Mycobacterium abscessus infection and potable water

Mycobacterium abscessus is a rapidly growing mycobacteria responsible for progressive pulmonary disease, soft tissue and wound infections, and can contaminate clinical specimens. Nontuberculous mycobacteria (NTM) are generally considered environmental organisms though M. abscessus has not featured frequently in environmental studies, particularly those examining potable water. In a study of Brisbane potable water, M. abscessus was isolate from ten different locations. The incidence of disease due to M. abscessus has been increasing in Queensland. Aim: To compare genotypically the M. abscessus isolates obtained from water to those obtained from human clinical specimens. Methods: From a study of Brisbane potable water between 2007 and 2009, ten isolates confirmed as M. abscessus were recovered. In addition, one strain was isolated from a rainwater tank of a patient with disease due to M. avium, and another from the swimming pool of a patient with M. intracellulare disease. A random sample of 74 clinical isolates referred to the QLD Mycobacterial reference laboratory during the same time period was available for comparison using repPCR strain typing (Diversilab). Results: The drinking water isolates formed two distinct strain patterns (A and B) that shared >90% similarity. The tankwater isolate (pattern C) shared >85% similarity with the potable water isolates, but the pool isolate (D) was distinctly different. Fifty-three clinical isolates clustered tightly (>95% similarity) with the Group A potable water isolates, 4 patients with Group B. Thirteen patient isolates clustered with the Rainwater tank isolate. One patient matched the pool isolate. There were a further 3 patient isolates that were unrelated to the water isolates. No differences were found between strain types in terms of geographic origin, gender, age, or site/type of infection. Conclusion: The high degree of similarity between strains of M. abscessus from potable water and strains causing infection in humans from the same area, strengthens the possibility that drinking water may be a source of infection in these patients.

Ureaplasma parvum serovar 3 multiple banded antigen size variation after chronic intra-amniotic infection/colonization

Ureaplasma species are the microorganisms most frequently associated with adverse pregnancy outcomes. The multiple banded antigen (MBA), a surface-exposed lipoprotein, is a key virulence factor of ureaplasmas. The MBA demonstrates size variation, which we have shown previously to be correlated with the severity of chorioamnion inflammation. We aimed to investigate U. parvum serovar 3 pathogenesis in vivo, using a sheep model, by investigating: MBA variation after long term (chronic) and short term (acute) durations of in utero ureaplasma infections, and the severity of chorioamnionitis and inflammation in other fetal tissues. Inocula of 2x107 colony-forming-units (CFU) of U. parvum serovar 3 (Up) or media controls (C) were injected intra-amniotically into pregnant ewes at one of three time points: day 55 (69d Up, n=8; C69, n=4); day 117 (7d Up, n=8; C7, n=2); and day 121 (3d Up, n=8; C3, n=2) of gestation (term=145-150d). At day 124, preterm fetuses were delivered surgically. Samples of chorioamnion, fetal lung, and umbilical cord were: (i) snap frozen for subsequent ureaplasma culture, and (ii) fixed, embedded, sectioned and stained by haematoxylin and eosin stain for histological analysis. Selected fetal lung clinical ureaplasma isolates were cloned and filtered to obtain cultures from a single CFU. Passage 1 and clone 2 ureaplasma cultures were tested by western blot to demonstrate MBA variation. In acute durations of ureaplasma infection no MBA variants (3d Up) or very few MBA variants (7d Up) were present when compared to the original inoculum. However, numerous MBA size variants were generated in vivo (alike within contiguous tissues, amniotic fluid and fetal lung, but different variants were present within chorioamnion), during chronic, 69d exposure to ureaplasma infection. For the first time we have shown that the degree of ureaplasma MBA variation in vivo increased with the duration of gestation.

