Counting Mycobacteria in Infected Human Cells and Mouse Tissue: A Comparison between qPCR and CFU

Pathak, Sharad; Awuh, Jane Atesoh; Leversen, Nils Anders; Flo, Trude Helen; Åsjö, Birgitta

doi:10.1371/journal.pone.0034931

Cited by 46 publications

(29 citation statements)

References 22 publications

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“…Although this variable does not only represent true bacterial viability, but also bacterial burden determined by the differences in culture and PCR sensitivity, the fact is that it might roughly indicate the amounts of live bacteria that are available for transmission according to PTB form. In any case, the results presented in this study would provide a reference of what can be expected in terms of infectivity if the lengthy and labor-intensive traditional isolation methods are replaced by the faster molecular PCR tests (Pathak et al, 2012). From a clinical and epidemiological perspective, the distribution of each of these two PTB forms through age seems to point out to different levels of production damage .…”

Section: Discussionmentioning

confidence: 78%

Latent infections are the most frequent form of paratuberculosis in slaughtered Friesian cattle

Vázquez¹,

Garrido²,

Molina³

et al. 2014

Span. j. agric. res.

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Paratuberculosis is a chronic mycobacterial infection causing granulomatous enteritis in ruminants, whose pathogenesis and epidemiology poses numerous challenges, including latency and reactivation. The most recent and complete classification of paratuberculosis immunopathological types in cattle recognized five categories. In this study, 1031 slaughtered Friesian cattle were submitted to serological, microbiological and pathological examinations with the aim of maximizing the rate of case detection. In most cases, infected animals had minimal lesions and almost no other proof of infection (38.9%), while the more characteristic types with the whole constellation of microbiological and immunological evidences accounted for a lower proportion (7.7%). As these findings in cattle suggest similarities with the epidemiology of tuberculosis in humans, we propose to re-group the original immunopathological types into two broader paratuberculosis epidemio-pathogenic forms or states: latent and patent. The former term would define infections with focal lesions and might constitute an apparent resilience status representing a difficult to detect reservoir of infection whose role could become critical if later immune-compromising factors lead to re-activation. The latter would group those cases with multifocal and diffuse inflammatory lesions with higher mycobacterial load and viability suggestive of a more immediate epidemiological risk. Interestingly, the relative frequency of presentation of each profile varied with age. The proportion of latent forms remained relatively constant between 33.8% and 54.3% through adulthood from 3 years of age, while patent forms were more frequent during the first years of age and tended to decrease among the oldest individuals.

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Section: Discussionmentioning

confidence: 78%

Latent infections are the most frequent form of paratuberculosis in slaughtered Friesian cattle

Vázquez¹,

Garrido²,

Molina³

et al. 2014

Span. j. agric. res.

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“…The total unbound lipid extracts from before the biphasic separation were subjected to MALDI-TOF MS. Two microliters of the lipid extract was dried on a steel MALDI-TOF MS sample target on a heat plate at 45°C. This was overlain with the matrix solution (1 l of saturated ␣-cyano-4-hydroxycinnamic acid [CHCA] in (18,19). We chose to do qPCR of the 16S rRNA gene because it is highly conserved and only a single copy exists in the M. abscessus genome.…”

Section: Multilocus Genotyping Of the Clinical Isolatesmentioning

confidence: 99%

Clonal Diversification and Changes in Lipid Traits and Colony Morphology in Mycobacterium abscessus Clinical Isolates

