Michael R. Barer

Viability and activity in readily culturable bacteria: a review and discussion of the practical issues

In microbiology the terms ‘viability’ and ‘culturability’ are often equated. However, in recent years the apparently self-contradictory expression ‘viable-but-nonculturable’ (‘VBNC’) has been applied to cells with various and often poorly defined physiological attributes but which, nonetheless, could not be cultured by methods normally appropriate to the organism concerned. These attributes include apparent cell integrity, the possession of some form of measurable cellular activity and the apparent capacity to regain culturability. We review the evidence relating to putative VBNC cells and stress our view that most of the reports claiming a return to culturability have failed to exclude the regrowth of a limited number of cells which had never lost culturability. We argue that failure to differentiate clearly between use of the terms ‘viability’ and ‘culturability’ in an operational versus a conceptual sense is fuelling the current debate, and conclude with a number of proposals that are designed to help clarify the major issues involved. In particular, we suggest an alternative operational terminology that replaces ‘VBNC’ with expressions that are internally consistent.

Acute exacerbations of chronic obstructive pulmonary disease: identification of biologic clusters and their biomarkers.

RATIONALE Exacerbations of chronic obstructive pulmonary disease (COPD) are heterogeneous with respect to inflammation and etiology. OBJECTIVES Investigate biomarker expression in COPD exacerbations to identify biologic clusters and determine biomarkers that recognize clinical COPD exacerbation phenotypes, namely those associated with bacteria, viruses, or eosinophilic airway inflammation. METHODS Patients with COPD were observed for 1 year at stable and exacerbation visits. Biomarkers were measured in sputum and serum. Viruses and selected bacteria were assessed in sputum by polymerase chain reaction and routine diagnostic bacterial culture. Biologic phenotypes were explored using unbiased cluster analysis and biomarkers that differentiated clinical exacerbation phenotypes were investigated. MEASUREMENTS AND MAIN RESULTS A total of 145 patients (101 men and 44 women) entered the study. A total of 182 exacerbations were captured from 86 patients. Four distinct biologic exacerbation clusters were identified. These were bacterial-, viral-, or eosinophilic-predominant, and a fourth associated with limited changes in the inflammatory profile termed “pauciinflammatory.” Of all exacerbations, 55%, 29%, and 28% were associated with bacteria, virus, or a sputum eosinophilia. The biomarkers that best identified these clinical phenotypes were sputum IL-1β, 0.89 (area under receiver operating characteristic curve) (95% confidence interval [CI], 0.83–0.95); serum CXCL10, 0.83 (95% CI, 0.70–0.96); and percentage peripheral eosinophils, 0.85 (95% CI, 0.78–0.93), respectively. CONCLUSIONS The heterogeneity of the biologic response of COPD exacerbations can be defined. Sputum IL-1β, serum CXCL10, and peripheral eosinophils are biomarkers of bacteria-, virus-, or eosinophil-associated exacerbations of COPD. Whether phenotype-specific biomarkers can be applied to direct therapy warrants further investigation.

Cytological and transcript analyses reveal fat and lazy persister-like bacilli in tuberculous sputum.

