Exhaled Pneumocystis jirovecii output and detection of asymptomatic exhalation by facemask sampling in HIV-uninfected, immunocompromised patients.

BACKGROUND: Pneumocystis jirovecii pneumonia (PJP) transmission is poorly defined. Previous studies have sampled air of rooms occupied by HIV-infected patients with PJP, while natural and direct exhalations of HIV-uninfected subjects remain under-investigated. Here, clinical facemasks were used to examine and quantify potential P. jirovecii exhalations from HIV-uninfected patients with suspected PJP and to determine whether pathogen exhalation was definable clinically or radiologically. METHODS: Forty-five patients in Leicester (England), highly suspected of having PJP based on European Conference on Infections in Leukaemia (ECIL-5) guidelines, each wore one facemask carrying a gelatine/PVA sampling matrix for 1 h while respiring normally. Mask contamination with P. jirovecii was assessed using a modified quantitative polymerase chain reaction targeting mitochondrial large subunit (MtLSU). Radiological findings on chest X-ray (CXR) and computed tomography (CT) were graded and analysed for correlation with P. jirovecii signals alongside relevant clinical and laboratory findings. RESULTS: P. jirovecii was detected in seven of 20 patients diagnosed with PJP and three of 19 patients with suspected but undiagnosed PJP. The median captured signal was 8.59 × 104 MtLSU copies/mask (interquartile range (IQR) = 3.01 × 105-1.81 × 104). Blood ß-D-glucan test results correlated with the mask detection data (r = 0.65; P<0.0001) but other clinical indices and radiological features did not. Five of the 10 P. jirovecii-exhalers exhibited normal CXR with a median exhalation burden 1.28 × 105 copies/mask (IQR = 1.51 × 105-2.27 × 104). Two P. jirovecii-exhalers (7.64 × 104 copies/mask) were asymptomatic. CONCLUSION: P. jirovecii was exhaled sufficiently during normal respiration to be detectable in facemasks worn by HIV-uninfected patients. Neither clinical nor radiological features correlated with P. jirovecii exhalation.

Batch process solar disinfection is an efficient means of disinfecting drinking water contaminated with Shigella dysenteriae type I.

AIMS: The mortality and morbidity rate caused by Shigella dysenteriae type I infection is increasing in the developing world each year. In this paper, the possibility of using batch process solar disinfection (SODIS) as an effective means of disinfecting drinking water contaminated with Sh. dysenteriae type I is investigated. METHODS: Phosphate-buffered saline contaminated with Sh. dysenteriae type I was exposed to simulated solar conditions and the inactivation kinetics of this organism was compared with that of Sh. flexneri, Vibrio cholerae and Salmonella typhimurium. SIGNIFICANCE: Recovery of injured Sh. dysenteriae type I may be improved by plating on medium supplemented with catalase or pyruvate. Sh. dysenteriae type I is very sensitive to batch process SODIS and is easily inactivated even during overcast conditions. Batch process SODIS is an appropriate intervention for use in developing countries during Sh. dysenteriae type I epidemics.

A comparitive study of ammonia-oxidizing bacteria in lab-scale industrial wastewater treatment reactors.

The diversity and community structure of the beta-proteobacterial ammonia oxidising bacteria (AOB) in a range of different lab-scale industrial wastewater treatment reactors were compared. Three of the reactors treat waste from mixed domestic and industrial sources whereas the other reactor treats waste solely of industrial origin. PCR with AOB selective primers was combined with denaturing gradient ge electrophoresis to allow comparative analysis of the dominant AOB populations and the phylogenetic affiliation of the dominant AOB was determined by cloning and sequencing or direct sequencing of bands excised from DGGE gels. Different AOB were found within and between different reactors. All AOB sequences identified were grouped within the genus Nitrosomonas. Within the lab-scale reactors there appeared to be selection for a low diversity of AOB and predominance of a single AOB population. Furthermore, the industrial input in both effluents apparently selected for salt tolerant AOB, most closely related to Nitrosococcus mobilis and Nitrosomonas halophila.

