Understanding the structure and dynamics of bacteria-phage infection networks

As rising levels of antibiotic resistance limit treatment options against bacterial infections, phage therapy offers a promising alternative. However, as bacteria and phage have co-evolved for millennia within natural microbial communities, where lytic phages naturally regulate bacterial density, a diverse set of phage resistance mechanisms exist. Here, I explore how the structure of bacteria-phage communities influences the evolution of phage resistance, and how the evolutionary principles which shape these communities may be exploited to improve the rational design of phage therapeutics. Using network analysis to assess how environmental conditions influence the structure of bacteria-phage communities, I showed that imbalances in selection pressure can destabilise bacteria-phage communities and drive phage to extinction. The community structure of microbial communities is underpinned by extensive gene-gene interactions between multiple pairs of co-evolving species. By determining the breadth of cross-resistance individual resistance mutations can promote, I characterised how cross-resistance can structure a collection of phage strains, and how these interactions determine the evolution of multiple resistances. Further, I characterised how this could be exploited to limit the evolution of resistance against phage cocktails, revealing that the evolution of multi-phage resistances are influenced by the order of phage exposure, such that sequential exposure promotes accumulation of multiple strong phage-specific resistances whereas simultaneous exposure to phage pairs promotes weaker resistances. Finally, by comparing the efficacy of phage combinations of increasing diversity, I assessed the relative contributions of phage diversity, functional diversity and cross-resistance structure on the efficacy of phage cocktails. This revealed that functionally diverse phage combinations (i.e. those targeting multiple adsorption receptors) make more effective phage cocktails. These results provide insight into the fundamental evolutionary processes which determine the efficacy of phage cocktails, revealing simple concepts which may be implemented to simplify the rational design of therapeutic phage treatments, such as the maximisation of functional diversity

The pilot and evaluation of a postnatal support Group for Iraqi Women in the year following the birth of their baby

The current study involved conducting a pilot test of a culturally sensitive support group program developed to assist Iraqi women in the year following the birth of their baby (CSSG-B) in Perth, Western Australia. The aim of this study was to evaluate the social validity of the program. It was hypothesized that women involved in the program would find the program to be socially valid and culturally appropriate, and will also report lower levels of depressive symptomatology and higher levels of social support, following the group intervention. Participants were 12 Iraqi Arabic speaking women, who had a child less than 12 months of age. The program was based on Iraqi women's explanatory models (Kleinman, 1978; Di Ciano et al., 2010) of the birth and motherhood experience. Social validity ratings were obtained during the implementation of the program in order to assess the level of acceptability of the intervention. A one-group pre-test-post-test design was used to determine if depressive symptoms had decreased during the course of the intervention and social support had increased. Results indicated that Iraqi Arabic speaking women found the support group intervention acceptable and relevant and there was a significant decrease in scores on the Edinburgh Postnatal Depression Scale (EPDS) from pre-test to post-test. These results that the culturally sensitive group intervention was culturally acceptable and was associated with decreased levels of depressive symptomatology

Plasmid carriage can limit bacteria–phage coevolution

Coevolution with bacteriophages is a major selective force shaping bacterial populations and communities. A variety of both environmental and genetic factors has been shown to influence the mode and tempo of bacteria–phage coevolution. Here, we test the effects that carriage of a large conjugative plasmid, pQBR103, had on antagonistic coevolution between the bacterium Pseudomonas fluorescens and its phage, SBW25ϕ2. Plasmid carriage limited bacteria–phage coevolution; bacteria evolved lower phage-resistance and phages evolved lower infectivity in plasmid-carrying compared with plasmid-free populations. These differences were not explained by effects of plasmid carriage on the costs of phage resistance mutations. Surprisingly, in the presence of phages, plasmid carriage resulted in the evolution of high frequencies of mucoid bacterial colonies. Mucoidy can provide weak partial resistance against SBW25ϕ2, which may have limited selection for qualitative resistance mutations in our experiments. Taken together, our results suggest that plasmids can have evolutionary consequences for bacteria that go beyond the direct phenotypic effects of their accessory gene cargo