Background Tuberculous sputum provides a sample of bacilli that must be eliminated by chemotherapy and that may go on to transmit infection. A preliminary observation that Mycobacterium tuberculosis cells contain triacylglycerol lipid bodies in sputum, but not when growing in vitro, led us to investigate the extent of this phenomenon and its physiological basis. Methods and Findings Microscopy-positive sputum samples from the UK and The Gambia were investigated for their content of lipid body–positive mycobacteria by combined Nile red and auramine staining. All samples contained a lipid body–positive population varying from 3% to 86% of the acid-fast bacilli present. The recent finding that triacylglycerol synthase is expressed by mycobacteria when they enter in vitro nonreplicating persistence led us to investigate whether this state was also associated with lipid body formation. We found that, when placed in laboratory conditions inducing nonreplicating persistence, two M. tuberculosis strains had lipid body levels comparable to those found in sputum. We investigated these physiological findings further by comparing the M. tuberculosis transcriptome of growing and nonreplicating persistence cultures with that obtained directly from sputum samples. Although sputum has traditionally been thought to contain actively growing tubercle bacilli, our transcript analyses refute the hypothesis that these cells predominate. Rather, they reinforce the results of the lipid body analyses by revealing transcriptional signatures that can be clearly attributed to slowly replicating or nonreplicating mycobacteria. Finally, the lipid body count was highly correlated (R2 = 0.64, p < 0.03) with time to positivity in diagnostic liquid cultures, thereby establishing a direct link between this cytological feature and the size of a potential nonreplicating population. Conclusion As nonreplicating tubercle bacilli are tolerant to the cidal action of antibiotics and resistant to multiple stresses, identification of this persister-like population of tubercle bacilli in sputum presents exciting and tractable new opportunities to investigate both responses to chemotherapy and the transmission of tuberculosis.

Blood Eosinophils to Direct Corticosteroid Treatment of Exacerbations of Chronic Obstructive Pulmonary Disease A Randomized Placebo-Controlled Trial

RATIONALE Exacerbations of chronic obstructive pulmonary disease (COPD) and responses to treatment are heterogeneous. OBJECTIVES Investigate the usefulness of blood eosinophils to direct corticosteroid therapy during exacerbations. METHODS Subjects with COPD exacerbations were entered into a randomized biomarker-directed double-blind corticosteroid versus standard therapy study. Subjects in the standard arm received prednisolone for 2 weeks, whereas in the biomarker-directed arm, prednisolone or matching placebo was given according to the blood eosinophil count biomarker. Both study groups received antibiotics. Blood eosinophils were measured in the biomarker-directed and standard therapy arms to define biomarker-positive and -negative exacerbations (blood eosinophil count > and ≤ 2%, respectively). The primary outcome was to determine noninferiority in health status using the chronic respiratory questionnaire (CRQ) and in the proportion of exacerbations associated with a treatment failure between subjects allocated to the biomarker-directed and standard therapy arms. MEASUREMENTS AND MAIN RESULTS There were 86 and 80 exacerbations in the biomarker-directed and standard treatment groups, respectively. In the biomarker-directed group, 49% of the exacerbations were not treated with prednisolone. CRQ improvement after treatment in the standard and biomarker-directed therapy groups was similar (0.8 vs. 1.1; mean difference, 0.3; 95% confidence interval, 0.0-0.6; P = 0.05). There was a greater improvement in CRQ in biomarker-negative exacerbations given placebo compared with those given prednisolone (mean difference, 0.45; 95% confidence interval, 0.01-0.90; P = 0.04). In biomarker-negative exacerbations, treatment failures occurred in 15% given prednisolone and 2% of those given placebo (P = 0.04). CONCLUSIONS The peripheral blood eosinophil count is a promising biomarker to direct corticosteroid therapy during COPD exacerbations, but larger studies are required.

Composition and diversity of ammonia‐oxidising bacterial communities in wastewater treatment reactors of different design treating identical wastewater

Autotrophic ammonia-oxidising bacteria (AOB) are a crucial component of the microbial communities of nitrifying wastewater treatment systems. Nitrification is known to occur in reactors of different configuration, but whether AOB communities are different in reactors of different design is unknown. We compared the diversity and community structure of the betaproteobacterial AOB in two full-scale treatment reactors - a biological aerated filter (BAF) and a trickling filter - receiving the same wastewater. Polymerase chain reaction (PCR) of 16S ribosomal RNA (rRNA) gene fragments with AOB-selective primers was combined with denaturing gradient gel electrophoresis (DGGE) to allow comparative analysis of the dominant AOB populations. The phylogenetic affiliation of the dominant AOB was determined by cloning and sequencing PCR-amplified 16S rRNA gene fragments. DGGE profiles were compared using a probability-based similarity index (Raup and Crick). The use of a probability-based index of similarity allowed us to evaluate if the differences and similarities observed in AOB community structure in different samples were statistically significant or could be accounted for by chance matching of bands in DGGE profiles, which would suggest random colonisation of the reactors by different AOB. The community structure of AOB was different in different sections of each of the reactors and differences were also noted between the reactors. All AOB-like sequences identified, grouped within the genus Nitrosomonas. A greater diversity of AOB was detected in the trickling filters than in the BAF though all samples analysed appeared to be dominated by AOB most closely related to Nitrosococcus mobilis. Numerical analysis of DGGE profiles indicated that the AOB communities in depth profiles from the filter beds were selected in a non-random manner.

