% vs. 13.0% (p = 0.481) and 23.1% vs. 17.6% (p = 0.481), upon stimulation with Mtb and BCG, respectively. Both groups had a predominant CD4 T cell response to Mtb (median rate = 82.5% vs. 79.7%; p = 0.796) and BCG (LPI = 84.3% vs. 83.0%; p = 0.853), which were significantly higher than the CD8, CD19 and γδ responses within both groups. CF patients tended to have a higher CD8 T cell response upon stimulation with the phytohemagglutinin mitogen than healthy controls (median rate = 42.8% vs. 31.7%, p = 0.075). CONCLUSION: The responses of BCG-vaccinated CF patients to Mtb and BCG are at least similar to those of healthy individuals. These are probably memory responses elicited by the BCG vaccination, which can cross-react with NTM and may explain the low frequency of NTM lung infection in our CF center.

Antibody response against Pseudomonas aeruginosa and its relationship with immune mediators in the upper and lower airways of cystic fibrosis patients.

BACKGROUND: The upper airways (UAW) are a niche and a reservoir of Pseudomonas aeruginosa strains that cause chronic infection of the lower airways (LAW) in cystic fibrosis (CF). Here, we assessed the role of anti-P. aeruginosa immunoglobulin A (IgA) and IgG antibodies in upper and lower airway infections in cystic fibrosis patients. METHODS: Nasal lavage fluid and induced sputum samples of 40 CF patients were microbiologically cultured. We searched for correlations between anti-P. aeruginosa IgA and IgG levels, measured by enzyme-linked immunosorbent assay (optical density), and unspecific immune mediators in both specimens. RESULTS: Anti-P. aeruginosa IgA (median optical density: 0.953 vs 0.298) and IgG (0.120 vs 0.059) were significantly higher in nasal lavage than in sputum, but not significantly different between patients with and without chronic P. aeruginosa infection in UAW. Matrix metallopeptidase-9 (MMP-9) in nasal lavage and neutrophil elastase (NE) in sputum were predictors of IgA in nasal lavage and IgA in sputum, respectively. IgA was a predictor of myeloperoxidase (MPO) in nasal lavage. Tissue inhibitor of metalloproteinases-1 (TIMP-1) was a predictor of IgG in sputum. IgG, TIMP-1, and NE in sputum were predictors of IgG in nasal lavage. CONCLUSION: The anti-P. aeruginosa IgA response was more prominent in CF patients' UAW, indicating a lower degree of inflammatory responses. Proteases may play a role in the anti-P. aeruginosa humoral response in the upper and LAW, and anti-P. aeruginosa IgG may be involved in the crosstalk between upper and lower airways in cystic fibrosis patients.

Nontuberculous mycobacterial infections in a Brazilian pediatric population: a seven-year survey.

The aim of this study was to describe the frequency of nontuberculous mycobacteria (NTM) isolation and their related outcomes among pediatric patients of a Brazilian university hospital from 2012 to 2019. NTM were identified in different clinical samples by microbiological culture and molecular-based methods. NTM were isolated from 14 patients, out of whom four (27%) were infected and were treated accordingly. Two were infected with Mycobacterium avium complex (MAC), two with M. abscessus complex (MABSC) and one with M. intracellulare. Two patients had cystic fibrosis-related lung disease and improved after successful NTM eradication. One patient was HIV-positive and died. One patient had severe combined immunodeficiency (SCID)-related pneumonia and is currently being followed-up. We conclude that NTM frequency in our center was low among pediatric patients. Whether this is inherent to Brazilian patients, due to the broad coverage of the Bacille Calmette-Guérin (BCG) vaccine in Brazil, or a result of underdiagnosis remains to be elucidated.

Draft Whole-Genome Sequences of Haemophilus influenzae Biogroup aegyptius Strains Isolated from Five Brazilian Purpuric Fever Cases and One Conjunctivitis Case.

Brazilian purpuric fever is a febrile hemorrhagic pediatric disease caused by Haemophilus influenzae biogroup aegyptius, a bacterium which was formerly associated with only self-limited purulent conjunctivitis. Here, we present draft genomes of strains from five Brazilian purpuric fever cases and one conjunctivitis case.

