**7. Epigenetics in the treatment of mesothelioma**

MPM inhibits tumor suppressor genes by promoting LOI and repression of CG genes by site-specific hypermethylation of DNA and/or polycomb repressor complexes in the context of hypomethylation of the genome. This "DNA methylation paradox" mimics epigenomic conditions in normal germ cells and lays the groundwork for epigenetic regimens that restore tumor suppressor gene expression and trigger growth arrest/apoptosis. Upregulation of CTAs, development of viral mimicry by derepression of endogenous retroviruses, and control of the tumor microenvironment all help to boost antitumor immunity [89, 90].

DNMTs are potential targets for MPM treatment because of their direct functions in suppressing tumor suppressor genes and maintaining pluripotency [91, 92]. Previous clinical attempts to inhibit DNMT activity in MPM, however, have failed miserably. Yogelzang et al. [93] showed a 17% objective response rate in 41 MPM patients who received 120 h of continuous dihydro-5-azacytidine infusions. Amazingly, 6 years following treatment, the single responder was diseasefree. The lack of efficacy of DNA hypomethylating drugs in solid tumors could be due to their usage at maximum tolerated doses, resulting in myelosuppression, rather than prolonged use at lower doses to obtain pharmacodynamic effects without systemic toxicity. The Phase I decitabine trial (DAC) clearly demonstrates that chronic exposures are required to achieve maximum gene induction effects in cancer tissues [94].

Furthermore, 5-AZA and DAC administered IV, SQ, or PO have short half-lives (less than 5 min) and poor biodistribution, limiting their potential utility in patients with solid tumors. Cytidine deaminase (CDA), which is found in practically all organs but mainly the gastrointestinal system, quickly inactivates these molecules [95, 96]. Documented toxicity increases Cmax and t1/2 (>50 nM and 4 h, respectively) as well as biodistribution of oral decitabine, decreasing inter-patient variability in drug levels significantly [95–98]. Significant increases in fetal hemoglobin, without neutropenia, thrombocytopenia, or lymphopenia, are indicative of hypomethylation of systemic DNA caused by oral DAC-THU. A phase II trial (NCT02664181) is currently underway at the Cleveland Clinic and NCI to examine whether DAC/THU can improve responses to nivolumab when given as second-line therapy to patients with non-small cell lung cancer. Despite encouraging preclinical data [26], efforts to target HDAC on MPM have also been disappointing.

As second- or third-line therapy, Krug et al. [99] randomized 661 MPM patients to receive the HDAC inhibitor vorinostat or placebo. Overall survival, as well as the drug's safety and tolerability, were the key outcomes. Vorinostat-treated patients had a median OS of 30.7 weeks (95% CI 26.7–36.1) compared to 27 weeks (95% CI 23.1– 31.9) for placebo-treated patients. Given the absence of evidence for HDAC upregulation in MPM and the limited antitumor effects of HDAC inhibitors alone in preclinical tests, the lack of efficacy of the single-agent vorinostat in patients with MPM is not surprising. Combinated techniques, such as using HDAC inhibitors to sensitize cells to TRAIL-mediated apoptosis or flavopiridol to boost romidepsin-mediated growth arrest and death, might be helpful for future clinical trials. Hypomethylating drugs, on the other hand, do not appear to lessen the incidence of mesothelioma after asbestos exposure. In fact, non-solid cancers such leukemias, lymphomas, and other myelodysplastic syndromes show the best benefits with this medicine.

It is feasible that BAP1 mutations could be used for MPM therapy in the future. BAP1 promotes the recruitment of the polychial deubiquitinase PR-DUB complex to *Epigenomics in Malignant Pleural Mesothelioma DOI: http://dx.doi.org/10.5772/intechopen.105408*

DNA damage sites by stabilizing BRCA-1 and promoting poly (ADP-Ribose) dependent recruitment of the polychial deubiquitinase PR-DUB complex to DNA damage sites. This activity is dependent on deubiquitinase activity and BAP1 phosphorylation. BAP1 mutations, which invariably show as a loss of function, cause BRCA-1 levels to drop and double-stranded DNA repair to be inhibited [100–102]. A BAP1 isoform including part of the catalytic domain sensitized MPM cells to the PARP1 inhibitor, according to Parotta et al. [102]. (Olaparib). Concomitant treatment with GDC0980, a dual PI3K-mTOR inhibitor that is downregulated by BRCA-1, could improve this sensitivity. These strategies could improve responses to cisplatin/pemetrexed in patients with BAP1 mutant MPM and should be evaluated in future clinical trials.

There is considerable interest in chromatographic remodeling agents with adoptive cell transfer or immune checkpoint inhibitors for cancer therapy, given the extensive preclinical studies showing DNA demethylating agents, HDAC inhibitors, and KMT inhibitors in the immunomodulatory effects of potentials [103].  The preclinical basis for combining gene induction regimens with cancer adoptive immunotherapy was established in these studies. Furthermore, novel microenvironmental data are likely to have a substantial impact on the outcomes of clinical trials for epigenetic treatments and immunotherapies [89].


**1. Introduction**

Esophageal cancer is an aggressive neoplasm with a higher prevalence among the male gender, associated with high rates of cancer-related mortality. The two

histological subtypes mainly described in the literature include squamous cell carcinoma and adenocarcinoma [1]. Esophageal cancer represents the eighth most common type of cancer and the sixth leading cause of mortality due to neoplasia, with a 5-year survival of less than 25% [2]. Management strategy for patients with esophageal cancer is multidisciplinary, depending on the neoplasia stage, and includes endoscopic procedures such as radiofrequency ablation, endoscopic mucosal resection, and endoscopic submucosal dissection and/or chemoradiotherapy (CRT) as an esophagus-preserving treatment for early esophageal cancer, while most cases are treated with surgical resection, combined with chemoradiotherapy [3]. The advent of minimally invasive esophageal surgery has led to lower rates of morbidity, mortality, shorter hospital stay, enhanced life quality, as well as better long-term oncological outcomes, compared with open esophagectomy [4, 5].

Commonly performed minimally invasive esophageal procedures include Ivor-Lewis, McKeown, and transhiatal esophagectomy. Ivor-Lewis esophagectomy has been associated with a shorter length of hospital stay, lower rate of postoperative complications, and lower readmission rates compared to the McKeown esophagectomy [5]. Moreover, advances in anastomotic leak management options, including endoscopic stent placement or endoscopic vacuum-assisted closure device placement, led to wider and more confident adoption of intrathoracic anastomosis, rendering minimally invasive Ivor-Lewis the most common approach performed in clinical practice [6]. Nowadays, robotic-assisted minimally invasive esophagectomy (RAMIE) has also emerged as an alternative approach to totally and hybrid minimally invasive esophagectomy (MIE), followed by enhanced tissue manipulation ability, better lymph node dissection, superior intraoperative image quality as well as reduced morbidity and mortality rates and improved postoperative outcomes [7]. However, RAMIE is also combined with lack of experience, longer operative times, and higher costs [7].

Anastomosis construction is considered the most crucial step during esophagectomy. Despite the advantages offered by the adoption of minimally invasive Ivor-Lewis esophagectomy and the development of high-tech anastomotic staplers, anastomotic leakage (AL) remains a serious and possibly fatal complication after esophagectomy, with incidence higher than the open approach in numerous studies [4, 8]. AL is defined as a "full-thickness gastrointestinal defect involving the esophagus, anastomosis, staple line, or conduit, irrespective of presentation or method of identification" by the Esophagectomy Complications Consensus Group and is classified into three types, depending on the management approach [9]. AL is the main cause of perioperative mortality, prolonged hospital stay, delayed oral feeding, need for reintervention, decreased overall survival, and increased risk of recurrence [10]. The steep learning curve of minimally invasive Ivor-Lewis esophagectomy is proposed as the main factor contributing to higher rates of AL, since AL incidence is limited to 4.5 % after the learning curve plateau had been achieved [4].