Resuscitation-promoting Factors Reveal an Occult Population of Tubercle Bacilli in Sputum

RATIONALE Resuscitation-promoting factors (Rpfs) are a family of secreted proteins produced by Mycobacterium tuberculosis (Mtb) that stimulate mycobacterial growth. Although mouse infection studies show that they support bacterial survival and disease reactivation, it is currently unknown whether Rpfs influence human infection. We hypothesized that tuberculous sputum might include a population of Rpf-dependent Mtb cells. OBJECTIVES To determine whether Rpf-dependent Mtb cells are present in human sputum and explore the impact of chemotherapy on this population. METHODS In tuberculous sputum samples we compared the number of cells detected by conventional agar colony-forming assay with that determined by limiting dilution, most-probable number assay in the presence or absence of Rpf preparations. MEASUREMENTS AND MAIN RESULTS In 20 of 25 prechemotherapy samples from separate patients, 80-99.99% of the cells demonstrated by cultivation could be detected only with Rpf stimulation. Mtb cells with this phenotype were not generated on specimen storage or by inoculating sputum samples with a selection of clinical isolates; moreover, Rpf dependency was lost after primary isolation. During chemotherapy, the proportion of Rpf-dependent cells was found to increase relative to the surviving colony-forming population. CONCLUSIONS Smear-positive sputum samples are dominated by a population of Mtb cells that can be grown only in the presence of Rpfs. These intriguing proteins are therefore relevant to human infection. The Rpf-dependent population is invisible to conventional culture and is progressively enhanced in relative terms during chemotherapy, indicating a form of phenotypic resistance that may be significant for both chemotherapy and transmission.

Targeting Persisters for Tuberculosis Control

ABSTRACT Mycobacterial persisters, the survivors from antibiotic exposure, necessitate the lengthy treatment of tuberculosis (TB) and pose a significant challenge for our control of the disease. We suggest that persisters in TB are heterogeneous in nature and comprise various proportions of the population depending on the circumstances; the mechanisms of their formation are complex and may be related to those required for persistence in chronic infection. Results from recent studies implicate multiple pathways for persister formation, including energy production, the stringent response, global regulators, the trans-translation pathway, proteasomal protein degradation, toxin-antitoxin modules, and transporter or efflux mechanisms. A combination of specifically persister-targeted approaches, such as catching them when active and susceptible either by stimulating them to “wake up” or by intermittent drug dosing, the development of new drugs, the use of appropriate drug combinations, and combined chemotherapy and immunotherapy, may be needed for more effective elimination of persisters and better treatment of TB. Variations in levels of persister formation and in host genetics can play a role in the outcome of clinical treatment, and thus, these may entail personalized treatment regimens.