Prevalence and clinical outcomes of nontuberculous mycobacteria in a Brazilian cystic fibrosis reference center.

Nontuberculous mycobacteria (NTM) have been well established as an opportunistic pathogenic bacterial group for cystic fibrosis (CF) patients, with a prevalence ranging from 3% to 23% worldwide. A myriad of factors can bias the prevalence rate in different CF centers, especially misdiagnosis as systematic screening for NTM are still lacking in a number of centers. Here, we evaluated the presence and clinical outcomes of NTM isolation in microbiological respiratory cultures from CF patients attending a Brazilian reference center after setting up a systematic diagnostic protocol. Of 117 patients with respiratory samples cultured for NTM research, we found seven patients (6%) with at least one positive result for NTM [four males (57.1%), median age = 21 years (9-58)]. These cases are reported one-by-one. Median FEV1 was 40%, all patients showed signs of lung deterioration, with a median number of pulmonary exacerbations of three per patient/year. However, the impact of NTM isolation remains unclear in our center as all patients were coinfected with other CF respiratory pathogens. Our NTM prevalence assimilates to the lowest levels reported in literature, which is possibly influenced by the routinely applied Bacille Calmette-Guérin vaccine.

The influence of glycerol upon L. reuteri activity against enteropathogens / A influência do glicerol sobre a atividade de L. Reuteri contra enteropatógenos

BACKGROUND: Probiotics containing L. reuteri are popular for treating and preventing bacterial gastrointestinal infections. L. reuteri, produces reuterin, an antibiotic that inhibits gram-negative bacteria. Reuterin production is the result of glycerol fermentation by L reuteri. Although L. reuteri is normally present in the gastrointestinal system, only small amounts of glycerol are usually available; therefore, the production of reuterin may not occur and this could reduce the effectiveness of the probiotic supplement. Our objective is to identify the minimum concentrations of glycerol required for L. reuteri to exert an inhibitory effect on enteropathogenic enterobacteriaceae. METHOD: Samples containing 10(8) colony forming units (CFU) of L. reuteri DSM17938 (Colikids®, Ache, Brazil/BioGaia, Sweden) were grown with varying concentrations of glycerol (0.05-5%). 10(6) CFU of E.coli (CDC0126/INCQS/FIOCRUZ), Shigella flexneri (ATCC/120022), S. enterica (ATCC6539) and Y. enterocolitica (ATCC9610) were inoculated with L. reuteri in the different glycerol concentrations. Each enterobacteria and glycerol 5% without L. reuteri cultures were used as positive control groups. RESULTS: All bacteria were completely inhibited at higher ranges of glycerol concentrations (0.2-5%) and grew at lower concentrations (0.05-0.1%). CONCLUSION: L. reuteri requires at least 0.2% of glycerol to completely inhibit enterobacterial growth. These preliminary findings may influence the current method of use of probiotic supplements. The antibiotic activity of L. reuteri may have potential clinical use against important enteropathogens.

CONTEXTO: Os probióticos que contêm L. reuteri são populares para tratar e prevenir infecções bacterianas do trato gastrointestinal. L. reuteri produz reuterina, um antibiótico que inibe bactérias gram-negativas. A produção de reuterina depende da presença de quantidades adequadas de glicerol, cuja fermentação resulta na produção do antibiótico. Embora L. reuteri esteja normalmente presente no sistema GI, apenas pequenas quantidades de glicerol estão geralmente disponíveis; portanto, a produção de reuterina pode não ocorrer o que poderia reduzir a eficácia do suplemento probiótico. Nosso objetivo é identificar as concentrações mínimas de glicerol necessárias para que L. reuteri exerça um efeito inibitório nas enterobacteriaceas enteropatogênicas. MÉTODO: 108 CFU de L. reuteri DSM17938 (Colikids®, Ache, Brasil / BioGaia, Suécia) cresceu com concentrações variáveis de glicerol (0,05-5%). Foram inoculadas 106 UFC de E. coli (CDC0126 / INCQS / FIOCRUZ), Shigella flexneri (ATCC / 120022), S. enterica (ATCC6539) e Y. enterocolitica (ATCC9610) com L. reuteri nas diferentes concentrações de glicerol. Cada enterobacteria e glicerol 5% sem culturas de L. reuteri foram utilizadas como grupos de controle positivo. RESULTADOS: Todas as bactérias foram completamente inibidas em maiores concentrações de glicerol (0.2-5%) e cresceram em concentrações mais baixas (0.05-0.1%). CONCLUSÃO: L. reuteri requer pelo menos 0,2% de glicerol para inibir completamente o crescimento de enterobacteria. Essas descobertas preliminares podem influenciar o método atual de uso de suplementos de probióticos. A atividade antibiótica de L. reuteri pode ter potencial uso clínico contra importantes enteropatógenos.