Mycobacterium abscessus isolated from municipal water : a potential source of human infection

Background Mycobacterium abscessus is a rapidly growing mycobacterium responsible for progressive pulmonary disease, soft tissue and wound infections. The incidence of disease due to M. abscessus has been increasing in Queensland. In a study of Brisbane drinking water, M. abscessus was isolated from ten different locations. The aim of this study was to compare genotypically the M. abscessus isolates obtained from water to those obtained from human clinical specimens. Methods Between 2007 and 2009, eleven isolates confirmed as M. abscessus were recovered from potable water, one strain was isolated from a rainwater tank and another from a swimming pool and two from domestic taps. Seventy-four clinical isolates referred during the same time period were available for comparison using rep-PCR strain typing (Diversilab). Results The drinking water isolates formed two clusters with ≥97% genetic similarity (Water patterns 1 and 2). The tankwater isolate (WP4), one municipal water isolate (WP3) and the pool isolate (WP5) were distinctly different. Patient isolates formed clusters with all of the water isolates except for WP3. Further patient isolates were unrelated to the water isolates. Conclusion The high degree of similarity between strains of M. abscessus from potable water and strains causing infection in humans from the same geographical area, strengthens the possibility that drinking water may be the source of infection in these patients.

Isolation of nontuberculous mycobacteria (NTM) from household water and shower aerosols in patients with pulmonary disease caused by NTM

It has been postulated that susceptible individuals may acquire infection with nontuberculous mycobacteria (NTM) from water and aerosol exposure. This study examined household water and shower aerosols of patients with NTM pulmonary disease. The mycobacteria isolated from clinical samples from 20 patients included M. avium (5 patients), M. intracellulare (12 patients), M. abscessus (7 patients), M. gordonae (1 patient), M. lentiflavum (1 patient), M. fortuitum (1 patient), M. peregrinum (1 patient), M. chelonae (1 patient), M. triplex (1 patient), and M. kansasii (1 patient). One-liter water samples and swabs were collected from all taps, and swimming pools or rainwater tanks. Shower aerosols were sampled using Andersen six-stage cascade impactors. For a subgroup of patients, real-time PCR was performed and high-resolution melt profiles were compared to those of ATCC control strains. Pathogenic mycobacteria were isolated from 19 homes. Species identified in the home matched that found in the patient in seven (35%) cases: M. abscessus (3 cases), M. avium (1 case), M. gordonae (1 case), M. lentiflavum (1 case), and M. kansasii (1 case). In an additional patient with M. abscessus infection, this species was isolated from potable water supplying her home. NTM grown from aerosols included M. abscessus (3 homes), M. gordonae (2 homes), M. kansasii (1 home), M. fortuitum complex (4 homes), M. mucogenicum (1 home), and M. wolinskyi (1 home). NTM causing human disease can be isolated from household water and aerosols. The evidence appears strongest for M. avium, M. kansasii, M. lentiflavum, and M. abscessus. Despite a predominance of disease due to M. intracellulare, we found no evidence for acquisition of infection from household water for this species.

Pulmonary innate immunity in children with protracted bacterial bronchitis

Objective To determine bronchoalveolar lavage (BAL) levels of 3 innate immunity components (human alpha-defensin-2 [hBD2], mannose-binding lectin [MBL], and surfactant protein-A [SP-A], the relationship with airway neutrophilia and infection, and cytokine production of stimulated BAL cells in children with current protracted bacterial bronchitis (PBB), children with resolved PBB (PBB well), and controls. Study design BAL of 102 children (mean age 2.8 years) fulfilling predefined criteria of current PBB (n=61), PBB well (n=20), and controls (n=21) was cultured (quantitative bacteriology) and viruses examined by polymerase chain reaction. hBD2, MBL, and SP-A were measured, and cytokine production of lipopolysaccharide-stimulated BAL cells were determined. Results Median hBD2 and MBL levels were significantly higher in the current PBB group (hBD2 = 164.4, IQR 0-435.5pg/mL; MBL = 1.7, 0.4-4ng/mL) than in the PBB well group (hBD2 = 0, IQR 0-85.2; MBL = 0.6, IQR 0.03-2.9) and controls (hBD2 = 3.6, IQR 0-126; MBL = 0.4, IQR 0.02-79). hBD2 was significantly higher in children with airway infection (n = 54; median 76.9, IQR 0-397.3) compared with those without (n = 48; 0, IQR 0-236.3), P=0.04. SP-A levels and cytokine production of stimulated BAL cells were similar between groups. Conclusion In children's airways, hBD2, but not MBL and SP-A, relates to inflammation and infection. In children with PBB, mechanisms involving airway hBD2 and MBL are augmented. These pulmonary innate immunity components and the ability of BAL cells to respond to stimuli are unlikely to be deficient.