Cross-resistance is modular in bacteria-phage interactions

Phages shape the structure of natural bacterial communities and can be effective therapeutic agents. Bacterial resistance to phage infection, however, limits the usefulness of phage therapies and could destabilize community structures, especially if individual resistance mutations provide cross-resistance against multiple phages. We currently understand very little about the evolution of cross-resistance in bacteria-phage interactions. Here we show that the network structure of cross-resistance among spontaneous resistance mutants of Pseudomonas aeruginosa evolved against each of 27 phages is highly modular. The cross-resistance network contained both symmetric (reciprocal) and asymmetric (non-reciprocal) cross-resistance, forming two cross-resistance modules defined by high within- but low between-module cross-resistance. Mutations conferring cross-resistance within-modules targeted either lipopolysaccharide or type-IV pilus biosynthesis, suggesting that the modularity of cross-resistance was structured by distinct phage receptors. In contrast, between-module cross-resistance was provided by mutations affecting the alternative sigma factor, RpoN, which controls many lifestyle-associated functions, including motility, biofilm formation and quorum sensing. Broader cross-resistance range was not associated with higher fitness costs or weaker resistance against the focal phage used to select resistance. However, mutations in rpoN, providing between-module cross-resistance, were associated with higher fitness costs than mutations associated with within-module cross-resistance, that is, in genes encoding either lipopolysaccharide or type-IV pilus biosynthesis. The observed structure of cross-resistance predicted both the frequency of resistance mutations and the ability of phage combinations to suppress bacterial growth. These findings suggest that the evolution of cross-resistance is common, is likely to play an important role in the dynamic structure of bacteria-phage communities, and could inform the design principles for phage therapy treatments

Plasmid fitness costs are caused by specific genetic conflicts

Plasmids play an important role in bacterial genome evolution by transferring genes between lineages. Fitness costs associated with plasmid acquisition are expected to be a barrier to gene exchange, but the causes of plasmid fitness costs are poorly understood. Single compensatory mutations are often sufficient to completely ameliorate plasmid fitness costs, suggesting that such costs are caused by specific genetic conflicts rather than generic properties of plasmids, such as their size, metabolic burden, or expression level. Here we show — using a combination of experimental evolution, reverse genetics, and transcriptomics — that fitness costs of two divergent large plasmids in Pseudomonas fluorescens are caused by inducing maladaptive expression of a chromosomal tailocin toxin operon. Mutations in single genes unrelated to the toxin operon, and located on either the chromosome or the plasmid, ameliorated the disruption associated with plasmid acquisition. We identify one of these compensatory loci, the chromosomal gene PFLU4242 , as the key mediator of the fitness costs of both plasmids, with the other compensatory loci either reducing expression of this gene or mitigating its deleterious effects by upregulating a putative plasmid-borne ParAB operon. The chromosomal mobile genetic element Tn6291, which uses plasmids for transmission, remained upregulated even in compensated strains, suggesting that mobile genetic elements communicate through pathways independent of general physiological disruption. Plasmid fitness costs caused by specific genetic conflicts are unlikely to act as a long-term barrier to horizontal gene transfer due to their propensity for amelioration by single compensatory mutations, explaining why plasmids are so common in bacterial genomes

Identification of a novel zinc metalloprotease through a global analysis of clostridium difficile extracellular proteins

Clostridium difficile is a major cause of infectious diarrhea worldwide. Although the cell surface proteins are recognized to be important in clostridial pathogenesis, biological functions of only a few are known. Also, apart from the toxins, proteins exported by C. difficile into the extracellular milieu have been poorly studied. In order to identify novel extracellular factors of C. difficile, we analyzed bacterial culture supernatants prepared from clinical isolates, 630 and R20291, using liquid chromatography-tandem mass spectrometry. The majority of the proteins identified were non-canonical extracellular proteins. These could be largely classified into proteins associated to the cell wall (including CWPs and extracellular hydrolases), transporters and flagellar proteins. Seven unknown hypothetical proteins were also identified. One of these proteins, CD630_28300, shared sequence similarity with the anthrax lethal factor, a known zinc metallopeptidase. We demonstrated that CD630_28300 (named Zmp1) binds zinc and is able to cleave fibronectin and fibrinogen in vitro in a zinc-dependent manner. Using site-directed mutagenesis, we identified residues important in zinc binding and enzymatic activity. Furthermore, we demonstrated that Zmp1 destabilizes the fibronectin network produced by human fibroblasts. Thus, by analyzing the exoproteome of C. difficile, we identified a novel extracellular metalloprotease that may be important in key steps of clostridial pathogenesis

Plasmid fitness costs are caused by specific genetic conflicts enabling resolution by compensatory mutation