Brazil's resolutions to regulate the sale of antibiotics: Impact on consumption and Escherichia coli resistance rates.

OBJECTIVE: The aim of this study was to determine the impact of two resolutions to restrict antibiotic use (RDCs no. 44/2010 and 20/2011) in the Campinas metropolitan area (Sao Paulo, Brazil) on antibiotic consumption, resistance rates, and trends in Escherichia coli-causing community-acquired urinary tract infection (UTI). METHODS: The annual retail sale information of antibiotics from drugstores in the Campinas metropolitan area between 2008 and 2012 were obtained through the Intercontinental Medical Statistics Health of Brazil. The daily-defined dose (DDD)/1000 inhabitants/day was calculated from these data to measure consumption. To examine resistance rates, we performed an observational retrospective study in a Campinas teaching hospital, where urinary cultures from outpatients with a clinical suspicion of UTI between October 2009 and September 2015 were analyzed. RESULTS: We observed an increase in rates of antibiotic sales from 2008 to 2011 (cephalosporin: 216.8%, quinolones: 170.9%, aminopenicillins: 140.9%), followed by a decrease in sales in 2012 (cephalosporin: 19.4%, quinolones: 12.7%, aminopenicillins: 11.1%). Sale of nitrofurans, however, did not significantly change during this period. In the retrospective analysis, we observed a significant increasing trend of E. coli resistance for all antibiotic classes, except nitrofurans and folate pathway inhibitors. CONCLUSIONS: We found changes in antibiotic consumption, with an initial increase, followed by a decrease in sales after implementation of the resolutions. However, bacterial resistance does not appear to be affected by the RDCs.

Secretory IgA response against Pseudomonas aeruginosa in the upper airways and the link with chronic lung infection in cystic fibrosis.

We assessed the diagnostic ability of an enzyme-linked immunosorbent assay test for measurement of specific secretory IgA (sIgA) in saliva to identify cystic fibrosis (CF) patients with Pseudomonas aeruginosa chronic lung infection and intermittent lung colonization. A total of 102 Brazilian CF patients and 53 healthy controls were included. Specific serum IgG response was used as a surrogate to distinguish CF patients according to their P. aeruginosa colonization/infection status. The rate of sIgA positivity was 87.1% in CF chronically infected patients (median value = 181.5 U/mL), 48.7% in intermittently colonized patients (median value = 45.8 U/mL) and 21.8% in free of infection patients (median value = 22.1 U/mL). sIgA levels in saliva were significantly associated with serum P. aeruginosa IgG and microbiological culture results. The sensitivity, specificity, PPV and NPV for differentiation between presence and absence of chronic lung infection were 87%, 63%, 51% and 92%, respectively. Measurement of sIgA in saliva may be used for screening patients in risk of developing P. aeruginosa chronic lung infection in CF and possibly also for paranasal sinusitis, and, most importantly, to efficiently rule out chronic P. aeruginosa lung infection.

Imported malaria in a non-endemic area: the experience of the university of Campinas hospital in the Brazilian Southeast.