Α thorough investigation of pathophysiology and risk factors for anastomotic leakage is necessary for the optimization of perioperative results as well as for the development of preventive strategies. The anatomic location of anastomosis has been proven as a factor affecting AL rate, since cervical anastomosis is followed by a higher rate of AL (10–25%), compared to intrathoracic anastomosis (<10%) [11]. Reasons leading to increased AL rate apart from cervical anastomosis include compromised perfusion of the fundus, increased risk of tension, local compression, and neoplasia characteristics, such as neoadjuvant radiation or extended resection [6]. The serosal status may also affect the rate of AL [12]. Preoperative irradiation has a conflicting effect on AL

*Prevention of Anastomotic Leak in Minimally Invasive Esophagectomy: The Role of Anastomotic… DOI: http://dx.doi.org/10.5772/intechopen.106041*

rate, while patient characteristics, including obese or underweight patients, preoperative malnutrition, cardiovascular comorbidities, diabetes mellitus, renal failure, and tobacco and steroid use have been associated with increased AL risk [6]. Finally, the anastomotic technique has not been proven to influence the AL rate [6]. ΑL related complications remain the main cause of perioperative morbidity and mortality, as well as poor quality of life after open and minimally invasive esophagectomy [13]. In addition, management of an intrathoracic anastomotic leak, contrary to the cervical anastomosis, demands more aggressive interventions, such as re-operation, thoracotomy, thoracoscopic drainage, or complete gastrointestinal diversion [10].

In conclusion, intrathoracic anastomosis, although more challenging technically, is associated with a series of advantages including lower anastomotic leak rate, lower stricture formation rate, decreased nerve injury rate, and improved oncological outcomes [10]. In addition, the emergence of interventional methods for the management of anastomotic leakage have led to wide adoption of intrathoracic anastomosis [14]. As a result, Ivor-Lewis MIE now ranks first as the most common approach to MIE used clinically [10]. No consensus has been achieved regarding the optimal esophageal anastomotic method. This chapter aims to provide a critical review of current strategies for intrathoracic anastomosis creation during minimally invasive Ivor-Lewis esophagectomy, as well as to discuss the methods proposed for minimizing anastomotic leakage.


**3.4.4 Distractions**

Similarly, the specific and uncoordinated reporting requirements of vertical programs/donors can lead to several forms being filled by a sole health worker for the same problem, distracting them from more productive uses of their time.

Although the Travis 2004 overview provided limited evidence, it concluded that:

*"Disease or service-specific strategies to strengthen health systems on their own are unlikely to bring about the improvements in health systems needed to achieve the MDGs. …Such an approach must be complemented by a substantial additional body of knowledge and action that takes the functioning of the health system as its core concern".*

#### **3.5 Can actions to strengthen disease-specific programs that are effective in one area be successfully spread within a country?**

There is some moderate evidence from research that such programmes can be successfully "scaled up". This means that more studies have found successful scale up than those which have found less successful scale up. However, there is a publication bias towards reporting the "scale ups" that were successful, and not reporting the unsuccessful ones.

One example is the strengthening strategy used to scale up NGO-CHW projects across one set of districts in Zambia. The original model was refined, and then a pilot scale up programme was made, which was then itself developed to allow spread in other regions. The HIV/AIDS CHW model was extended to provide programmes including malaria control, and immunization (Øvretveit, 2008) .

There is evidence that success appears to depend on how the implementation is carried out, and on certain enabling factors in the environment, as well as on the type of disease specific programmes: more complex multiple-component programmes appear to be less successful in scale up, but this may be because the capacity was not there to ensure continual coordination in some cases. Research based guidance for scale-up is given in Øvretveit , 2006.

#### **3.6 Are many strengthening strategies together more successful?**

The evidence (WB, 2005) is that they can be, but often are not because of a lack of resources and management capacity at different levels to coordinate and implement the different strategies. There is some evidence that multiple compatible strategies, where different changes reinforce each other, are likely to have a more significant, long-term effect than single-action strategies alone (e.g. integrated delivery of health service, multiple component healthcare reforms). But the risks of failed implementation are higher because:

Strengthening Health Systems in Yemen:

circumstances.

than salaried employees.

Review of Evidence and Implications for Effective Actions for the Poor 291

There is also research into strategies used for scale-up which have proven successful. There are useful frameworks for scale up of successful pilot strengthening approaches in Yemen including one by Cooley & Kohl 2005. Their tested framework gives a three-step process to carry out ten key tasks which their study suggests were needed for effective scaling up. Key choices to be made in deciding how to apply a strengthening intervention more widely (e.g. in scale-up) are: the sequencing of elements of the strengthening programme; and the pace

of spread (e.g. rapid or phased); the areas to spread to and the sequence of areas.

(Nyonator et al., 2006) gives some possible lessons for how to carry out a strengthening strategy in Yemen. The model aimed to reorient primary health care from clinics to communities, by relocated nurses to live and work in community-constructed clinics and using volunteers to mobilizing traditional social institutions to get community support. The scale up strategy

The study notes actions which helped to overcome constraints to scale-up by comparing slow and faster implementing districts. One was to use "peer exchange" to discuss the details of practical changes which would be needed and to use the original pilot as a demonstration model for visits. This is combined with training for upgrading clinical skills, new referral arrangements, quality

The study notes that once the initiative gets started in one or two zones there is spread of the new approach within districts, but spread is slow across district boundaries because of staff exchanges. So within- and across- district involvement of leaders from neighbouring communities was necessary. It also notes a resource constraints problem to scale up where often fewer resources are available than

The action taken to address some of the problems of nurses working in areas they were not from, was a "community engaged" approach to decentralized training. Communities select nurse trainees, who are sent to a local training centre where fees are paid by the districts and communities to be served by the trainees. On graduation, nurses return home, rather than to a post in a distant location.

The study provides an analysis of issues and principles some of which may apply in other settings and for other strengthening-strategies. The first highlighted was the role of research: not just evidence from a district which replicated the pilot, both of which convinced policy-makers and others that the pilot would work elsewhere, but to identify problems and guide the scale-up. The second was the need for specific guidelines about parts of the programme that needed to be changed, the steps needed to get the operational change, and for monitoring whether change was taking place. The third observation was that the pilot was useful as a demonstration of the model and as a training centre. There was a need to resource the pilot founding implementation team to

communicating the evidence and progress as well as sustaining the effort: newsletters documented community and worker experience with the programme and conferences, demonstration exchanges, and staff meetings. The report notes that "CHPS is thus a complex story. Its core strategy is based on a complex experiment, multiple replication efforts, and diverse sources of evidence. But, its core

Fig. 1. Example of lessons from research from a health service strengthening strategy

pass on their experience and motivation. A fourth item was the value of many ways of

agenda is quite simple for stakeholders to understand and embrace".

were used in the pilot. The "faster" districts had found additional funds, usually not from government, for example "private practitioners" –paramedics who are community financed rather

A Community-based Health Planning and Services (CHPS) initiative in Ghana

used decentralized planning to adapt the operational details to local

assurance, and community-based health management.

#### **3.6.1 Consensus and support**

It is more difficult to obtain consensus and support for each component of a multiple-action strategy than for a single approach.

### **3.6.2 Management and oversight**

More complex multiple-action strategies demand greater management capacity if the actions are to be mutually-reinforcing. Persistent oversight is needed for effective consensus building, planning, coordination, review, and readjustment. Management capacity may not be able to provide these.

### **3.6.3 Timing**

Because of limited resources and capacity, the specific actions for multiple-action strategies will need to be phased-in at different times so that these resources and capacity are not overwhelmed by the demands of many actions at one time.

## **3.6.4 Implementation of non-mutually reinforcing actions**

There is a possibility that specific actions will not be implemented, or may be implemented in ways that undermine other components of the strategy. For example, incentives to provide specific services (e.g. special payments for immunization), can reduce incentives to provide other services for which there is greater need (Øvretveit, 2006).