Plasmids play an important role in bacterial genome evolution by transferring genes between lineages. Fitness costs associated with plasmid carriage are expected to be a barrier to gene exchange, but the causes of plasmid fitness costs are poorly understood. Single compensatory mutations are often sufficient to completely ameliorate plasmid fitness costs, suggesting that such costs are caused by specific genetic conflicts rather than generic properties of plasmids, such as their size, metabolic burden, or gene expression level. By combining the results of experimental evolution with genetics and transcriptomics, we show here that fitness costs of 2 divergent large plasmids in Pseudomonas fluorescens are caused by inducing maladaptive expression of a chromosomal tailocin toxin operon. Mutations in single genes unrelated to the toxin operon, and located on either the chromosome or the plasmid, ameliorated the disruption associated with plasmid carriage. We identify one of these compensatory loci, the chromosomal gene PFLU4242, as the key mediator of the fitness costs of both plasmids, with the other compensatory loci either reducing expression of this gene or mitigating its deleterious effects by up-regulating a putative plasmid-borne ParAB operon. The chromosomal mobile genetic element Tn6291, which uses plasmids for transmission, remained up-regulated even in compensated strains, suggesting that mobile genetic elements communicate through pathways independent of general physiological disruption. Plasmid fitness costs caused by specific genetic conflicts are unlikely to act as a long-term barrier to horizontal gene transfer (HGT) due to their propensity for amelioration by single compensatory mutations, helping to explain why plasmids are so common in bacterial genomes.</jats:p

Evaluation of the Stability of Bacteriophages in Different Solutions Suitable for the Production of Magistral Preparations in Belgium

In Belgium, the incorporation of phages into magistral preparations for human application has been permitted since 2018. The stability of such preparations is of high importance to guarantee quality and efficacy throughout treatments. We evaluated the ability to preserve infectivity of four different phages active against three different bacterial species in five different buffer and infusion solutions commonly used in medicine and biotechnological manufacturing processes, at two different concentrations (9 and 7 log pfu/mL), stored at 4 °C. DPBS without Ca2+ and Mg2+ was found to be the best option, compared to the other solutions. Suspensions with phage concentrations of 7 log pfu/mL were unsuited as their activity dropped below the effective therapeutic dose (6-9 log pfu/mL), even after one week of storage at 4 °C. Strong variability between phages was observed, with Acinetobacter baumannii phage Acibel004 being stable in four out of five different solutions. We also studied the long term storage of lyophilized staphylococcal phage ISP, and found that the titer could be preserved during a period of almost 8 years when sucrose and trehalose were used as stabilizers. After rehydration of the lyophilized ISP phage in saline, the phage solutions remained stable at 4 °C during a period of 126 days

Ecological conditions determine extinction risk in co-evolving bacteria-phage populations.

BACKGROUND: Antagonistic coevolution between bacteria and their viral parasites, phage, drives continual evolution of resistance and infectivity traits through recurrent cycles of adaptation and counter-adaptation. Both partners are vulnerable to extinction through failure of adaptation. Environmental conditions may impose unequal abiotic selection pressures on each partner, destabilising the coevolutionary relationship and increasing the extinction risk of one partner. In this study we explore how the degree of population mixing and resource supply affect coevolution-induced extinction risk by coevolving replicate populations of Pseudomonas fluorescens SBW25 with its associated lytic phage SBW25Ф2 under four treatment regimens incorporating low and high resource availability with mixed or static growth conditions. RESULTS: We observed an increased risk of phage extinction under population mixing, and in low resource conditions. High levels of evolved bacterial resistance promoted phage extinction at low resources under both mixed and static conditions, whereas phage populations could survive when phage susceptible bacterial genotypes rose to high frequency. CONCLUSIONS: These findings demonstrate that phage extinction risk is influenced by multiple abiotic conditions, which together act to destabilise the bacteria-phage coevolutionary relationship. The risk of coevolution-induced extinction is therefore dependent on the ecological context

Multicenter European Prevalence Study of Neurocognitive Impairment and Associated Factors in HIV Positive Patients

We conducted a cross-sectional study in 448 HIV positive patients attending five European outpatient clinics to determine prevalence of and factors associated with neurocognitive impairment (NCI) using computerized and pen-and-paper neuropsychological tests. NCI was defined as a normalized Z score ≤-1 in at least 2 out of 5 cognitive domains. Participants' mean age was 45.8 years; 84% male; 87% white; 56% university educated; median CD4 count 550 cells/mm(3); 89% on antiretroviral therapy. 156 (35%) participants had NCI, among whom 26 (17%; 5.8% overall) reported a decline in activities of daily living. Prevalence of NCI was lower in those always able to afford basic needs (adjusted prevalence ratio [aPR] 0.71, 95% confidence interval [CI] 0.54-0.94) or with a university education (aPR 0.72, 95% CI 0.54-0.97) and higher in those with severe depressive symptoms (aPR 1.53, 95% CI 1.09-2.14) or a significant comorbid condition (aPR 1.40, 95% CI 1.03-1.90)