BACKGROUND: Although malaria in Brazil almost exclusively occurs within the boundaries of the Amazon Region, some concerns are raised regarding imported malaria to non-endemic areas of the country, notably increased incidence of complications due to delayed diagnoses. However, although imported malaria in Brazil represents a major health problem, only a few studies have addressed this subject. METHODS: A retrospective case series is presented in which 263 medical charts were analysed to investigate the clinical and epidemiological characterization of malaria cases that were diagnosed and treated at Hospital & Clinics, State University of Campinas between 1998 and 2011. RESULTS: Amongst all medical charts analysed, 224 patients had a parasitological confirmed diagnosis of malaria. Plasmodium vivax and Plasmodium falciparum were responsible for 67% and 30% of the infections, respectively. The majority of patients were male (83%) of a productive age (median, 37 years old). Importantly, severe complications did not differ significantly between P. vivax (14 cases, 9%) and P. falciparum (7 cases, 10%) infections. CONCLUSIONS: Severe malaria cases were frequent among imported cases in Brazil outside of the Amazon area. The findings reinforce the idea that P. vivax infections in Brazil are not benign, regardless the endemicity of the area studied. Moreover, as the hospital is located in a privileged site, it could be used for future studies of malaria relapses and primaquine resistance mechanisms. Finally, based on the volume of cases treated and the secondary complications, referral malaria services are needed in the non-endemic areas of Brazil for a rapid and efficient and treatment.

Complete Genome Sequence of an F8-Like Lytic Myovirus ({varphi}SPM-1) That Infects Metallo-ß-Lactamase-Producing Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an important cause of infection, especially in immunocompromised patients. In this regard, strains producing carbapenemases, mainly metallo-ß-lactamases (MBLs), have become a significant public health concern. Here, we present the complete annotated genome sequence (65.7 kb) of an F8-related lytic myovirus (Pbunalikevirus genus) that infects MBL-producing P. aeruginosa strains.

National prevalence survey in Brazil to evaluate the quality of microbiology laboratories: the importance of defining priorities to allocate limited resources.

This report describes a survey of microbiology laboratories (n = 467) serving Brazilian hospitals with ≥10 intensive care beds and/or involved in the government health care adverse event reporting system. Coordinators were interviewed and laboratories classified as follows: Level 0 (no minimal functioning conditions-85.4% of laboratories); Level 1 (minimal functioning conditions but inadequate execution of basic routine-6.7%); Level 2 (minimal functioning conditions and adequate execution of basic routine but no adequate procedures for quality control-5.8%); Level 3 (minimal functioning conditions, adequate execution of basic routine, and adequate procedures for quality control, but no direct communication with the infection control department-0.9%); Level 4 (minimal functioning conditions, adequate execution of basic routine, adequate procedures for quality control, and direct communication with infection control, but no available advanced resources-none); and Level 5 (minimal functioning conditions, adequate execution of basic routine, adequate procedures for quality control, direct communication with infection control, and available advanced resources-0.9%). Twelve laboratories did not perform Ziehl-Neelsen staining; 271 did not have safety cabinets; and >30% without safety cabinets had automated systems. Low quality was associated with serving hospitals not participating in government adverse-event program; private hospitals; nonteaching hospitals; and those outside state capitals. Results may reflect what occurs in many other countries where defining priorities is important due to limited resources.

National prevalence survey in Brazil to evaluate the quality of microbiology laboratories: the importance of defining priorities to allocate limited resources / Encuesta de prevalencia nacional para evaluar la calidad de los laboratorios de microbiología en el Brasil: importancia de la definición de prioridades para asignar recursos limitados

This report describes a survey of microbiology laboratories (n = 467) serving Brazilian hospitals with >10 intensive care beds and/or involved in the government health care adverse event reporting system. Coordinators were interviewed and laboratories classified as follows: Level 0 (no minimal functioning conditions-85.4% of laboratories); Level 1 (minimal functioning conditions but inadequate execution of basic routine-6.7%); Level 2 (minimal functioning conditions and adequate execution of basic routine but no adequate procedures for quality control-5.8%); Level 3 (minimal functioning conditions, adequate execution of basic routine, and adequate procedures for quality control, but no direct communication with the infection control department-0.9%); Level 4 (minimal functioning conditions, adequate execution of basic routine, adequate procedures for quality control, and direct communication with infection control, but no available advanced resources-none); and Level 5 (minimal functioning conditions, adequate execution of basic routine, adequate procedures for quality control, direct communication with infection control, and available advanced resources-0.9%). Twelve laboratories did not perform Ziehl-Neelsen staining; 271 did not have safety cabinets; and >30% without safety cabinets had automated systems. Low quality was associated with serving hospitals not participating in government adverse-event program; private hospitals; nonteaching hospitals; and those outside state capitals. Results may reflect what occurs in many other countries where defining priorities is important due to limited resources.