Overall, the evidence shows that, the more complex the strengthening strategy (e.g. many changes, phased changes, with a large overall change), the more support is required (expert facilitators, external training and supervision). Multiple component and sophisticated strengthening strategies can be more effective only if properly implemented – it is costly to provide this support nationally and some level of ongoing support or supervision is often required (Øvretveit, 2006).

#### **3.7 Is how the strategy implemented more important than the type of strategy?**

One conclusion from this review of research is that that almost any strategy might be possible to implement, if certain conditions and implementation methods are present. There is positive evidence for this from successful implementation, as well as negative evidence from the failed implementations which did not have supportive conditions or were not wellmanaged.

A systematic review of 150 studies using high quality experimental designs (Øvretveit et al., 2008) noted that many of the strategies studied had significant amounts and types of resources to ensure full implementation: similar results could only be expected if the resources or conditions were repeated. One common element in the few studies with many positive outcomes was efforts to assess needs and constraints. In these studies 'constraint reduction plans' were found in 66% of the randomized interventions. However, many interventions that did not use this approach also had positive outcomes. Also, the research often did not describe to what extent these constraint reduction plans were implemented.

Overall there is evidence, that, of all the fully implemented strategies, some were effective in strengthening service delivery for poor people. What appears to be important is targeting poor people, ensuring regular measurement of impact, and oversight to ensure the poor benefit.

It is more difficult to obtain consensus and support for each component of a multiple-action

More complex multiple-action strategies demand greater management capacity if the actions are to be mutually-reinforcing. Persistent oversight is needed for effective consensus building, planning, coordination, review, and readjustment. Management capacity may not

Because of limited resources and capacity, the specific actions for multiple-action strategies will need to be phased-in at different times so that these resources and capacity are not

There is a possibility that specific actions will not be implemented, or may be implemented in ways that undermine other components of the strategy. For example, incentives to provide specific services (e.g. special payments for immunization), can reduce incentives to

Overall, the evidence shows that, the more complex the strengthening strategy (e.g. many changes, phased changes, with a large overall change), the more support is required (expert facilitators, external training and supervision). Multiple component and sophisticated strengthening strategies can be more effective only if properly implemented – it is costly to provide this support nationally and some level of ongoing support or supervision is often

**3.7 Is how the strategy implemented more important than the type of strategy?**

One conclusion from this review of research is that that almost any strategy might be possible to implement, if certain conditions and implementation methods are present. There is positive evidence for this from successful implementation, as well as negative evidence from the failed implementations which did not have supportive conditions or were not well-

A systematic review of 150 studies using high quality experimental designs (Øvretveit et al., 2008) noted that many of the strategies studied had significant amounts and types of resources to ensure full implementation: similar results could only be expected if the resources or conditions were repeated. One common element in the few studies with many positive outcomes was efforts to assess needs and constraints. In these studies 'constraint reduction plans' were found in 66% of the randomized interventions. However, many interventions that did not use this approach also had positive outcomes. Also, the research often did not describe to what extent these constraint reduction plans

Overall there is evidence, that, of all the fully implemented strategies, some were effective in strengthening service delivery for poor people. What appears to be important is targeting poor people, ensuring regular measurement of impact, and oversight to ensure the poor

**3.6.1 Consensus and support**

strategy than for a single approach.

**3.6.2 Management and oversight**

overwhelmed by the demands of many actions at one time.

**3.6.4 Implementation of non-mutually reinforcing actions**

provide other services for which there is greater need (Øvretveit, 2006).

be able to provide these.

required (Øvretveit, 2006).

managed.

were implemented.

benefit.

**3.6.3 Timing**

There is also research into strategies used for scale-up which have proven successful. There are useful frameworks for scale up of successful pilot strengthening approaches in Yemen including one by Cooley & Kohl 2005. Their tested framework gives a three-step process to carry out ten key tasks which their study suggests were needed for effective scaling up. Key choices to be made in deciding how to apply a strengthening intervention more widely (e.g. in scale-up) are: the sequencing of elements of the strengthening programme; and the pace of spread (e.g. rapid or phased); the areas to spread to and the sequence of areas.

A Community-based Health Planning and Services (CHPS) initiative in Ghana (Nyonator et al., 2006) gives some possible lessons for how to carry out a strengthening strategy in Yemen. The model aimed to reorient primary health care from clinics to communities, by relocated nurses to live and work in community-constructed clinics and using volunteers to mobilizing traditional social institutions to get community support. The scale up strategy used decentralized planning to adapt the operational details to local circumstances.

The study notes actions which helped to overcome constraints to scale-up by comparing slow and faster implementing districts. One was to use "peer exchange" to discuss the details of practical changes which would be needed and to use the original pilot as a demonstration model for visits. This is combined with training for upgrading clinical skills, new referral arrangements, quality assurance, and community-based health management.

The study notes that once the initiative gets started in one or two zones there is spread of the new approach within districts, but spread is slow across district boundaries because of staff exchanges. So within- and across- district involvement of leaders from neighbouring communities was necessary. It also notes a resource constraints problem to scale up where often fewer resources are available than were used in the pilot. The "faster" districts had found additional funds, usually not from government, for example "private practitioners" –paramedics who are community financed rather than salaried employees.

The action taken to address some of the problems of nurses working in areas they were not from, was a "community engaged" approach to decentralized training. Communities select nurse trainees, who are sent to a local training centre where fees are paid by the districts and communities to be served by the trainees. On graduation, nurses return home, rather than to a post in a distant location.

The study provides an analysis of issues and principles some of which may apply in other settings and for other strengthening-strategies. The first highlighted was the role of research: not just evidence from a district which replicated the pilot, both of which convinced policy-makers and others that the pilot would work elsewhere, but to identify problems and guide the scale-up. The second was the need for specific guidelines about parts of the programme that needed to be changed, the steps needed to get the operational change, and for monitoring whether change was taking place. The third observation was that the pilot was useful as a demonstration of the model and as a training centre. There was a need to resource the pilot founding implementation team to pass on their experience and motivation. A fourth item was the value of many ways of communicating the evidence and progress as well as sustaining the effort: newsletters documented community and worker experience with the programme and conferences, demonstration exchanges, and staff meetings. The report notes that "CHPS is thus a complex story. Its core strategy is based on a complex experiment, multiple replication efforts, and diverse sources of evidence. But, its core agenda is quite simple for stakeholders to understand and embrace".

Fig. 1. Example of lessons from research from a health service strengthening strategy

Strengthening Health Systems in Yemen:

likelihood of implementation of the strategy (Travis, 2004).

health workers and capable managers (WB, 2005).

and persistence to make the changes needed.

motivation and retention in most situations).

including volunteer services.

**most countries?**

**resources?**

**strategies**

Review of Evidence and Implications for Effective Actions for the Poor 293

**3.10 Are there strategies which are less dependent on the environment, which work in**

There was no strong evidence from the review of research that some strategies were more "robust" than others, and less influenced by some of the conditions which appeared to affect implementation of all the strategies. A number of studies reported success where implementation had included constraints assessment and actions to reduce constraints (table 1 at end of this review). The implications are that, if decision makers take action to ensure that as many of these conditions as possible are met, this would increase the

The research reviewed shows that one condition profoundly influencing all strengthening strategies are whether there are adequate resources for the change, for example as indicated by average per-capita income of the country, health care expenditure, and availability of

Findings from the WB, 2005 HSS case studies show that some strategies were not implemented because of lack of resources initially, or a reduction in available resources later, typically when donor or project finance ceased. There was evidence from the cases that availability of finance was a necessary but not sufficient condition for health service strengthening: some human resource strategies had financial resources, but a shortage of

**4. Conclusions from the review of research into health service strengthening**

There is evidence of high levels of unmet health needs in Yemen, and of the potential for health services to prevent and alleviate suffering, especially of poor people. There is evidence of a number of deficiencies in the allocation of services, their performance and accessibility. This evidence suggests that changing the allocation of resources and increasing efficiency could do much to meet existing needs. However, the changes will not be easy to make, will take time, and will need capable management and incentives for change at all levels. Central and local government will need support to build the capacity, commitment

Research in Yemen has found vertical programmes have been effective, but possibly at the expense of generic primary health care and district services. Research found that PHC could provide EPI successfully on an outreach basis, and this "integration" model has recently been used for Malaria, TB, IMCI, nutrition, and bilharzias in a GAVI programme. Evidence shows that some PHC and districts can successfully provide prevention and care for these diseases and clients, but others need additional strengthening so as to be able to do so. Research from other lower income countries shows the strength of service delivery (amount, accessibility for those most in need and quality) is most strongly influenced by the resources available for the service. This, in turn, depends on the amount of finance from government and private (individual and other), the number and skills of health workers, the facilities and supplies (especially drugs), and participation of the community in different ways

Other specific factors have a greater or lesser influence on the strength of service delivery in different situations (e.g. pay and conditions of government workers can be critical for

**3.11 Should we implement a strategy if we are uncertain if we have the right**

health workers prevented full implementation (e.g. in Ethiopia, Afghanistan).

