Inference of symbiotic adaptations in nature using experimental evolution.

Microbes must adapt to the presence of other species, but it can be difficult to recreate the natural context for these interactions in the laboratory. We describe a method for inferring the existence of symbiotic adaptations by experimentally evolving microbes that would normally interact in an artificial environment without access to other species. By looking for changes in the fitness effects microbes adapted to isolation have on their partners, we can infer the existence of ancestral adaptations that were lost during experimental evolution. The direction and magnitude of trait changes can offer useful insight as to whether the microbes have historically been selected to help or harm one another in nature. We apply our method to the complex symbiosis between the social amoeba Dictyostelium discoideum and two intracellular bacterial endosymbionts, Paraburkholderia agricolaris and Paraburkholderia hayleyella. Our results suggest P. hayleyella-but not P. agricolaris-has generally been selected to attenuate its virulence in nature, and that D. discoideum has evolved to antagonistically limit the growth of Paraburkholderia. The approach demonstrated here can be a powerful tool for studying adaptations in microbes, particularly when the specific natural context in which the adaptations evolved is unknown or hard to reproduce.

A chronic strain of the cystic fibrosis pathogen Pandoraea pulmonicola expresses a heterogenous hypo-acylated lipid A.

Pandoraea sp. is an emerging Gram-negative pathogen in cystic fibrosis causing severe and persistent inflammation and damage of the lungs. The molecular mechanisms underlying the high pathogenicity of Pandoraea species are still largely unknown. As Gram-negatives, Pandoraea sp. express lipopolysaccharides (LPS) whose recognition by the host immune system triggers an inflammatory response aimed at the bacterial eradication from the infected tissues. The degree of the inflammatory response strongly relies on the fine structure of the LPS and, in particular, of its glycolipid moiety, i.e. the lipid A. Here we report the structure of the lipid A isolated from the LPS of a chronic strain of P. pulmonicola (RL 8228), one of the most virulent identified so far among the Pandoraea species. Our data demonstrated that the examined chronic strain produces a smooth-type LPS with a complex mixture of hypoacylated lipid A species displaying, among other uncommon characteristics, the 2-hydroxylation of some of the acyl chains and the substitution by an additional glucosamine on one or both the phosphate groups.

Paraburkholderia Symbionts Display Variable Infection Patterns That Are Not Predictive of Amoeba Host Outcomes.

Symbiotic interactions exist within a parasitism to mutualism continuum that is influenced, among others, by genes and context. Dynamics of intracellular invasion, replication, and prevalence may underscore both host survivability and symbiont stability. More infectious symbionts might exert higher corresponding costs to hosts, which could ultimately disadvantage both partners. Here, we quantify infection patterns of diverse Paraburkholderia symbiont genotypes in their amoeba host Dictyostelium discoideum and probe the relationship between these patterns and host outcomes. We exposed D. discoideum to thirteen strains of Paraburkholderia each belonging to one of the three symbiont species found to naturally infect D. discoideum: Paraburkholderia agricolaris, Paraburkholderia hayleyella, and Paraburkholderia bonniea. We quantified the infection prevalence and intracellular density of fluorescently labeled symbionts along with the final host population size using flow cytometry and confocal microscopy. We find that infection phenotypes vary across symbiont strains. Symbionts belonging to the same species generally display similar infection patterns but are interestingly distinct when it comes to host outcomes. This results in final infection loads that do not strongly correlate to final host outcomes, suggesting other genetic factors that are not a direct cause or consequence of symbiont abundance impact host fitness.

Microbiology of Cystic Fibrosis Airway Disease.

Although survival of individuals with cystic fibrosis (CF) has been continuously improving for the past 40 years, respiratory failure secondary to recurrent pulmonary infections remains the leading cause of mortality in this patient population. Certain pathogens such as Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and species of the Burkholderia cepacia complex continue to be associated with poorer clinical outcomes including accelerated lung function decline and increased mortality. In addition, other organisms such as anaerobes, viruses, and fungi are increasingly recognized as potential contributors to disease progression. Culture-independent molecular methods are also being used for diagnostic purposes and to examine the interaction of microorganisms in the CF airway. Given the importance of CF airway infections, ongoing initiatives to promote understanding of the epidemiology, clinical course, and treatment options for these infections are needed.

Pneumonia due to Pandoraea Apista after evacuation of traumatic intracranial hematomas:a case report and literature review.

BACKGROUND: Pandoraea species is a newly described genus, which is multidrug resistant and difficult to identify. Clinical isolates are mostly cultured from cystic fibrosis (CF) patients. CF is a rare disease in China, which makes Pandoraea a total stranger to Chinese physicians. Pandoraea genus is reported as an emerging pathogen in CF patients in most cases. However, there are few pieces of evidence that confirm Pandoraea can be more virulent in non-CF patients. The pathogenicity of Pandoraea genus is poorly understood, as well as its treatment. The incidence of Pandoraea induced infection in non-CF patients may be underestimated and it's important to identify and understand these organisms. CASE PRESENTATION: We report a 44-years-old man who suffered from pneumonia and died eventually. Before his condition deteriorated, a Gram-negative bacilli was cultured from his sputum and identified as Pandoraea Apista by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). CONCLUSION: Pandoraea spp. is an emerging opportunistic pathogen. The incidences of Pandoraea related infection in non-CF patients may be underestimated due to the difficulty of identification. All strains of Pandoraea show multi-drug resistance and highly variable susceptibility. To better treatment, species-level identification and antibiotic susceptibility test are necessary.