Lipid domains of mycobacteria studied with fluorescent molecular probes

The complex mycobacterial cell envelope is recognized as a critical factor in our failure to control tuberculosis, leprosy and other non‐tuberculous pathogens. Although its composition has been extensively determined, many details regarding the organization of the envelope remain uncertain. This is particularly so for the non‐covalently bound lipids, whose natural distribution may be disrupted by conventional biochemical or cytological techniques. In order to study the native organization of lipid domains in the mycobacterial envelope, we have applied a range of fluorescent lipophilic probes to live mycobacteria, including Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium gadium and Mycobacterium aurum, and analysed the resultant signals by fluorescence microscopy and digital image processing. Five key features were observed: (i) the presence of both envelope and intracellular lipid domains; (ii) differential localization of probes into these domains influenced predominantly by their hydrophobicity, as modelled by their calculated octanol:water partition coefficients and by their amphiphilicities; (iii) uneven distribution of lipophilic material in the envelope; (iv) selective labelling of septal regions of the envelope; and (v) modification of labelling patterns by additional treatments such as fluorescence quenching antibodies, detergents and solvents. Using this last approach, a coherent cell envelope lipid domain was demonstrated outside the cytoplasmic membrane and, for the first time, the proposed covalently linked mycolyl‐arabinogalactan‐peptidoglycan macromolecular complex was imaged directly. The use of fluorescent probes and high‐resolution fluorescence microscopy has enabled us to obtain a coherent view of distinct lipid domains in mycobacteria. Further application of this approach will facilitate understanding of the role of lipids in the physiology of these organisms.

Procalcitonin and C-Reactive Protein in Hospitalized Adult Patients With Community-Acquired Pneumonia or Exacerbation of Asthma or COPD

Background: Antibiotic overuse in respiratory illness is common and is associated with drug resistance and hospital-acquired infection. Biomarkers that can identify bacterial infections may reduce antibiotic prescription. We aimed to compare the usefulness of the biomarkers procalcitonin and C-reactive protein (CRP) in patients with pneumonia or exacerbations of asthma or COPD. Methods: Patients with a diagnosis of community-acquired pneumonia or exacerbation of asthma or COPD were recruited during the winter months of 2006 to 2008. Demographics, clinical data, and blood samples were collected. Procalcitonin and CRP concentrations were measured from available sera. Results: Sixty-two patients with pneumonia, 96 with asthma, and 161 with COPD were studied. Serum procalcitonin and CRP concentrations were strongly correlated (Spearman rank correlation coefficient [rs] = 0.56, P < .001). Patients with pneumonia had increased procalcitonin and CRP levels (median [interquartile range] 1.27 ng/mL [2.36], 191 mg/L [159]) compared with those with asthma (0.03 ng/mL [0.04], 9 mg/L [21]) and COPD (0.05 ng/mL [0.06], 16 mg/L [34]). The area under the receiver operating characteristic curve (95% CI) for distinguishing between patients with pneumonia (antibiotics required) and exacerbations of asthma (antibiotics not required), for procalcitonin and CRP was 0.93 (0.88-0.98) and 0.96 (0.93-1.00). A CRP value > 48 mg/L had a sensitivity of 91% (95% CI, 80%-97%) and specificity of 93% (95% CI, 86%-98%) for identifying patients with pneumonia. Conclusions: Procalcitonin and CRP levels can both independently distinguish pneumonia from exacerbations of asthma. CRP levels could be used to guide antibiotic therapy and reduce antibiotic overuse in hospitalized patients with acute respiratory illness.

A deletion defining a common Asian lineage of Mycobacterium tuberculosis associates with immune subversion.

Six major lineages of Mycobacterium tuberculosis appear preferentially transmitted amongst distinct ethnic groups. We identified a deletion affecting Rv1519 in CH, a strain isolated from a large outbreak in Leicester U.K., that coincidentally defines the East African-Indian lineage matching a major ethnic group in this city. In broth media, CH grew less rapidly and was less acidic and H2O2-tolerant than reference sequenced strains (CDC1551 and H37Rv). Nevertheless, CH was not impaired in its ability to grow in human monocyte-derived macrophages. When compared with CDC1551 and H37Rv, CH induced less protective IL-12p40 and more antiinflammatory IL-10 and IL-6 gene transcription and secretion from monocyte-derived macrophages. It thus appears that CH compensates microbiological attenuation by skewing the innate response toward phagocyte deactivation. Complementation of Rv1519, but none of nine additional genes absent from CH compared with the type strain, H37Rv, reversed the capacity of CH to elicit antiinflammatory IL-10 production by macrophages. The Rv1519 polymorphism in M. tuberculosis confers an immune subverting phenotype that contributes to the persistence and outbreak potential of this lineage.