et al. 2015

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fThe smooth-to-rough colony morphology shift in Mycobacterium abscessus has been implicated in loss of glycopeptidolipid (GPL), increased pathogenicity, and clinical decline in cystic fibrosis (CF) patients. However, the evolutionary phenotypic and genetic changes remain obscure. Serial isolates from nine non-CF patients with persistent M. abscessus infection were characterized by colony morphology, lipid profile via thin-layer chromatography and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), sequencing of eight genes in the GPL locus, and expression level of fadD23, a key gene involved in the biosynthesis of complex lipids. All 50 isolates were typed as M. abscessus subspecies abscessus and were clonally related within each patient. Rough isolates, all lacking GPL, predominated at later disease stages, some showing variation within rough morphology. While most (77%) rough isolates harbored detrimental mutations in mps1 and mps2, 13% displayed previously unreported mutations in mmpL4a and mmpS4, the latter yielding a putative GPL precursor. Two isolates showed no deleterious mutations in any of the eight genes sequenced. Mixed populations harboring different GPL locus mutations were detected in 5 patients, demonstrating clonal diversification, which was likely overlooked by conventional acid-fast bacillus ( MABSC causes serious chronic pulmonary infections in those with underlying conditions such as bronchiectasis or cystic fibrosis (CF). In the United States and some other parts of the world, it is the second most frequent pulmonary nontuberculous mycobacterial (NTM) infection (28%), after Mycobacterium avium complex, and its prevalence is increasing (6, 7). MABSC causes significant mortality in the CF population. In Scandinavian countries, one-fourth of CF patients who have persistently positive MABSC sputum cultures have received lung transplantation or died (8).Glycopeptidolipid (GPL) is the most abundant glycolipid in the outer cell envelope of MABSC, and its synthesis and transport are controlled by the GPL locus (9). A growing number of studies have linked the rough colony morphology in MABSC to loss of GPL, increased pathogenicity, and clinical decline in CF patients (10)(11)(12)(13)(14)(15)(16). While most studies of persistent MABSC infection focused on CF patients, less is known about MABSC in the non-CF population regarding presence of mixed populations, clonal relatedness, and phenotypic and genotypic changes of strains collected over time.We have very limited understanding of the evolutionary phenotypic and genetic changes occurring in MABSC during persistent human lung infection. We lack the fundamental knowledge needed to identify selective pressures associated with rough morphology and, once identified, how to reverse them. To address these questions, we collected serial clinical isolates of M. abscessus from non-CF patients with persistent pulmonary infection who were followed for 5 years or more and assessed strain relatedness and diversity, colony...

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“…However, only platable, replicating bacteria are detected. Additionally, the plating method has considerable technical variation, and other methods need to be considered for enumerating bacteria in complex samples (149). In infected cells the multiplicity of infection varies with infection efficiency and replication post-infection, and mycobacterial load varies considerably in human lesions (150).…”

Section: Direct Quantification Of Mycobacterial Proteins In Infected mentioning

confidence: 99%

Mycobacterium tuberculosisin the Proteomics Era

et al. 2014

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The emerging field of proteomics has contributed greatly to improving our understanding of the human pathogen Mycobacterium tuberculosis over the last two decades. In this chapter we provide a comprehensive overview of mycobacterial proteome research and highlight key findings. First, studies employing a combination of two-dimensional gel electrophoresis and mass spectrometry (MS) provided insights into the proteomic composition, initially of the whole bacillus and subsequently of subfractions, such as the cell wall, cytosol, and secreted proteins. Comparison of results obtained under various culture conditions, i.e., acidic pH, nutrient starvation, and low oxygen tension, aiming to mimic facets of the intracellular lifestyle of M. tuberculosis, provided initial clues to proteins relevant for intracellular survival and manipulation of the host cell. Further attempts were aimed at identifying the biological functions of the hypothetical M. tuberculosis proteins, which still make up a quarter of the gene products of M. tuberculosis, and at characterizing posttranslational modifications. Recent technological advances in MS have given rise to new methods such as selected reaction monitoring (SRM) and data-independent acquisition (DIA). These targeted, cutting-edge techniques combined with a public database of specific MS assays covering the entire proteome of M. tuberculosis allow the simple and reliable detection of any mycobacterial protein. Most recent studies attempt not only to identify but also to quantify absolute amounts of single proteins in the complex background of host cells without prior sample fractionation or enrichment. Finally, we will discuss the potential of proteomics to advance vaccinology, drug discovery, and biomarker identification to improve intervention and prevention measures for tuberculosis.

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Counting Mycobacteria in Infected Human Cells and Mouse Tissue: A Comparison between qPCR and CFU

Cited by 46 publications

References 22 publications

Latent infections are the most frequent form of paratuberculosis in slaughtered Friesian cattle

Latent infections are the most frequent form of paratuberculosis in slaughtered Friesian cattle

Clonal Diversification and Changes in Lipid Traits and Colony Morphology in Mycobacterium abscessus Clinical Isolates

Mycobacterium tuberculosisin the Proteomics Era

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