Este artículo describe una encuesta realizada en Brasil en laboratorios de microbiología (n = 467) que prestaban servicio a hospitales que contaban al menos con 10 camas de cuidados intensivos. Se entrevistó a los coordinadores y los laboratorios se clasificaron de la siguiente manera: nivel 0 (sin condiciones de funcionamiento mínimas: 85,4% de los laboratorios), nivel 1 (condiciones de funcionamiento mínimas pero ejecución inadecuada del trabajo habitual básico: 6,7%), nivel 2 (condiciones de funcionamiento mínimas y ejecución adecuada del trabajo habitual básico, pero sin procedimientos de control de calidad apropiados: 5,8%), nivel 3 (condiciones de funcionamiento mínimas, ejecución adecuada del trabajo habitual básico y procedimientos de control de calidad apropiados, pero sin comunicación directa con el departamento de control de infecciones: 0,9%), nivel 4 (condiciones de funcionamiento mínimas, ejecución adecuada del trabajo habitual básico, procedimientos de control de calidad apropiados y comunicación directa con el departamento de control de infecciones, pero sin recursos avanzados disponibles: ningún laboratorio) y nivel 5 (condiciones de funcionamiento mínimas, ejecución adecuada del trabajo habitual básico, procedimientos de control de calidad apropiados, comunicación directa con el departamento de control de infecciones y recursos avanzados disponibles: 0,9%). Doce laboratorios no realizaban la tinción de Ziehl-Neelsen, 271 no contaban con cámaras de seguridad biológica, y más de 30% de los laboratorios que carecían de cámaras de seguridad biológica tenían sistemas automatizados. La escasa calidad se asoció a la falta de participación en el programa gubernamental de notificación de acontecimientos adversos, a los hospitales privados, a los hospitales no docentes y a la ubicación de los hospitales fuera de las capitales de los estados. Los resultados pueden reflejar lo que ocurre en muchos otros países con recursos limitados, donde es importante definir las prioridades.

A heat-stable cytotoxic factor produced by Achromobacter xylosoxidans isolated from Brazilian patients with CF is associated with in vitro increased proinflammatory cytokines.

BACKGROUND: Recently, Achromobacter xylosoxidans has been related to chronic lung diseases in patients suffering from cystic fibrosis (CF), but its involvement has not been elucidated. Some virulence properties of A. xylosoxidans isolated from Brazilian patients with CF were revealed in this work. METHODS: This study examined the production of a cytotoxic factor of A. xylosoxidans capable of stimulating the secretion of inflammatory cytokines (IL-6 and IL-8) from lung mucoepidermoid carcinoma cells (NCI-H292). The cytokines were measured using enzyme-linked immunosorbent (ELISA) assays. To investigate whether the cytotoxic factors may be endotoxins, they were treated with polymyxin B. RESULTS: The culture supernatants of all A. xylosoxidans produced a heat stable, active cytotoxin in NCI-H292 cells capable of leading to intracellular vacuoles and subsequent cell contact loss, chromatin condensation, a picnotic nucleus and cell death. There was a higher concentration of proinflammatory cytokines in the NCI-H292 cells after 24 h of incubation, with the fraction greater than 50 kDa from the culture supernatant. The cytotoxin activity remained even after treatment with polymyxin B, which suggested that the release of IL-6 and IL-8 was not stimulated by lipopolysaccharide (LPS). CONCLUSION: The cytotoxic factor produced by A. xylosoxidans may represent an important virulence factor, which when associated with CF chronic lung inflammation, may cause tissue damage and decline of lung function.