Revealing bacteria-phage interactions in human microbiome through the CRISPR-Cas immune systems.

The human gut microbiome is composed of a diverse consortium of microorganisms. Relatively little is known about the diversity of the bacteriophage population and their interactions with microbial organisms in the human microbiome. Due to the persistent rivalry between microbial organisms (hosts) and phages (invaders), genetic traces of phages are found in the hosts' CRISPR-Cas adaptive immune system. Mobile genetic elements (MGEs) found in bacteria include genetic material from phage and plasmids, often resultant from invasion events. We developed a computational pipeline (BacMGEnet), which can be used for inference and exploratory analysis of putative interactions between microbial organisms and MGEs (phages and plasmids) and their interaction network. Given a collection of genomes as the input, BacMGEnet utilizes computational tools we have previously developed to characterize CRISPR-Cas systems in the genomes, which are then used to identify putative invaders from publicly available collections of phage/prophage sequences. In addition, BacMGEnet uses a greedy algorithm to summarize identified putative interactions to produce a bacteria-MGE network in a standard network format. Inferred networks can be utilized to assist further examination of the putative interactions and for discovery of interaction patterns. Here we apply the BacMGEnet pipeline to a few collections of genomic/metagenomic datasets to demonstrate its utilities. BacMGEnet revealed a complex interaction network of the Phocaeicola vulgatus pangenome with its phage invaders, and the modularity analysis of the resulted network suggested differential activities of the different P. vulgatus' CRISPR-Cas systems (Type I-C and Type II-C) against some phages. Analysis of the phage-bacteria interaction network of human gut microbiome revealed a mixture of phages with a broad host range (resulting in large modules with many bacteria and phages), and phages with narrow host range. We also showed that BacMGEnet can be used to infer phages that invade bacteria and their interactions in wound microbiome. We anticipate that BacMGEnet will become an important tool for studying the interactions between bacteria and their invaders for microbiome research.

Diversity and dynamics of the CRISPR-Cas systems associated with Bacteroides fragilis in human population.

BACKGROUND: CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) systems are adaptive immune systems commonly found in prokaryotes that provide sequence-specific defense against invading mobile genetic elements (MGEs). The memory of these immunological encounters are stored in CRISPR arrays, where spacer sequences record the identity and history of past invaders. Analyzing such CRISPR arrays provide insights into the dynamics of CRISPR-Cas systems and the adaptation of their host bacteria to rapidly changing environments such as the human gut. RESULTS: In this study, we utilized 601 publicly available Bacteroides fragilis genome isolates from 12 healthy individuals, 6 of which include longitudinal observations, and 222 available B. fragilis reference genomes to update the understanding of B. fragilis CRISPR-Cas dynamics and their differential activities. Analysis of longitudinal genomic data showed that some CRISPR array structures remained relatively stable over time whereas others involved radical spacer acquisition during some periods, and diverse CRISPR arrays (associated with multiple isolates) co-existed in the same individuals with some persisted over time. Furthermore, features of CRISPR adaptation, evolution, and microdynamics were highlighted through an analysis of host-MGE network, such as modules of multiple MGEs and hosts, reflecting complex interactions between B. fragilis and its invaders mediated through the CRISPR-Cas systems. CONCLUSIONS: We made available of all annotated CRISPR-Cas systems and their target MGEs, and their interaction network as a web resource at https://omics.informatics.indiana.edu/CRISPRone/Bfragilis . We anticipate it will become an important resource for studying of B. fragilis, its CRISPR-Cas systems, and its interaction with mobile genetic elements providing insights into evolutionary dynamics that may shape the species virulence and lead to its pathogenicity.

Implementing a successful proactive telephone breastfeeding peer support intervention: volunteer recruitment, training, and intervention delivery in the RUBY randomised controlled trial.

BACKGROUND: The RUBY randomised controlled trial demonstrated the benefit of proactive telephone peer support in promoting breastfeeding continuation in a setting with high breastfeeding initiation, where typically this is difficult to achieve. This paper describes the implementation and delivery of the peer support intervention with a focus on recruitment, training, and support of peer volunteers, and includes a description of the key components of the calls. METHODS: Data collection occurred between December 2012 and June 2016 in Melbourne, Australia. Volunteers completed enrolment forms at the training session and recorded data related to each call in a Call Log maintained for each mother supported. Data were summarised using descriptive statistics and responses to open-ended questions analysed using content analysis. RESULTS: A total of 693 women expressed interest in the peer support role, with 246 completing training, that is, 95% of whom supported at least one mother. Each supported a mean of two mothers (range 1 to 11). Training session topics included respecting individual values, using positive language, confidence building, active listening, empathetic support, and normal baby behaviour. There were 518 periods of support where at least one call was made between a volunteer and a mother to whom she was allocated. Of the 518 periods of support, 359 Call Logs (69%) were returned. The 359 call logs recorded a total of 2398 calls between peers and mothers. Call length median duration was 12 min (range 1 to 111 min). Volunteers perceived the most valued aspects of the calls were the provsion of 'general emotional support' (51%) and 'general information/discussion about breastfeeding' (44%). During the first call, mothers raised questions about 'nipple pain/ damage' (24%) and 'general breastfeeding information' (23%). At ≥12 weeks postpartum, issues raised related to 'normal infant behaviour' (22%), 'feed frequency' (16%), and 'general breastfeeding information' (15%). Volunteers referred women to other resources during 28% of calls, most commonly to the Australian Breastfeeding Association. CONCLUSIONS: Our findings demonstrate that the RUBY trial was feasible and sustainable in terms of recruiting volunteers who were willing to participate in training and who proceeded to provide peer support. Call content was responsive to the evolving breastfeeding information needs of mothers and the provision of emotional support was perceived by volunteers to be important. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry, ACTRN 12612001024831 .

