The Opportunistic Pathogen Propionibacterium acnes: Insights into Typing, Human Disease, Clonal Diversification and CAMP Factor Evolution

We previously described a Multilocus Sequence Typing (MLST) scheme based on eight genes that facilitates population genetic and evolutionary analysis of P. acnes. While MLST is a portable method for unambiguous typing of bacteria, it is expensive and labour intensive. Against this background, we now describe a refined version of this scheme based on two housekeeping (aroE; guaA) and two putative virulence (tly; camp2) genes (MLST) that correctly predicted the phylogroup (IA, IA, IB, IC, II, III), clonal complex (CC) and sequence type (ST) (novel or described) status for 91% isolates (n = 372) via cross-referencing of the four gene allelic profiles to the full eight gene versions available in the MLST database (http://pubmlst.org/pacnes/). Even in the small number of cases where specific STs were not completely resolved, the MLST method still correctly determined phylogroup and CC membership. Examination of nucleotide changes within all the MLST loci provides evidence that point mutations generate new alleles approximately 1.5 times as frequently as recombination; although the latter still plays an important role in the bacterium's evolution. The secreted/cell-associated 'virulence' factors tly and camp2 show no clear evidence of episodic or pervasive positive selection and have diversified at a rate similar to housekeeping loci. The co-evolution of these genes with the core genome might also indicate a role in commensal/normal existence constraining their diversity and preventing their loss from the P. acnes population. The possibility that members of the expanded CAMP factor protein family, including camp2, may have been lost from other propionibacteria, but not P. acnes, would further argue for a possible role in niche/host adaption leading to their retention within the genome. These evolutionary insights may prove important for discussions surrounding camp2 as an immunotherapy target for acne, and the effect such treatments may have on commensal lineages. © 2013 McDowell et al.

Within-host evolution of Burkholderia pseudomallei over a twelve-year chronic carriage infection

UNLABELLED: Burkholderia pseudomallei causes the potentially fatal disease melioidosis. It is generally accepted that B. pseudomallei is a noncommensal bacterium and that any culture-positive clinical specimen denotes disease requiring treatment. Over a 23-year study of melioidosis cases in Darwin, Australia, just one patient from 707 survivors has developed persistent asymptomatic B. pseudomallei carriage. To better understand the mechanisms behind this unique scenario, we performed whole-genome analysis of two strains isolated 139 months apart. During this period, B. pseudomallei underwent several adaptive changes. Of 23 point mutations, 78% were nonsynonymous and 43% were predicted to be deleterious to gene function, demonstrating a strong propensity for positive selection. Notably, a nonsense mutation inactivated the universal stress response sigma factor RpoS, with pleiotropic implications. The genome underwent substantial reduction, with four deletions in chromosome 2 resulting in the loss of 221 genes. The deleted loci included genes involved in secondary metabolism, environmental survival, and pathogenesis. Of 14 indels, 11 occurred in coding regions and 9 resulted in frameshift mutations that dramatically affected predicted gene products. Disproportionately, four indels affected lipopolysaccharide biosynthesis and modification. Finally, we identified a frameshift mutation in both P314 isolates within wcbR, an important component of the capsular polysaccharide I locus, suggesting virulence attenuation early in infection. Our study illustrates a unique clinical case that contrasts a high-consequence infectious agent with a long-term commensal infection and provides further insights into bacterial evolution within the human host.

IMPORTANCE: Some bacterial pathogens establish long-term infections that are difficult or impossible to eradicate with current treatments. Rapid advances in genome sequencing technologies provide a powerful tool for understanding bacterial persistence within the human host. Burkholderia pseudomallei is considered a highly pathogenic bacterium because infection is commonly fatal. Here, we document within-host evolution of B. pseudomallei in a unique case of human infection with ongoing chronic carriage. Genomic comparison of isolates obtained 139 months (11.5 years) apart showed a strong signal of adaptation within the human host, including inactivation of virulence and immunogenic factors, and deletion of pathways involved in environmental survival. Two global regulatory genes were mutated in the 139-month isolate, indicating extensive regulatory changes favoring bacterial persistence. Our study provides insights into B. pseudomallei pathogenesis and, more broadly, identifies parallel evolutionary mechanisms that underlie chronic persistence of all bacterial pathogens.

A spatio-temporal contrast of the predatory impact of an invasive freshwater crustacean

Aim: Impacts of invasive species may vary across invasion gradients, owing to trait-based sorting of individuals through dispersal: those aggregating at invasion fronts may be more aggressive and voracious. We examine, in the field and laboratory, variation in the predatory impacts of an invasive Ponto-Caspian crustacean Hemimysis anomala G.O. Sars, 1907 at two sites along a spatio-temporal gradient of invasion.

Location: Republic of Ireland.

Methods: We used reciprocal transplant field-deployed mesocosms to compare predation rates of invasion front and well-established H. anomala on natural zooplankton assemblages. In the laboratory, we measured the functional response (relationship between predation rate and prey supply) of H. anomala from both sites, for a per capita mechanistic comparison of predation efficiency. We also assessed prey selectivity of H. anomala in the mesocosm experiments to further compare feeding behaviour. Finally, we used a correlative approach to assess the community impact of H. anomala across sites, including a nearby uninvaded site, by comparing zooplankton diversities and densities.

Results: Invasion front H. anomala had higher predation rates than well-established H. anomala at high in situ zooplankton densities. Invasion front H. anomala also had higher functional responses - in particular showing higher 'attack rates' - indicating a heightened ability to locate and capture prey. Prey selectivity was consistent across the spatio-temporal contrast, with positive selection for cladocerans. Zooplankton diversity and density declined with time since H. anomala invasion, both being maximal at the uninvaded site.