#### **3.8 How important is it to adapt the strategy to fit the situation, and to continue to adapt it?**

There was some evidence from the review that adaption – taking a strategy and adapting it to the country and local conditions - was associated with fuller strategy implementation. In addition, that continuous strategy adaption in scale up led to fuller implementation and that adaptation was easier in small-scale interventions. In Ghana, a scale-up of a child and maternal health service strengthening pilot was made using an approach adapted for the situation using peer demonstration, diffusion, and teamwork (Phillips et al, 2006).

There are different examples of intervention approaches which decisions makers can use. A two-phased approach involves a pilot, then feedback, and then further modification of the intervention, followed by regional or national dissemination. A three-phased approach may prove to be even more effective. If time and resources allow, the initial pilot may be followed by additional pilots at the same time within different country contexts, allowing for more detailed guidance for decision-makers. There is evidence that implementation effectiveness is increased by providing continuous feedback to the strategy team and leaders about health service needs, constraints, implementation progress, and health service impact. This can be done by independent researchers.

Data from the WB, 2005 HSS case studies show that the planning of all the strategies in each country included some type of assessment of constraints and adaptation of ideas to the country situation. There was great variation after initial national planning in, continual adaptation (e.g. whether annual reviews and re-planning were carried out to adjust the strategy to changing circumstances), and also in adaptation by lower levels to the situation and needs of local areas.

There is evidence that strategies with not only initial, but also continual and local adaptation were more successful from the examples of decentralization in Ethiopia, Ghana Uganda, and Zambia (Øvretveit et al., 2008).

#### **3.9 Is stakeholder involvement and consultation necessary to effective implementation?**

Overall there is some limited evidence from the research that consulting or involving those who make the change, or who can stop it, is necessary for implementation. But there are also counter examples from authoritarian governance situations such as China where success was due to strong implementation structures without consultation (Kaufman, et al 2006). The research can help decision makers be more aware of the different approaches to consultation and involvement of different parties and levels of the health system, and of examples where this has been done. Research does not show if involvement and consultation is always necessary or which type is most effective in which situations.

The research reports adaptation by decision makers or implementers alone, after consultation. It also reports consultation and stakeholder involvement with little adaptation, and as pre-implementation preparation or as a form of education (Fajans, 2006)

The evidence suggests that some pre-implementation consultation can increase the speed and depth to which a strategy is implemented, but much depends on the country's history and culture. Many studies refer to lack of stakeholder consultation, or of lack of involvement and commitment as one explanation for less successful implementation. Where there has been successful implementation, widespread involvement in a process for agreeing the strategy is often reported as building commitment and as a key factor explaining successful implementation.

There was some evidence from the review that adaption – taking a strategy and adapting it to the country and local conditions - was associated with fuller strategy implementation. In addition, that continuous strategy adaption in scale up led to fuller implementation and that adaptation was easier in small-scale interventions. In Ghana, a scale-up of a child and maternal health service strengthening pilot was made using an approach adapted for the

There are different examples of intervention approaches which decisions makers can use. A two-phased approach involves a pilot, then feedback, and then further modification of the intervention, followed by regional or national dissemination. A three-phased approach may prove to be even more effective. If time and resources allow, the initial pilot may be followed by additional pilots at the same time within different country contexts, allowing for more detailed guidance for decision-makers. There is evidence that implementation effectiveness is increased by providing continuous feedback to the strategy team and leaders about health service needs, constraints, implementation progress, and health service impact.

Data from the WB, 2005 HSS case studies show that the planning of all the strategies in each country included some type of assessment of constraints and adaptation of ideas to the country situation. There was great variation after initial national planning in, continual adaptation (e.g. whether annual reviews and re-planning were carried out to adjust the strategy to changing circumstances), and also in adaptation by lower levels to the situation

There is evidence that strategies with not only initial, but also continual and local adaptation were more successful from the examples of decentralization in Ethiopia, Ghana Uganda,

Overall there is some limited evidence from the research that consulting or involving those who make the change, or who can stop it, is necessary for implementation. But there are also counter examples from authoritarian governance situations such as China where success was due to strong implementation structures without consultation (Kaufman, et al 2006). The research can help decision makers be more aware of the different approaches to consultation and involvement of different parties and levels of the health system, and of examples where this has been done. Research does not show if involvement and

The research reports adaptation by decision makers or implementers alone, after consultation. It also reports consultation and stakeholder involvement with little adaptation,

The evidence suggests that some pre-implementation consultation can increase the speed and depth to which a strategy is implemented, but much depends on the country's history and culture. Many studies refer to lack of stakeholder consultation, or of lack of involvement and commitment as one explanation for less successful implementation. Where there has been successful implementation, widespread involvement in a process for agreeing the strategy is often reported as building commitment and as a key factor explaining successful

consultation is always necessary or which type is most effective in which situations.

and as pre-implementation preparation or as a form of education (Fajans, 2006)

**3.9 Is stakeholder involvement and consultation necessary to effective**

**3.8 How important is it to adapt the strategy to fit the situation, and to continue to**

situation using peer demonstration, diffusion, and teamwork (Phillips et al, 2006).

This can be done by independent researchers.

and needs of local areas.

**implementation?**

implementation.

and Zambia (Øvretveit et al., 2008).

**adapt it?**

#### **3.10 Are there strategies which are less dependent on the environment, which work in most countries?**

There was no strong evidence from the review of research that some strategies were more "robust" than others, and less influenced by some of the conditions which appeared to affect implementation of all the strategies. A number of studies reported success where implementation had included constraints assessment and actions to reduce constraints (table 1 at end of this review). The implications are that, if decision makers take action to ensure that as many of these conditions as possible are met, this would increase the likelihood of implementation of the strategy (Travis, 2004).

#### **3.11 Should we implement a strategy if we are uncertain if we have the right resources?**

The research reviewed shows that one condition profoundly influencing all strengthening strategies are whether there are adequate resources for the change, for example as indicated by average per-capita income of the country, health care expenditure, and availability of health workers and capable managers (WB, 2005).

Findings from the WB, 2005 HSS case studies show that some strategies were not implemented because of lack of resources initially, or a reduction in available resources later, typically when donor or project finance ceased. There was evidence from the cases that availability of finance was a necessary but not sufficient condition for health service strengthening: some human resource strategies had financial resources, but a shortage of health workers prevented full implementation (e.g. in Ethiopia, Afghanistan).

### **4. Conclusions from the review of research into health service strengthening strategies**

There is evidence of high levels of unmet health needs in Yemen, and of the potential for health services to prevent and alleviate suffering, especially of poor people. There is evidence of a number of deficiencies in the allocation of services, their performance and accessibility. This evidence suggests that changing the allocation of resources and increasing efficiency could do much to meet existing needs. However, the changes will not be easy to make, will take time, and will need capable management and incentives for change at all levels. Central and local government will need support to build the capacity, commitment and persistence to make the changes needed.