Clinical factors associated with the humoral immune response to influenza vaccination in chronic obstructive pulmonary disease

Background and objective Individuals with chronic obstructive pulmonary disease (COPD) are at a high risk of developing significant complications from infection with the influenza virus. It is therefore vital to ensure that prophylaxis with the influenza vaccine is effective in COPD. The aim of this study was to assess the immunogenicity of the 2010 trivalent influenza vaccine in persons with COPD compared to healthy subjects without lung disease, and to examine clinical factors associated with the serological response to the vaccine. Methods In this observational study, 34 subjects (20 COPD, 14 healthy) received the 2010 influenza vaccine. Antibody titers at baseline and 28 days post-vaccination were measured using the hemagglutination inhibition assay (HAI) assay. Primary endpoints included seroconversion (≥4-fold increase in antibody titers from baseline) and the fold increase in antibody titer after vaccination. Results Persons with COPD mounted a significantly lower humoral immune response to the influenza vaccine compared to healthy participants. Seroconversion occurred in 90% of healthy participants, but only in 43% of COPD patients (P=0.036). Increasing age and previous influenza vaccination were associated with lower antibody responses. Antibody titers did not vary significantly with cigarette smoking, presence of other comorbid diseases, or COPD severity. Conclusion The humoral immune response to the 2010 influenza vaccine was lower in persons with COPD compared to non-COPD controls. The antibody response also declined with increasing age and in those with a history of prior vaccination.

Short-term nutritional supplementation during management of pulmonary exacerbations in cystic fibosis: A controlled study, including effects of protein turnover

Effects of nutritional supplements on minimizing weight loss and abnormalities of protein turnover during pulmonary exacerbations in cystic fibrosis (CF) were studied by controlled trial. Patients received pulmonary therapy and either standard diet (n = 10) or adjunctive enteral supplements (n = 12). Initial protein turnover, measured by [15N]glycine kinetics, showed alterations of protein synthesis (P Syn) and catabolism (P Cat), which correlated with the degree of underweight, and negligible net protein deposition (P Dep). With treatment both groups had significant increases in mean body weight and forced expiratory volume in 1 s, expressed as percent predicted value for height (FEV1) by 3 wk, but a significant correlation between initial underweight and subsequent weight gain was observed only in supplemented patients. Mean P Syn and P Dep increased significantly (p < 0.001) only in the supplemented group. Pulmonary exacerbations in CF have important adverse effects on body-protein metabolism, similar to changes in protein-energy malnutrition and infection. These effects are reversed by short-term nutritional support. Strategic nutritional intervention should thus be considered in management, especially in malnourished patients.

Expression profiling of lymph nodes in tuberculosis patients reveal inflammatory milieu at site of infection

Extrapulmonary manifestations constitute 15 to 20% of tuberculosis cases, with lymph node tuberculosis (LNTB) as the most common form of infection. However, diagnosis and treatment advances are hindered by lack of understanding of LNTB biology. To identify host response, Mycobacterium tuberculosis infected lymph nodes from LNTB patients were studied by means of transcriptomics and quantitative proteomics analyses. The selected targets obtained by comparative analyses were validated by quantitative PCR and immunohistochemistry. This approach provided expression data for 8,728 transcripts and 102 proteins, differentially regulated in the infected human lymph node. Enhanced inflammation with upregulation of T-helper1-related genes, combined with marked dysregulation of matrix metalloproteinases, indicates tissue damage due to high immunoactivity at infected niche. This expression signature was accompanied by significant upregulation of an immunoregulatory gene, leukotriene A4 hydrolase, at both transcript and protein levels. Comparative transcriptional analyses revealed LNTB-specific perturbations. In contrast to pulmonary TB-associated increase in lipid metabolism, genes involved in fatty-acid metabolism were found to be downregulated in LNTB suggesting differential lipid metabolic signature. This study investigates the tissue molecular signature of LNTB patients for the first time and presents findings that indicate the possible mechanism of disease pathology through dysregulation of inflammatory and tissue-repair processes.