Standardized staining methods: Feulgen-Rossenbeck reaction for desoxyribonucleic acid and periodic acid-Schiff (PAS) procedure.

A project group working under the European Confederation of Laboratory Medicine (ECLM) presents recommendations for standardized procedures for the Feulgen-Rossenbeck-Schiff and the periodic acid-Schiff (PAS) reactions on cytological and histological material. The advantages and disadvantages of such standardized procedures are presented here in a preamble. Both users and manufacturers are encouraged to give their opinions with a view to achieving consensus on these procedures and on how further work on these lines may proceed.

A comparison of autotrophic ammonia-oxidizing bacteria in full- and laboratory-scale wastewater treatment reactors.

Lab-scale reactors are commonly used to simulate full-scale plants as they permit the effects of defined experimental perturbations to be evaluated. Ideally, lab- and full-scale reactors should possess similar microbial populations. To determine this we compared the diversity of the beta-proteobacterial autotrophic ammonia-oxidising bacteria (AOB) in a full-scale and lab-scale biological aerated filter (BAF) using PCR with AOB selective primers combined with denaturing gradient gel electrophoresis (DGGE). PCR amplified 16S rRNA gene fragments from the nitrification unit of the lab-and full-scale BAF were subjected to cloning and sequencing to determine the phylogenetic affiliation of the AOB. A high degree of comparability between the lab-and full-scale BAF was observed with respect to AOB populations. However minor differences were apparent. The importance of these minor constituents in the overall performance of the reactor is unknown. Nonetheless the lab-scale reactor in this study did appear to reflect the dominant AOB community within the full-scale equivalent.

Histopathological methods for the investigation of microbial communities associated with disease lesions in reef corals.

AIMS: To determine the spatial structure of microbial communities associated with disease lesions of reef corals (Scleractinia). METHODS AND RESULTS: Agarose pre-embedding preserved the structure of the disease lesion and surrounding tissues prior to demineralization of the carbonate exoskeleton and embedding in resin. Fluorescence in situ hybridization (FISH) was used to localize bacteria in the lesions of various diseases. CONCLUSIONS: The techniques successfully preserved the in situ spatial structure of degenerated coral tissues. In one case (white plague disease), significant bacterial populations were found only in fragmented remnants of degenerated coral tissues at the lesion boundary that would not have been detected using conventional histopathological techniques. SIGNIFICANCE AND IMPACT OF THE STUDY: Determining the composition, spatial structure and dynamics of microbial communities within the disease lesions is necessary to understand the process of disease progression. The methods described may be applicable to a wide range of diseases involving necrotic lesion formation and requiring extensive tissue processing, such as skeleton demineralization.

Fluorescence and Brightfield Cytology of Live M. tuberculosis Cells.

Although light microscopy fell out of favor as a research tool in prokaryotic biology in the 1980s, advances in the reagents available for cell labeling (staining) and in the user-friendliness of microscopes were underpinning a revolution in eukaryotic cell biology. The development of epifluorescence hardware, particularly confocal microscopy and low-light imaging systems, and computational deblurring and video enhancement methodologies, substantially extended the range of potential applications. These developments now enable us to detect weaker signals at higher levels of resolution than was previously possible. Finally the personal computer and related software developments have brought image analysis within affordable range for many laboratories and facilitate quantitation of cellular properties on an objective basis. We have sought to apply these advances across a range of prokaryotic applications and here we describe the methods we have applied to live Mycobacterium tuberculosis cells. Although we have principally been concerned with two applications, the determination of viability at the cellular level (see Note 1) and the nature and distribution of lipid domains, more general aspects of light microscopic cytological analyses are discussed below.

Effects of simulated solar disinfection of water on infectivity of Salmonella typhimurium.