Research in Yemen has found vertical programmes have been effective, but possibly at the expense of generic primary health care and district services. Research found that PHC could provide EPI successfully on an outreach basis, and this "integration" model has recently been used for Malaria, TB, IMCI, nutrition, and bilharzias in a GAVI programme. Evidence shows that some PHC and districts can successfully provide prevention and care for these diseases and clients, but others need additional strengthening so as to be able to do so.

Research from other lower income countries shows the strength of service delivery (amount, accessibility for those most in need and quality) is most strongly influenced by the resources available for the service. This, in turn, depends on the amount of finance from government and private (individual and other), the number and skills of health workers, the facilities and supplies (especially drugs), and participation of the community in different ways including volunteer services.

Other specific factors have a greater or lesser influence on the strength of service delivery in different situations (e.g. pay and conditions of government workers can be critical for motivation and retention in most situations).

Strengthening Health Systems in Yemen:

**5.3 Strengthening management and systems**

"change champions," and implementation teams.

**5.4 Reduce constraints to health service strengthening**

**Factor Description**

with adequate management systems are:

performance and accountability

implementation of most types of strategies:

**Enabling/Hindering**

Resources for the strengthening strategy

Management and governance capacity

Political stability and support

Source: Øvretveit, 2006

Table 1. Enabling/Hindering Factor

management information and human resource management.

Review of Evidence and Implications for Effective Actions for the Poor 295

For these health service strengthening actions to be carried out, actions to strengthen the health system will be needed. These are actions which increase the capability of managers at all levels and how they work together, and improve the management systems such as for

For most strategies, managers at all levels need to be developed and given time to plan and implement strengthening interventions (rather than solely manage routine operations), with some managers dedicated full-time to implementing the strengthening intervention. Aspects of leadership associated with successful strengthening changes include: a clearly communicated mandate from top management that gives authority, resources, and accountability to leaders and teams throughout the organization, as well as respected

The evidence suggests that some strategies which require stronger management capacity should not be pursued on more than a pilot basis until the capacity has been developed. Strategies which might be considered later, and which have proven to have some success

1. Performance based contracting or other ways of linking financing to measures of

2. Payment of incentives to health workers to increase the quantity and quality of services.

In planning and implementing a strategy, decision makers would be advised to assess and address the following factors which have been reported in research to enable/hinder

> Funding for the strengthening strategy Number of personnel engaged in carrying out the strengthening intervention Competence of managerial and front-line personnel (e.g. professionalism, skills, expertise in change processes), particularly their ability to adapt the intervention to local circumstances (i.e. through an assessment of constraints, opportunities, resources)

> Ability/power of each management level to prompt the level below to take action Ability of each management level to hold others accountable (i.e. impose rewards, sanctions) Degree of corruption Degree of local community participation and assistance in the implementation process

> Frequency of changes in government, or leadership implementing the strategy Degree and consistency of support by powerful interest groups Degree of consensus among powerful interest groups Degree and consistency of popular support


**3. Discussion**

While supplementation with LAM was previously shown to be effective in improving pig performance from weaning until day 14 post-weaning [5], the results of this study suggest that, under good sanitary conditions, there is no additional benefit on performance to continued supplementation with either LAM or FUC supplementation up to day 35 post-weaning. It is important to note, however, that pigs fed the LAM supplemented diet had increased colonic butyrate production and positive alterations in the colonic microbiome characterized by reduced *Campylobacter* and increases in the genus *Roseburia* and *Faecalbacterium*. Such effects could contribute to a better host immune response to any potential environmental/pathogen challenge during this time period.

There were substantial differences in the microbiome profile between the three experimental groups. There was a contrasting effect between the two SWE supplemented treatments, particularly in relation to changes in the phylum Proteobacteria. The pigs fed the LAM supplemented diet had a decrease in this phylum, while pigs fed the FUC supplemented diet had an increase in the abundance of this phyla compared to pigs fed the basal diet. These differences were supported at both family and genus taxonomic levels with differences in the family *Campylobacteraceae* and the genus *Campylobacter.* The magnitude of the differences was interesting as pigs fed LAM had 23 times lower *Campylobacter* compared to pigs fed the basal diet, while pigs fed the FUC supplemented diet had 4 times higher *Campylobacter* compared to pigs fed the control diet. *Campylobacter* is a major foodborne pathogen often solely linked to infections from broilers through *C. jejuni*, however, *C. coli* is implicated with infections originating from swine [9]. A potential anti-bacterial effect following the addition of LAM is potentially of major benefit to the pig industry [10]. While *Campylobacter* is often considered to form part of the commensal flora of pigs and cause no negative impact on animal performance, there is some debate as high counts of *Campylobacter* are often observed in conjunction with other bacteria during incidences of diarrhea [11]. From either the aspect of reducing a zoonotic pathogen or improving intestinal health, the observed effects on *Campylobacter* by the addition of LAM can be considered a positive finding while the large increase in *Campylobacter* following the addition of FUC is likely to have negative connotations.

Coinciding with the lowest abundance of Proteobacteria in this study, pigs fed the LAM supplemented diet had the greatest abundance of the phylum Firmicutes. This increase was predominantly related to increases in the *Ruminococcaceae* and the *Lachnospiraceae* families. At genus level this coincided with differences in *Faecalbacterium* and *Roseburia.* Within the genus *Faecalbacterium* only one species has been identified in the literature, which is *Faecalbacterium prausnitzi*, implying that this change at genus level relates to a change in abundance of this species. This genus has been associated with increased intestinal health based on a low incidence of post-weaning diarrhea in pigs [12]. In humans, *Faecalbacterium prausnitzi* is used as an indicator of intestinal health and is also considered to be a very promising probiotic for human use [13,14]. The end products of carbohydrate fermentation by *Faecalbacterium prausnitzi* are formate, lactate and substantial quantities of butyrate [15]. Further corroborating the potential improved gut health in pigs fed LAM are an increase in the genus *Roseburia*. *Roseburia* is a member of the Firmicutes phylum and is associated with the production of butyrate [8]. Therefore, the increase in *Faecalbacterium and Roseburia* could be associated with the increase in butyric acid identified in pigs from this study fed the LAM supplemented diet. Butyrate is produced from bacterial fermentation to provide energy to colonocytes. It also has functions as a cellular mediator, regulating multiple functions of gut cells and beyond, including gene expression, cell differentiation, gut tissue development, immune modulation, oxidative stress reduction and diarrhea control [16]. These changes suggest an improvement in gut health in pigs fed the LAM supplemented diet.

In contrast to the clear beneficial effects of supplementation with LAM on members of the Firmicutes and Proteobacteria phyla, the increase in abundance of the Spirochaetes phylum are less clear. Within this phylum associated changes were identified with increases in the family *Spirochaetaceae* and the genus *Treponema* were also identified, with pigs fed the LAM supplemented diet having greater abundance than both the two other experimental groups. The genus contains both pathogenic as well as commensal bacteria [17]. Pathogenic bacteria include *Treponema pallidum*, and others such as *T. denticola* (Tde), *T. putidum* (Tpu) *T. pedis* (Tpe), *T. brennaborense* (Tbr), and *T. paraluiscuniculi* (Tpar) which are the causative agents of various disease in both humans and animals [18]. Porcine intestinal Spirochaetes form a diverse group of organisms including the beta-hemolytic *Serpulina*/*Treponema hyodysenteriae* which is the main etiological agent of swine dysentery [19]. While the classification of OTUs at species level is unreliable, in this study, species classified as part of the genus Treponema include *berlinense*, *bryantii*, *succinifaciens*, *suis* and *porcinum*. While data related to these species is limited, *Treponema porcinum*, *Treponema succinifaciens*, *Treponema berlinense* and *Treponema bryantii* are associated with improved feed efficiency in pigs [20,21]. Further analysis such as shotgun metagenomics will need to be conducted to better understand the impact of changes in this genus have on the function of the microbiome. However, based on the other positive effects on gut health in pigs fed the LAM supplemented diet and the lack of negative impacts on gut health, it seems the changes in the Spirochaetes phylum are not negatively impacting the pigs.