Comparison of CRISPR-Cas Immune Systems in Healthcare-Related Pathogens.

The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) and Clostridium difficile have been identified as the leading global cause of multidrug-resistant bacterial infections in hospitals. CRISPR-Cas systems are bacterial immune systems, empowering the bacteria with defense against invasive mobile genetic elements that may carry the antimicrobial resistance (AMR) genes, among others. On the other hand, the CRISPR-Cas systems are themselves mobile. In this study, we annotated and compared the CRISPR-Cas systems in these pathogens, utilizing their publicly available large numbers of sequenced genomes (e.g., there are more than 12 thousands of S. aureus genomes). The presence of CRISPR-Cas systems showed a very broad spectrum in these pathogens: S. aureus has the least tendency of obtaining the CRISPR-Cas systems with only 0.55% of its isolates containing CRISPR-Cas systems, whereas isolates of C. difficile we analyzed have CRISPR-Cas systems each having multiple CRISPRs. Statistical tests show that CRISPR-Cas containing isolates tend to have more AMRs for four of the pathogens (A. baumannii, E. faecium, P. aeruginosa, and S. aureus). We made available all the annotated CRISPR-Cas systems in these pathogens with visualization at a website (https://omics.informatics.indiana.edu/CRISPRone/pathogen), which we believe will be an important resource for studying the pathogens and their arms-race with invaders mediated through the CRISPR-Cas systems, and for developing potential clinical applications of the CRISPR-Cas systems for battles against the antibiotic resistant pathogens.

Proactive Peer (Mother-to-Mother) Breastfeeding Support by Telephone (Ringing up About Breastfeeding Early [RUBY]): A Multicentre, Unblinded, Randomised Controlled Trial.

BACKGROUND: Breastfeeding rates are suboptimal internationally, and many infants are not receiving any breast milk at all by six months of age. Few interventions increase breastfeeding duration, particularly where there is relatively high initiation. The effect of proactive peer (mother-to-mother) support has been found to increase breastfeeding in some contexts but not others, but if it is shown to be effective would be a potentially sustainable model in many settings. We aimed to determine whether proactive telephone-based peer support during the postnatal period increases the proportion of infants being breastfed at six months of age. METHODS: RUBY (Ringing Up about Breastfeeding earlY) was a multicentre, two-arm un-blinded randomised controlled trial conducted in three hospitals in Victoria, Australia. First-time mothers intending to breastfeed were recruited after birth and prior to hospital discharge, and randomly assigned (1:1) to usual care or usual care plus proactive telephone-based breastfeeding support from a trained peer volunteer for up to six months postpartum. A computerised random number program generated block sizes of four or six distributed randomly, with stratification by site. Research midwives were masked to block size, but masking of allocation was not possible. The primary outcome was the proportion of infants receiving any breast milk at six months of age. Analyses were by intention to treat; data were collected and analysed masked to group. The trial is registered with ACTRN, number 12612001024831. FINDINGS: Women were recruited between Feb 14, 2013 and Dec 15, 2015 and randomly assigned to peer support (n = 574) or usual care (n = 578). Five were not in the primary analysis [5 post-randomisation exclusions]. Infants of women allocated to telephone-based peer support were more likely than those allocated to usual care to be receiving breast milk at six months of age (intervention 75%, usual care 69%; Adj. RR 1·10; 95% CI 1·02, 1·18). There were no adverse events. INTERPRETATION: Providing first time mothers with telephone-based support from a peer with at least six months personal breastfeeding experience is an effective intervention for increasing breastfeeding maintenance in settings with high breastfeeding initiation. FUNDING: The Felton Bequest, Australia, philanthropic donation and La Trobe University grant.

Ringing Up about Breastfeeding: a randomised controlled trial exploring early telephone peer support for breastfeeding (RUBY) - trial protocol.

BACKGROUND: The risks of not breastfeeding for mother and infant are well established, yet in Australia, although most women initiate breastfeeding many discontinue breastfeeding altogether and few women exclusively breastfeed to six months as recommended by the World Health Organization and Australian health authorities. We aim to determine whether proactive telephone peer support during the postnatal period increases the proportion of infants who are breastfed at six months, replicating a trial previously found to be effective in Canada. DESIGN/METHODS: A two arm randomised controlled trial will be conducted, recruiting primiparous women who have recently given birth to a live baby, are proficient in English and are breastfeeding or intending to breastfeed. Women will be recruited in the postnatal wards of three hospitals in Melbourne, Australia and will be randomised to peer support or to 'usual' care. All women recruited to the trial will receive usual hospital postnatal care and infant feeding support. For the intervention group, peers will make two telephone calls within the first ten days postpartum, then weekly telephone calls until week twelve, with continued contact until six months postpartum. Primary aim: to determine whether postnatal telephone peer support increases the proportion of infants who are breastfed for at least six months. HYPOTHESIS: that telephone peer support in the postnatal period will increase the proportion of infants receiving any breast milk at six months by 10% compared with usual care (from 46% to 56%).Outcome data will be analysed by intention to treat. A supplementary multivariate analysis will be undertaken if there are any baseline differences in the characteristics of women in the two groups which might be associated with the primary outcomes. DISCUSSION: The costs and health burdens of not breastfeeding fall disproportionately and increasingly on disadvantaged groups. We have therefore deliberately chosen trial sites which have a high proportion of women from disadvantaged backgrounds. This will be the first Australian randomised controlled trial to test the effectiveness and cost effectiveness of proactive peer telephone support for breastfeeding. TRIAL REGISTRATION: Australian and New Zealand Clinical Trials Registry ACTRN12612001024831.