To determine whether cells of Salmonella typhimurium rendered nonculturable by simulated solar disinfection retain infectivity for mice. Bacteria suspended in water were exposed to UVA irradiation for up to 8 h. Culturability, determined by colony forming unit and Most Probable Number counts, fell by six log10 units, while cellular activity determined by the Kogure cell elongation test was retained by approximately 5% of the cells present after 8 h. Intraperitoneal doses of nonculturable cells and active but nonculturable (ABNC) cells exceeding the LD50 of the test organism and BALB/c mouse host, respectively, by 4 and 3 orders of magnitude failed to produce detectable infections. Culturable cells that had been irradiated for 1.5 h were less infective (virulent) than their nonirradiated counterparts. Nonculturable and ABNC cells of Salm. typhimurium produced by UVA irradiation do not retain infectivity for mice. Although ABNC cells could be produced by low cost solar disinfection systems, they do not appear to pose a potential infection hazard.

Toxin gene expression by shiga toxin-producing Escherichia coli: the role of antibiotics and the bacterial SOS response.

Toxin synthesis by Shiga toxin-producing Escherichia coli (STEC) appears to be coregulated through induction of the integrated bacteriophage that encodes the toxin gene. Phage production is linked to induction of the bacterial SOS response, a ubiquitous response to DNA damage. SOS-inducing antimicrobial agents, particularly the quinolones, trimethoprim, and furazolidone, were shown to induce toxin gene expression in studies of their effects on a reporter STEC strain carrying a chromosome-based stx2::lacZ transcriptional fusion. At antimicrobial levels above those required to inhibit bacterial replication, these agents are potent inducers (up to 140-fold) of the transcription of type 2 Shiga toxin genes (stx2); therefore, they should be avoided in treating patients with potential or confirmed STEC infections. Other agents (20 studied) and incubation conditions produced significant but less striking effects on stx2 transcription; positive and negative influences were observed. SOS-mediated induction of toxin synthesis also provides a mechanism that could exacerbate STEC infections and increase dissemination of stx genes. These features and the use of SOS-inducing antibiotics in clinical practice and animal husbandry may account for the recent emergence of STEC disease.

Density gradient separation of active and non-active cells from natural environments.

We present a method for the selective, physical separation of active and non-active bacterial cells from natural communities. The method exploits the reduction of tetrazolium salts to form insoluble formazan crystals intracellularly in response to the addition of different oxidisable substrates. The intracellular deposition of formazan alters the bouyant density of active cells enabling them to be separated by density gradient centrifugation. The method has been successfully applied to the fractionation and collection of large whole cell sub-populations of active and non-active cells from sea-water samples. Removal of the bands from the density gradient, followed by PCR amplification and DGGE analyses showed distinct differences in the PCR amplicon diversity associated with the active and non-active cell fractions; an indication of changes in bacterial community structure in response to the addition of oxidisable substrate. Thus, based on their in situ respiration potential, the approach enables the cytochemical enrichment and molecular characterisation of mixed bacterial populations in natural environments.

Relationships between culturability, activity and virulence in pathogenic bacteria.

While it is clear that the cells of many culturable pathogenic bacteria may become nonculturable but retain some cytological indices of activity and integrity, the potential for such cells to cause human disease is far from certain. Here we discuss both results and practical considerations relating to this issue. We conclude that there are no available cytological tests that satisfactorily predict whether cells have infective potential. In contrast, we recognize that nonculturable cells of pathogenic bacteria can retain substantial physiological activity, including the capacity to synthesize toxins. However, the clinical significance of these phenomena is yet to be established.

Bacterial viability and culturability.

Renewed interest in the relationships between viability and culturability in bacteria stems from three sources: (1) the recognition that there are many bacteria in the biosphere that have never been propagated or characterized in laboratory culture; (2) the proposal that some readily culturable bacteria may respond to certain stimuli by entering a temporarily non-culturable state termed 'viable but non-culturable' (VBNC) by some authors; and (3) the development of new techniques that facilitate demonstration of activity, integrity and composition of non-culturable bacterial cells. We review the background to these areas of interest emphasizing the view that, in an operational context, the term VBNC is self-contradictory (Kell et al., 1998) and the likely distinctions between temporarily non-culturable bacteria and those that have never been cultured. We consider developments in our knowledge of physiological processes in bacteria that may influence the outcome of a culturability test (injury and recovery, ageing, adaptation and differentiation, substrate-accelerated death and other forms of metabolic self-destruction, prophages, toxin-antitoxin systems and cell-to-cell communication). Finally, we discuss whether it is appropriate to consider the viability of individual bacteria or whether, in some circumstances, it may be more appropriate to consider viability as a property of a community of bacteria.