Pigs fed both the LAM and the FUC supplemented diets had reduced bacterial diversity based on the Observed, Chao1, ACE and Fisher measures of alpha diversity. While there are conflicting opinions in the literature, lower bacterial diversity is generally considered to have negative connotations as greater microbial diversity is directly associated with ecosystem stability [22]. For example, in humans, reductions in diversity is associated with a range of health issues and diseases [23]. Therefore, while a reduced diversity in the pigs fed the FUC supplemented agrees with the changes in the microbiome and performance for this group, the reduction in diversity in pigs fed the LAM supplemented diet is surprising. The changes in the microbiome in this group particularly in the reduction in *Campylobacter* suggest improved gut health and therefore, it is unlikely that a reduction in diversity is having a negative influence in this group of pigs.

In relation to the gene expression data in this study, there are contrasting effects between the diets supplemented with the seaweed extracts. In relation to supplementation with LAM only minor changes in gene expression were identified relative to the control while more substantial changes were identified in pigs fed the FUC supplemented diet relative to pigs fed the control diet. However, there was region specific differences. In the duodenum, pigs fed the FUC supplemented diet had increased expression of SLC6A19 and CNDP1 compared to pigs fed the basal and also the pigs fed the diet supplemented with LAM. The two SWE supplemented groups had reduced expression of SLC16A10 compared to pigs fed the basal non-supplemented diet. These three transporters are all involved in the transport of amino acids [24]. This suggests there is increased availability and transport of amino acids in the duodenum of pigs fed the diets containing seaweed extracts compared to pigs fed the control. However, nutrient transporters are regulated by dietary substrate levels meaning the increased expression of protein transporters is a response to an increase in nutrient availability in the duodenum which can be impacted by a number of factors such as feed intake, but also may be a response to changes in intestinal architecture such as reductions in villus height [25]. A further influence of the SWE on nutrient transport was identified in the jejunum with pigs fed the FUC supplemented diets having increased expression of MCT1/SLC16A. SLC16A1 is responsible for the transport of monocarboxylates such as L-lactate and ketone bodies and also with the transport of butyrate [26,27]. These data, while speculative suggest an increased bacterial fermentation in the jejunum with the addition of FUC leading to increased production of butyrate and subsequent absorption with MCT1. While the influence of bacterial fermentation in small intestine is not as pronounced as the large intestine, the production and utilisation of butyrate in the small intestine and in particular the jejunum, as is the case in this study is important in the modulation of balance between apoptosis and proliferation [28]. In contrast to the upregulation of expression of genes related to nutrient digestion and absorption in the duodenum and jejunum, in the ileum, FUC supplementation led to a reduction in the expression of SLC2A2, GCG, FABP2, SGLT1 and SI in the ileum. Similar, results were identified where the feeding of a similar level of FUC in the post-weaning period had a similar influence on nutrient transporters suggesting that the addition of FUC has an impact in nutrient availability [6]. In the study of Rattigan et al. [6] the authors attributed the reduction in gene expression to the presence of alginates and fucoidan in the extract. Both FUC and alginates have been attributed with increasing the viscosity of digestive contents, reducing the flow of digesta and reducing the mixing of digestive contents leading to lower rates of nutrient breakdown [29,30]. The impact of these contrasting effects in different regions of the small intestine are difficult to quantify particularly due to the fact ileal digestibility's

was not measured. Unfortunately, the material required to conduct an analysis of nutrient digestibility was unavailable due to the volume of digesta in the digestive tract at the time of slaughter. Further research will be required to further understand the impacts of these extracts and nutrient breakdown and absorption.

Significant effects were identified between experimental groups in both the expression of targets related to barrier function and the expression of cytokines. Pigs fed the FUC supplemented diet had reductions in the expression of *OCLN* in both the jejunum and ileum compared to pigs fed the basal diet, while a tendency towards reduced expression was also identified in the duodenum. In agreement with these data is a reduction in the expression of *CLDN3* and *CLDN5* in the ileum compared to pigs fed the control diet. Proteins in the claudin and occludin families are a main component of tight junctions and form a seal that modulates paracellular transport in the intestinal epithelium [31]. Reduced expression of claudins such as 3 and 5 is associated with intestinal inflammatory disorders [32]. This is in agreement with effect identified on the expression of IL8/CXCL8 in the jejunum and the increased expression of *TGFB1* and *TNF* in the ileum of pigs fed the diet supplemented with FUC compared to pigs fed the basal unsupplemented diet. For the pigs fed the FUC supplemented diet the changes in gene expression are likely indicators of detrimental effects of the extract on gut health with increased markers of inflammation and reduced gene expression of targets associated with barrier function. In conjunction with the changes in the microbiome in the large intestine these data are possible explanations for the reduction in performance of pigs fed FUC. The pigs fed LAM had reductions in the expression of *MUC1* compared to pigs fed the basal and also pigs fed the diet supplanted with LAM. MUC1 provides a barrier against potential pathogens while also modulating the expression of inflammatory cytokines [33]. Therefore, the increase in expression of this gene is surprising as pigs fed the diet supplemented with LAM had no increases in inflammation, a healthier microbial profile and improved performance. Further research will need to be conducted to establish the reasons for this increase in expression such as a quantification of protein abundance as changes in expression are not always a true reflection of the production of the associated protein.

The aim of this study was to determine if the continued supplementation of two SWEs past the initial post-weaning period continues to be beneficial to performance. It is interesting to note that while SWE supplementation past the immediate post-weaning period up to d 35 in this study did not result in improved performance in healthy pigs under good sanitary experimental conditions, there was evidence to suggest that LAM supplementation had positive effects on gut health. This is significant as previous studies have identified that SWE supplementation can provide substantial performance and health benefits when the pigs become challenged. Heim at al [34] identified that SWE supplemented pigs had lower faecal scores and better performance than basal fed pigs following an ETEC challenge. Rattigan et al. [6] identified that LAM supplementation improves performance when pigs are housed in unsanitary conditions. While the mode of action is not completely understood, the evidence would suggest that bioactives in the SWE can influence both the host immune cells and gut microbiome cells. For instance, bioactives such as laminarin, can be internalized by intestinal epithelial cells and gut associated lymphoid cells [35]. This internalization upregulates the expression of pattern recognition receptors, increasing protective cytokine expression and induces protection against infectious challenge [36]. Hence, this effect would not be evident until the pigs undergo some type of environmental or pathogenic challenge.

#### **4. Materials and Methods**

All experimental procedures described in this work were approved under the University College Dublin Animal Research Ethics Committee (AREC-17-19-O'Doherty, 11 May 2017) and were conducted in accordance with Irish legislation (SI no. 543/2012) and the EU directive 2010/63/EU for animal experimentation.

#### *4.1. Experimental Design and Animal Management*

72 weaned pigs (progeny of Landrace boars × (Large White × Landrace) sows) were sourced from sows from the same farrowing house. Blocking of piglets was done on the basis of initial live weight (8.4 kg, sd 1.05 kg), sex, litter of birth and pigs were assigned to one of three groups: 1) basal diet; 2) basal diet + 250 ppm fucoidan rich extract (FUC); 3) basal diet + 300 ppm of a laminarin (LAM) rich extract with a total of 8 replicates per treatment.

The pigs were penned in groups of three and housed on fully slatted floors (1.68 × 1.22 m) with the initial temperature set at 30 ◦C and subsequently reduced each week by 2 ◦C with the humidity maintained at 65%. Pig weights were recorded on day 0 (weaning) and subsequently on days 7, 14, 21, 28 and 35. Feed and water were available ad libitum from four-space feeders and nipple drinkers, respectively, throughout the experiment.