Main conclusions: Our study, for the first time, (1) reveals differences in predatory per capita effects and associated behavioural traits between two sites along a spatio-temporal invasion gradient and (2) shows a negative community-level impact of the invasive H. anomala in natural water bodies. Further spatio-temporal comparisons of predatory per capita effects of invaders are needed to assess the generality of these results.

Stock structure of Atlantic herring Clupea harengus in the Norwegian Sea and adjacent waters

The genetic structure of Atlantic herring Clupea harengus L. was investigated in its north-easterly distribution in the Norwegian Sea and adjacent waters, using 23 neutral and one non-neutral (Cpa111) microsatellite loci. Fish from the suspected 2 main populations - the Norwegian spring-spawning herring (NSSH) and the Icelandic summer-spawning herring (ISSH) - were collected at spawning locations in their respective spawning seasons from 2009 to 2012. Samples were also collected from Norwegian autumn spawning locations, from different local Norwegian fjords such as the inner part of Trondheimsfjorden, Lindås pollene, Landvikvannet and Lusterfjorden, as well as from suspected Faroese spawning components. The observed level of genetic differentiation was significant but low (F_ST = 0.007) and mostly attributable to the differentiation of the local Norwegian fjord populations. The locus Cpa111, which was detected to putatively be under positive selection, exhibited the highest FST value (0.044). The observed genetic patterns were robust to exclusion of this locus. Landvikvannet herring was also genetically distinguishable from the 3 other fjord populations. In addition, the present study does not support genetic structuring among the ISSH and the NSSH.

Comparison of techniques to screen and characterize bacteria-specific hybridomas for high-quality monoclonal antibodies selection

Antibodies are are very important materials for diagnostics. A rapid and simple hybridoma screening method will help in delivering specific monoclonal antibodies. In this study, we systematically developed the first antibody array to screen for bacteria-specific monoclonal antibodies using Listeria monocytogenes as a bacteria model. The antibody array was developed to expedite the hybridoma screening process by printing hybridoma supernatants on a glass slide coated with an antigen of interest. This screening method is based on the binding ability of supernatants to the coated antigen. The bound supernatants were detected by a fluorescently labeled anti-mouse immunoglobulin. Conditions (slide types, coating, spotting, and blocking buffers) for antibody array construction were optimized. To demonstrate its usefulness, antibody array was used to screen a sample set of 96 hybridoma supernatants in comparison to ELISA. Most of the positive results identified by ELISA and antibody array methods were in agreement except for those with low signals that were undetectable by antibody array. Hybridoma supernatants were further characterized with surface plasmon resonance to obtain additional data on the characteristics of each selected clone. While the antibody array was slightly less sensitive than ELISA, a much faster and lower cost procedure to screen clones against multiple antigens has been demonstrated. (C) 2011 Elsevier Inc. All rights reserved.

Variable selection via RIVAL and its application in nuclear material detection

In many situations, the number of data points is fixed, and the asymptotic convergence results of popular model selection tools may not be useful. A new algorithm for model selection, RIVAL (removing irrelevant variables amidst Lasso iterations), is presented and shown to be particularly effective for a large but fixed number of data points. The algorithm is motivated by an application of nuclear material detection where all unknown parameters are to be non-negative. Thus, positive Lasso and its variants are analyzed. Then, RIVAL is proposed and is shown to have some desirable properties, namely the number of data points needed to have convergence is smaller than existing methods.

Using interviewer random effects to remove selection bias from HIV prevalence estimates

Background: Selection bias in HIV prevalence estimates occurs if non-participation in testing is correlated with HIV status. Longitudinal data suggests that individuals who know or suspect they are HIV positive are less likely to participate in testing in HIV surveys, in which case methods to correct for missing data which are based on imputation and observed characteristics will produce biased results. Methods: The identity of the HIV survey interviewer is typically associated with HIV testing participation, but is unlikely to be correlated with HIV status. Interviewer identity can thus be used as a selection variable allowing estimation of Heckman-type selection models. These models produce asymptotically unbiased HIV prevalence estimates, even when non-participation is correlated with unobserved characteristics, such as knowledge of HIV status. We introduce a new random effects method to these selection models which overcomes non-convergence caused by collinearity, small sample bias, and incorrect inference in existing approaches. Our method is easy to implement in standard statistical software, and allows the construction of bootstrapped standard errors which adjust for the fact that the relationship between testing and HIV status is uncertain and needs to be estimated. Results: Using nationally representative data from the Demographic and Health Surveys, we illustrate our approach with new point estimates and confidence intervals (CI) for HIV prevalence among men in Ghana (2003) and Zambia (2007). In Ghana, we find little evidence of selection bias as our selection model gives an HIV prevalence estimate of 1.4% (95% CI 1.2% – 1.6%), compared to 1.6% among those with a valid HIV test. In Zambia, our selection model gives an HIV prevalence estimate of 16.3% (95% CI 11.0% - 18.4%), compared to 12.1% among those with a valid HIV test. Therefore, those who decline to test in Zambia are found to be more likely to be HIV positive. Conclusions: Our approach corrects for selection bias in HIV prevalence estimates, is possible to implement even when HIV prevalence or non-participation is very high or very low, and provides a practical solution to account for both sampling and parameter uncertainty in the estimation of confidence intervals. The wide confidence intervals estimated in an example with high HIV prevalence indicate that it is difficult to correct statistically for the bias that may occur when a large proportion of people refuse to test.