Transcriptional analysis of persistent Chlamydia pneumoniae infection in vitro

Chlamydia pneumoniae can cause acute respiratory infections including pneumonia. Repeated and persistent Chlamydia infections occur and persistent C. pneumoniae infection may have a role in the pathogenesis of atherosclerosis and coronary heart disease and may also contribute to the development of chronic inflammatory lung diseases like chronic obstructive pulmonary disease (COPD) and asthma. In this thesis in vitro models for persistent C. pneumonia infection were established in epithelial and monocyte/macrophage cell lines. Expression of host cell genes in the persistent C. pneumoniae infection model of epithelial cells was studied by microarray and RT-PCR. In the monocyte/macrophage infection model expression of selected C. pneumoniae genes were studied by RT-PCR and immunofluorescence microscopy. Chlamydia is able to modulate host cell gene expression and apoptosis of host cells, which may assist Chlamydia to evade the host cells' immune responses. This, in turn, may lead to extended survival of the organism inside epithelial cells and promote the development of persistent infection. To simulate persistent C. pneumoniae infection in vivo, we set up a persistent infection model exposing the HL cell cultures to IFN-gamma. When HL cell cultures were treated with moderate concentration of IFN-gamma, the replication of C. pneumoniae DNA was unaffected while differentiation into infectious elementary bodies (EB) was strongly inhibited. By transmission electron microscopy small atypical inclusions were identified in IFN-gamma treated cultures. No second cycle of infection was observed in cells exposed to IFN-gamma , whereas C. pneumoniae was able to undergo a second cycle of infection in unexposed HL cells. Although monocytic cells can naturally restrict chlamydial growth, IFN-gamma further reduced production of infectious C. pneumoniae in Mono Mac 6 cells. Under both studied conditions no second cycle of infection could be detected in monocytic cell line suggesting persistent infection in these cells. As a step toward understanding the role of host genes in the development and pathogenesis of persistent C. pneumoniae infection, modulation of host cell gene expression during IFN-gamma induced persistent infection was examined and compared to that seen during active C. pneumoniae infection or IFN-gamma treatment. Total RNA was collected at 6 to 150 h after infection of an epithelial cell line (HL) and analyzed by a cDNA array (available at that time) representing approximately 4000 human transcripts. In initial analysis 250 of the 4000 genes were identified as differentially expressed upon active and persistent chlamydial infection and IFN-gamma treatment. In persistent infection more potent up-regulation of many genes was observed in IFN-gamma induced persistent infection than in active infection or in IFN-gamma treated cell cultures. Also sustained up-regulation was observed for some genes. In addition, we could identify nine host cell genes whose transcription was specifically altered during the IFN-gamma induced persistent C. pneumoniae infection. Strongest up-regulation in persistent infection in relation to controls was identified for insulin like growth factor binding protein 6, interferon-stimulated protein 15 kDa, cyclin D1 and interleukin 7 receptor. These results suggest that during persistent infection, C. pneumoniae reprograms the host transcriptional machinery regulating a variety of cellular processes including adhesion, cell cycle regulation, growth and inflammatory response, all of which may play important roles in the pathogenesis of persistent C. pneumoniae infection. C. pneumoniae DNA can be detected in peripheral blood mononuclear cells indicating that the bacterium can also infect monocytic cells in vivo and thereby monocytes can assist the spread of infection from the lungs to other anatomical sites. Persistent infection established at these sites could promote inflammation and enhance pathology. Thus, the mononuclear cells are in a strategic position in the development of persistent infection. To investigate the intracellular replication and fate of C. pneumoniae in mononuclear cells we analyzed the transcription of 11 C. pneumoniae genes in Mono Mac 6 cells during infection by real time RT-PCR. Our results suggest that the transcriptional profile of the studied genes in monocytes is different from that seen in epithelial cells and that IFN-gamma has a less significant effect on C. pneumoniae transcription in monocytes. Furthermore, our study shows that type III secretion system (T3SS) related genes are transcribed and that Chlamydia possesses a functional T3SS during infection in monocytes. Since C. pneumoniae infection in monocytes has been implicated to have reduced antibiotic susceptibility, this creates opportunities for novel therapeutics targeting T3SS in the management of chlamydial infection in monocytes.

HBHA-specific IFN-{gamma} Synthesis at the Site of Infection during Active Tuberculosis in Humans.