#### *4.2. Preperation of Experimental Diets and Characterisation of Extracts*

Pigs in this experiment were offered one of three diets: (1) basal diet; (2) basal diet + 250 ppm FUC; (3) basal diet + 300 ppm LAM. The basal diet contained 15.3 MJ/kg digestible energy, 190 g/kg crude protein and 13.5 g/kg total lysine. The basal diet was comprised of wheat (34%), extruded full fat soya (17%), flaked wheat (13%) soya bean meal (48% crude protein) (10.5%), flaked maize (7%) whey powder (5%), provisoy (soya protein concentrate) (6.5%), soya oil (3%) with the remainder compromised of mineral and vitamin supplements (Table 7). Lysine was used a reference for calculation of the other amino acid requirements (NRC, 2012). The extraction procedures and methodology were described in previous publications but are further outlined below [5,6,37].

A laminarin-rich extract (BioAtlantis Ltd., Clash Industrial Estate, Tralee, Ireland) was obtained from *Laminaria Digitata* using a hydrothermal-assisted extraction and preoptimized conditions for maximum yield of laminarin as described previously [38,39]. Briefly, dried and milled seaweed was suspended of 0.1N HCl maintaining a solid to liquid ratio of 1:21 (g/mL). The mixture was thoroughly agitated to ensure uniformity and then subjected to a temperature of 100 ◦C for 30 min. The subsequent crude extract was partially purified to increase the relative polysaccharide content and to remove or reduce other constituents; proteins, polyphenols, mannitol and alginate. This was achieved through mixing the crude extract with pure ethanol (to remove polyphenols) followed by water (to remove protein) and calcium chloride (to remove alginates).

A fucoidan-rich extract (BioAtlantis Ltd., Clash Industrial Estate, Tralee, Ireland) was obtained from *Ascophyllum nodosum* using a hydrothermal-assisted extraction and preoptimized conditions for maximum yield of fucoidan [39]. The crude extract was partially purified to increase the relative polysaccharide content and to remove or reduce other constituents such as proteins, polyphenols, mannitol and alginate. This was achieved through mixing the crude extract with pure ethanol (to remove polyphenols) followed by water (to remove protein) and calcium chloride (to remove alginates). Fractions were separated based on molecular weight by employing a molecular weight cut-off centrifugal concentrator (100 KDa cut-off).

#### *4.3. Post Slaughter Sample Collection*

On d35 following slaughter, tissue sections of 1cm2 were cut from the duodenum, jejunum and ileum and washed using sterile PBS (Oxoid). The overlying smooth muscle was removed before storage in 5 mL RNAlater solution (Applied Biosystems, Dublin, Ireland) overnight at 4 ◦C which was subsequently removed before long term storage at −80 ◦C. Colonic digesta was removed and stored in sterile containers (Sarstedt, Wexford, Ireland) and frozen ( −20 ◦C) for subsequent 16s rRNA sequencing and VFA analyses.

**Table 7.** Ingredient and chemical composition of basal diet \*.

\* Treatments were as follows: (1) basal diet; (2) basal diet; basal diet + 250 parts per million (ppm) fucoidan; (3) basal diet + 300 ppm of a laminarin rich extract basal diet; † Values calculated based on tabulated nutritional composition [40]. The basal diet was formulated to provide: Cu, 100; Fe, 140; Mn, 47; Zn, 120; I, 0.6; Se, 0.3; retinol, 1.8; cholecalciferol, 0.025; α-tocopherol, 67; phytylmenaquinone, 4; cyanocobalamin, 0.01; riboflavin, 2; nicotinic acid, 12; pantothenic acid, 10; choline chloride, 250; thiamine, 2; pyridoxine, 0.015 (mg/kg diet). Celite was also included at 300 mg/kg.

#### *4.4. Volatile Fatty Acid Analysis*

The VFA concentrations in the digesta were determined using gas liquid chromatography (GLC) as reported by Clarke et al. [41]. 1 g of digesta was mixed with distilled water (2.5 × sample weight) and centrifuged at 1400 g for 10 min (Sorvall GLC-2B laboratory centrifuge, DuPont, Wilmington, DE, USA). One mL of the subsequent supernatant and 1 mL of internal standard (0.05% 3-methyl-n-valeric acid in 0.15 M oxalic acid dihydrate) were mixed with 3 mL of distilled water. The reaction mixture was centrifuged at 500 g for 10 min, and the supernatant was filtered through 0.45 PTFE syringe filter into a chromatographic sample vial. An injection volume of 1 μL was injected into a Varian 3800 GC equipped with a EC ™ 1000 Grace column (15 m × 0.53 mm I.D) with 1.20 μm film thickness. The temperature was set at 75–95 ◦C increasing by 3 ◦C/min, 95–200 increasing by 20 ◦C per minute, which was held for 0.50 min. The detector and injector temperature were 280 ◦C and 240 ◦C, respectively, while the total analysis time was 12.42 min.


2.3.5. X-ray Diffraction (XRD)

X-ray diffraction (XRD) measurements were performed using a Bruker D8-Advance diffractometer equipped with Cu Kα1 radiation (λ = 1.54060 Å). The diffraction patterns were recorded with a step size of 0.15087◦, and dwell time of 5s.


**3. Changing blue carbon capture and storage rates in polar seas**

Despite the relative constancy in many oceanographic parameters over geological time, the polar regions are quite dynamic in fluctuation between ice ages, the duration and rate of change to interglacial periods and within these, the position of the marginal sea ice zone, water masses and ice shelf extent. All of these can directly alter the biomass of organisms, their carbon capture and storage rates as well as direct carbon dioxide uptake and release by oceanic storage. Section 2 has highlighted that sediment carbonate levels [8] and organism blue carbon capture and storage rates [10, 15] all vary considerably between and within regions. Measuring any change over time necessarily must have georeferenced baselines to measure against but most 'long term monitoring' programmes are relatively young. One of the most notable multidecadal data sets is that for zooplankton, focused on krill and salps. Analysis of this was one of the first to show change in polar ecosystems (krill reductions) in response to climate [24]. However these organisms are mobile and ice edge associated, which highlight both problems in measurement and interpretation – are the less krill in there survey areas because there are less overall or because they are somewhere-else? Crustacean zooplankton, such as Krill, are important to blue carbon capture and storage rates [5, 6] and may be important to sequestration rates as well [4].

We know little about blue carbon capture, storage and sequestration rates for the vast majority of the seabed, and there are a tiny number of sites which have been monitored regularly for more than a decade. Recently a series of ice shelf disintegrations along the Antarctic Peninsula and some elsewhere have been accompanied by major increases in primary [18] and secondary [19] production. These new and increased stocks of seabed blue carbon there have been estimated to constitute ~7x105 tons of carbon per year equivalent to 10,000 hectares of tropical rainforest [19]. These ice shelf collapses have formed an increasing number of giant icebergs, which have also increased carbon capture in the water column through ocean fertilization [20]. Duprat et al. [20] estimated the increases in water column blue carbon of a number of such icebergs. Those estimates were later built upon in terms of their total blue carbon impact (trade-offs of creating new sink areas and ocean fertilization versus scouring potential) to show a 5000 km2 iceberg contributes a net positive of 106 tons of carbon per year [17]. Ice shelf losses, iceberg production and arctic forest increases [2] are not the only sources of blue carbon change around the polar regions.

within and between regions, organisms, environments and time. Partitioning out the causes and effects of variability is key to meaningful estimates of blue carbon stocks and how they are likely to change. For example, young animals are likely to have higher specific growth rates (thus have high carbon capture but low storage values) whereas older animals would typically be larger but grow slower (and thus be associated with low carbon capture but high carbon storage values). Thus an event influencing population demographics (e.g. iceberg scour) could change carbon capture relative to storage rates, and this could alter depend on

Recent work in the Ross, Weddell and Bellingshausen seas have shown rapid growth rates, and changes in growth rates, are possible in polar organisms. Such blue carbon change has happened in response to wind-driven or ice shelf collapse promoted increased food availability [18, 19, 23] respectively, or increase in temperature [21]. With carbon sinks, sources and flux values being so important to global climate as well as projecting trends and predicting future scenarios it is clear that quantification of blue carbon has an important role in this, and the polar regions are the most poorly known. Understanding biological response to polar change has become even more important since it has become apparent that amongst the most