The Variability of the Order Burkholderiales Representatives in the Healthcare Units.

BACKGROUND AND AIM: The order Burkholderiales became more abundant in the healthcare units since the late 1970s; it is especially dangerous for intensive care unit patients and patients with chronic lung diseases. The goal of this investigation was to reveal the real variability of the order Burkholderiales representatives and to estimate their phylogenetic relationships. METHODS: 16S rDNA and genes of the Burkholderia cenocepacia complex (Bcc) Multi Locus Sequence Typing (MLST) scheme were used for the bacteria detection. RESULTS: . A huge diversity of genome size and organization was revealed in the order Burkholderiales that may prove the adaptability of this taxon's representatives. The following variability of the Burkholderiales in Russian healthcare units has been revealed: Burkholderiaceae (Burkholderia, Pandoraea, and Lautropia), Alcaligenaceae (Achromobacter), and Comamonadaceae (Variovorax). The Burkholderia genus was the most diverse and was represented by 5 species and 16 sequence types (ST). ST709 and 728 were transmissible and often encountered in cystic fibrosis patients and in hospitals. A. xylosoxidans was estimated by 15 genotypes. The strains of first and second ones were the most numerous. CONCLUSIONS: Phylogenetic position of the genus Lautropia with smaller genome is ambiguous. The Bcc MLST scheme is applicable for all Burkholderiales representatives for resolving the epidemiological problems.

Virulence of an emerging respiratory pathogen, genus Pandoraea, in vivo and its interactions with lung epithelial cells.

Pandoraea species have emerged as opportunistic pathogens among cystic fibrosis (CF) and non-CF patients. Pandoraea pulmonicola is the predominant Pandoraea species among Irish CF patients. The objective of this study was to investigate the pathogenicity and potential mechanisms of virulence of Irish P. pulmonicola isolates and strains from other Pandoraea species. Three patients from whom the P. pulmonicola isolates were isolated have since died. The in vivo virulence of these and other Pandoraea strains was examined by determining the ability to kill Galleria mellonella larvae. The P. pulmonicola strains generally were the most virulent of the species tested, with three showing a comparable or greater level of virulence in vivo relative to another CF pathogen, Burkholderia cenocepacia, whilst strains from two other species, Pandoraea apista and Pandoraea pnomenusa, were considerably less virulent. For all Pandoraea species, whole cells were required for larval killing, as cell-free supernatants had little effect on larval survival. Overall, invasive Pandoraea strains showed comparable invasion of two independent lung epithelial cell lines, irrespective of whether they had a CF phenotype. Pandoraea strains were also capable of translocation across polarized lung epithelial cell monolayers. Although protease secretion was a common characteristic across the genus, it is unlikely to be involved in pathogenesis. In conclusion, whilst multiple mechanisms of pathogenicity may exist across the genus Pandoraea, it appears that lung cell invasion and translocation contribute to the virulence of P. pulmonicola strains.

Chronic colonization with Pandoraea apista in cystic fibrosis patients determined by repetitive-element-sequence PCR.

Pandoraea apista is recovered with increasing frequency from the lungs of patients with cystic fibrosis (CF) and may represent an emerging pathogen (I. M. Jorgensen et al., Pediatr. Pulmonol. 36:439-446, 2003). We identified two CF patients from our hospital whose sputum specimens were culture positive for P. apista over the course of several years. Repetitive-element-sequence PCR was employed to determine whether sequential isolates that were recovered from these patients represented a single clone and whether each patient had been chronically colonized with the same strain. Banding patterns generated with ERIC primers, REP primers, and BOX primers showed that individual patient isolates had a high degree of similarity (>97%) and were considered identical. However, only the banding patterns from the ERIC primers and BOX primers were able to show that the strains from patients I and II were unique (similarity indices of 79.8% and 70.0%, respectively). We concluded that all strains of P. apista from patient I were identical, as were all strains from patient II, establishing chronic colonization. Only two of the three methods employed indicate that the strains from the two patients are distinct. This implied that the organism was not transferred from one patient to the other, suggesting that the choice of methodology could generate misleading results when examining person-to-person transmission regarding this organism.

Bleeding sap and old wood are the two main sources of contamination of merging organs of vine plants by Xylophilus ampelinus, the causal agent of bacterial necrosis.

The spatial distribution of vine plants contaminated by Xylophilus ampelinus, the agent responsible for bacterial necrosis, was studied over a 5-year period within two vineyards in the Cognac area. Both vineyards were planted with Vitis vinifera cv. Ugni blanc but were different in age and agronomic location. The emission of X. ampelinus in contaminated bleeding sap was observed during vine sprouting. Contaminated bleeding sap is an important source of inoculum for external contamination due to the high susceptibility of young merging shoots to the pathogen. X. ampelinus emission by bleeding sap was not affected by the age of the plants or the location of the vineyards. However, its emission was irregular with time, and it varied between two fruit canes from individual plants and between plants as well as between years. Moreover, the two vineyards appeared to be entirely contaminated. Consequently, the behavior of the pathogen is not predictable. The distribution of the pathogen inside vine plant organs was analyzed through the four growing seasons. The old wood was contaminated throughout the year and constituted a stock inoculum for endophytic contamination of crude sap during the winter and the spring. Despite the fact that most of the young green shoots were contaminated in May, X.ampelinus was not found in green shoots in June and September, refuting the hypothesis of an epiphytic life of the pathogen under natural conditions. Although all plants were entirely contaminated in both vineyards, symptoms were rare and were observed on different plants each year.