RATIONALE: Tuberculosis (TB) remains a major cause of mortality. A better understanding of the immune responses to mycobacterial antigens may be helpful to develop improved vaccines and diagnostics. OBJECTIVE: The mycobacterial antigen heparin-binding-hemagglutinin (HBHA) induces strong interferon-gamma (IFN-gamma) responses by circulating lymphocytes from Mycobacterium tuberculosis latently infected subjects, and low responses associated with CD4(+) regulatory T (Treg) cells in TB patients. Here, we investigated HBHA-specific IFN-gamma responses at the site of the TB disease. METHODS: Bronchoalveolar lavages, pleural fluids and blood were prospectively collected from 61 patients with a possible diagnosis of pulmonary and/or pleural TB. HBHA-specific IFN-gamma production was analyzed by flow cytometry and ELISA. The suppressive effect of pleural Treg cells was investigated by depletion experiments. MEASUREMENTS AND MAIN RESULTS: The percentages of HBHA-induced IFN-gamma(+) alveolar and pleural lymphocytes were higher for pulmonary (P<0.0001) and for pleural (P<0.01) TB than for non-TB controls. Local CD4(+) and CD8(+) T cells produced the HBHA-specific IFN-gamma. This local secretion was not suppressed by Treg lymphocytes, contrasting with previously reported data on circulating lymphocytes. CONCLUSION: TB patients display differential effector and regulatory T cell responses to HBHA in local and circulating lymphocytes with a predominant effector CD4(+) and CD8(+) response locally, compared to a predominant Treg response among circulating lymphocytes. These findings may be helpful for the design of new vaccines against TB, and the detection of HBHA-specific T cells at the site of the infection may be a promising tool for the rapid diagnosis of active TB.

Elective versus symptomatic antibiotic treatment in cystic fibrosis patients with chronic Pseudomonas infection of the lungs

BACKGROUND:
A previous retrospective study suggested that a policy of regular anti-pseudomonal antibiotic treatment improved pulmonary function and increased survival in patients with cystic fibrosis chronically infected with Pseudomonas species. The results of a prospective multicentre study to compare the effects on pulmonary function and mortality of three monthly elective anti-pseudomonal antibiotic treatment with conventional symptomatic treatment are reported.

METHODS:
Sixty patients with cystic fibrosis, chronically infected with P aeruginosa, were randomised to the two treatment arms (elective or symptomatic) and followed clinically at yearly reviews. The major end points were changes in forced expiratory volume in one second (FEV(1)) and forced vital capacity (FVC). Survival was a secondary end point.

RESULTS:
Patients in the symptomatic group received a mean of three antibiotic treatments each year and those in the elective group received four antibiotic treatments during each year of the study. No significant differences in FEV(1) and FVC were found between the two groups after three years. There was a statistically non-significant higher rate of deaths in the elective group (n = 4), three of which were associated with B cepacia infection, compared with the symptomatic group (n = 0).

CONCLUSIONS:
This study did not demonstrate an advantage of a policy of elective antibiotic treatment over symptomatic treatment in patients with cystic fibrosis chronically infected with Pseudomonas species.

The effect of treatment of cystic fibrosis pulmonary exacerbations on airways and systemic inflammation

Background: Chronic infection in cystic fibrosis (CF) and airway inflammation leads to progressive lung injury Neutrophils are considered to be responsible for the onset and promotion of the inflammatory response within the CF lung. The relationship between infection and inflammation is complex but circulating inflammatory markers may not truly reflect the local inflammatory response in the lung. The aims of this study were to investigate the change of inflammatory biomarkers and cells within sputum and blood before and after intravenous antibiotics for a pulmonary exacerbation of CF Methods: Assays included neutrophil elastase (NE) and complex, interleukin-8 (IL-8) and soluble intercellular adhesion molecule-1 (sICAM-1), fas ligand (FAS-L), and TNFr-1. Analysis of sputum cell differential and absolute cell counts and immunocytochemistry (CD11b and CD95) on sputum and isolated blood neutrophils were carried out. Results: There were no significant differences in absolute or differential sputum cell counts or sputum sol measurements following antibiotics. There was a significant increase in the percentage of blood neutrophils with minimal CD11b staining, 28 (4.1) mean percentage (SEM) versus41 (2.9) and a decrease in the percentage showing maximal staining 30 (0.5) versus 15 (2.5). There was a significant increase in the percentage of blood neutrophils without CD95 staining, 43 (5.4) mean percentage versus 52 (5.1). Conclusion: These data suggest a modifiable systemic response to IV antibiotics but a local sustained inflammatory response in the lung.

Implementation of European standards of care for cystic fibrosis - Control and treatment of infection

Background: Several guidelines oil infection control and treatment of infection exist for cystic fibrosis (CF) caregivers, although the extent of implementation is variable.