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.

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.

A sandwich hybridization assay employing enzyme amplification for termination of specific ribosomal RNA from unpurified cell lysates.

We have employed the power of the cyclic NAD-based enzyme amplification system to the determination of 16S rRNA. This generally applicable system employs two oligonucleotide probes, one of which is captured on a microtiter well surface and the other labeled with alkaline phosphatase. The detection of very low levels of hybridization of the capture probe is then achieved by the means of the ultrasensitive enzyme-amplified assay system, resulting in a highly sensitive, convenient, and rapid technology which can be directly employed on unpurified samples. We have been able to demonstrate the detection of 20 amol (10(7) molecules) of pure rRNA, and specific signals from as few as 2000 bacterial cells have also been demonstrated. The total procedural time can be short-5 to 18 h-depending on the dynamic range and sensitivity required. RNA target in the range of 10(12)-10(8) molecules can be assayed within 5 h. Extending the substrate incubation time enables between 10(11) and 10(7) molecules to be determined within 18 h. The system has great potential use with respect to studying the distribution and physiological states of cellular organisms.

Transient increases in colony counts observed in declining populations of Campylobacter jejuni held at low temperature.

Colony forming unit counts of Campylobacter jejuni were serially determined in a variety of microcosms in which growth was not expected. Unremitting decline in colony counts occurred in nutrient-free systems, however, transient increases were observed in human faecal emulsions and nutrient media on storage at between 1 and 25 degrees C. The phenomenon, which was more pronounced at lower temperatures, could not be attributed to sampling errors, cell clumping or the influence of minor fluctuations in experimental conditions. C. jejuni is capable of either growth at low temperatures or transition between temporarily nonculturable and culturable states.

Biofilms in drinking water systems: a possible reservoir for Helicobacter pylori.

A laboratory model system was utilised to investigate the persistence of Helicobacter pylori in mixed-species heterotrophic biofilms. A single-stage continuous culture vessel was linked to a modified-Robbins device (mRD) incorporating removable stainless steel coupons. The system was innoculated with H. pylori (NCTC 11637) and the fate of the organism monitored by polymerase chain reaction (PCR) analysis. Helicobacter pylori was detected in biofilm material for a period of up to 192 h. Theoretical washout would have occurred at around 48 h thus detection of H. pylori for a prolonged period after theoretical washout suggested that the organism possessed the ability to persist in the mixed-species heterotrophic biofilm. Preliminary studies using heat-inactivated H. pylori showed that the organism was not detected in biofilm material at any time post-challenge suggesting that the persistence of H. pylori in such material was a phenomenon requiring the organism to be in a viable state. Further investigations to assess the biological basis for the association of H. pylori with drinking water biofilms and the risk that this may pose to public health are being undertaken.

A highly discriminatory method for the direct comparison of two closely related bacterial populations by pyrolysis mass spectrometry.

The ability of pyrolysis mass spectrometry (Py-MS) to discriminate between paired groups of closely related bacteria has been examined. The technique was challenged with increasing levels of biological difference; from that between different subcultures of the same isolate of Staphylococcus aureus, through that between separate isolates of the same strain and eventually to that between different staphylococcal species and Streptococcus pyogenes. By using an analytical method in which the spectral data from any two groups can be directly compared at a time, it was shown that Py-MS was capable of measuring statistically significant differences between staphylococci and Streptococcus pyogenes, between different Staphylococcus species and between two isolates of the same strain of S. aureus from different sources. Two subcultures of the same isolate were found to be indistinguishable. The differing magnitude of the Py-MS-derived differences corresponded to the "biological differences", being much larger between staphylococci and streptococci than between staphylococcal species and only just statistically significant between isolates of the same strain from different sources. The technique described allows the assessment of the magnitude and significance of Py-MS-derived differences between any two bacterial populations.