Despite the relative constancy in many oceanographic parameters over geological time, the polar regions are quite dynamic in fluctuation between ice ages, the duration and rate of change to interglacial periods and within these, the position of the marginal sea ice zone, water masses and ice shelf extent. All of these can directly alter the biomass of organisms, their carbon capture and storage rates as well as direct carbon dioxide uptake and release by oceanic storage. Section 2 has highlighted that sediment carbonate levels [8] and organism blue carbon capture and storage rates [10, 15] all vary considerably between and within regions. Measuring any change over time necessarily must have georeferenced baselines to measure against but most 'long term monitoring' programmes are relatively young. One of the most notable multidecadal data sets is that for zooplankton, focused on krill and salps. Analysis of this was one of the first to show change in polar ecosystems (krill reductions) in response to climate [24]. However these organisms are mobile and ice edge associated, which highlight both problems in measurement and interpretation – are the less krill in there survey areas because there are less overall or because they are somewhere-else? Crustacean zooplankton, such as Krill, are important to blue carbon capture and storage rates [5, 6] and

We know little about blue carbon capture, storage and sequestration rates for the vast majority of the seabed, and there are a tiny number of sites which have been monitored regularly for more than a decade. Recently a series of ice shelf disintegrations along the Antarctic Peninsula and some elsewhere have been accompanied by major increases in primary [18] and secondary [19] production. These new and increased stocks of seabed blue carbon there have been

rainforest [19]. These ice shelf collapses have formed an increasing number of giant icebergs,

tons of carbon per year equivalent to 10,000 hectares of tropical

severe physical changes have been associated with these areas.

may be important to sequestration rates as well [4].

estimated to constitute ~7x105

**3. Changing blue carbon capture and storage rates in polar seas**

which season it occurred in.

48 Carbon Capture, Utilization and Sequestration

Sea ice extent, particularly 'fast ice' (the freezing of the sea surface, anchored to land) has been one of the most drastic physical changes in the polar regions, particularly throughout the Arctic. Sea ice changes and primary production responses have been more complex around Antarctica [25], but crucially most sea ice losses have been over productive continental shelf whereas most of the sea ice gains have been over deeper slope and abyssal ocean depths [16]. Historical expedition zoobenthic collections and modern samples of longer lived animals with relevant information in skeletons has shown that blue carbon capture rates may have doubled over the last 25 years around West Antarctica [26]. The mechanism for this seems to be that reduced extent (in time and space) of sea ice leads to longer (but not necessarily larger biomass) phytoplankton blooms, resulting in longer meal times for primary consumers resulting in more carbon storage as growth (**Figure 7**). The total blue carbon increases driven by sea ice losses [17, 26] probably greatly exceed those caused by ice shelf collapse/giant iceberg formation [17–20]. However, from what we currently know, change in polar blue carbon is a complex of increases

**Figure 7.** Schematic showing influence of ozone losses on phytoplankton carbon capture and zoobenthic carbon storage on polar seabeds.

[18], sea ice loss [25] and iceberg production [20]. It also seems likely that polar macroalgal production could increase spatial and temporal extent with exposure of new habitats, sea ice reduction and increased light energy reaching the shallows. These negatively feedback

depending on how much reaches the seabed and how much is recycled in the microbial loop and reworked following bioturbation. All natural carbon sequestration is via burial, mainly at the seabed, where zoobenthic assemblages (consumers) live. They are an important part of the negative feedback on climate, as new and longer availability of phytoplankton is converted into increased growth (organic carbon to tissues and inorganic carbon to skeletons). The feedback value is complicated to measure because it is dynamic in space and time but also because of simultaneous positives and negatives. For example ice shelf loss leads to more open water, a) reducing albedo, thus potential to absorb more heat; b) reduces buttressing of ice sheets, thus potential for this to accelerate coastwards, c) increasing potential for gas exchange, d) generating new phytoplankton blooms, e) opening new habitat for zoobenthos and f) generating giant icebergs with ocean fertilization potential [17–20]. Even the latter components themselves each contain contrasting feedbacks on climate, for example calving of an

giant iceberg such as that to break off Larsen C in 2017 may scour and recycle 4x104

factors such as increased heat absorption and less than expected growth gains.

benthic carbon but algal capture and seabed zoobenthic storage of new carbon contributes

probably similar to that of Arctic Taiga expansion [2], although this too has also complicating

see http://nsidc.org/) so biological responses to these are the largest measured natural negative feedback on climate change. These are dwarfed as an organic carbon store by tropical forests, but these are not increasing as a result of climate change and thus not a negative feedback (their genuine sequestration potential is also low, as burial rates of carbon are very small except for water logged swamp forests). The magnitude of polar blue carbon negative feedback from sea ice losses depends on whether the carbon is calculated from primary production, secondary production, immobilized carbon or sequestered carbon. The sequestration value is considered to be as low as two orders of magnitude different along the cascade from algal production to buried sequestered benthos (**Figure 9**). Scaling up from regional samples suggests that between 2002 and 2015 the zoobenthic blue carbon negative

in terms of sequestration [16] along the West Antarctic Peninsula continental shelf alone.

continental shelves, such as the Kerguelen Plateau doubles this [17], equivalent to 1–2% of global anthropogenic output. It is clear this feedback is dynamic, polar blue carbon storage has demonstrably increased in coincidence with climate-forced sea ice changes, at least around West Antarctica [26]. Global climate change, ozone losses and other indirect (e.g. non indigenous species invasions) or direct (e.g. harvesting) anthropogenic pressures have the potential to have major impacts on marine biodiversity [27], and thus considerably increase

tons of carbon per year [17]. The magnitude of this negative feedback is

whereas ice shelf losses approximate to ~30,000 km2

T C in immobilization or 1.6x106

T C yr.−1 but including outer Subantarctic

km2

with increasing atmospheric

Blue Carbon on Polar and Subpolar Seabeds http://dx.doi.org/10.5772/intechopen.78237

are genuinely sequestered,

tCyr−1 of

51

tons C

) the annual zoobenthic

(mitigate) on climate change through increased capture of CO2

content. Only very small proportions of this captured CO2

of CO2

a net positive of 106

Sea-ice loss areas exceed 1,000,000 km2

feedback averaged ~107 T C in production, 4.5x106

blue carbon feedback is estimated at 30-80x106

or decrease polar blue carbon.

Scaled up to the whole Antarctic continental shelf area (4.4x106

**Figure 8.** Trends in zoobenthic blue carbon accumulation around the Southern Ocean. The key to cell (3x3 degrees) colors are red (biggest increase) to blue (biggest decrease) [17]. Cells with question marks are samples awaiting analysis (from the Antarctic circumnavigation expedition and future British Antarctic survey scientific cruises.

and decreases (**Figure 8**). Around the Southern Ocean blue carbon increases are most associated with West Antarctic seas and decreases with the East Antarctic coasts [17] but the vast majority of all shelf carbon stocks and change is unknown. It seems likely that the biggest blue carbon changes are near coast caused by ice shelf collapse [18–20], iceberg scour [19] and glacier retreat driven sedimentation [22] but there could also be significant offshore change associated with a shifting seasonal sea ice margin [4, 24, 25]. Given the higher potential ectotherm growth performances at slightly higher sea temperatures [21] it also seems likely that the Arctic and subpolar regions are key areas to quantify blue carbon budgets for. Quantifying these becomes one of the key steps in estimating biotic feedbacks on climate change.


**1. Introduction**

Enterococci are Gram-positive, non-spore forming and facultative anaerobic cocci. They are indigenous flora of the intestinal tract, oral cavity and vagina in healthy persons. The genus comprises 54 species which are ubiquitously present in nature [1]. Enterococci have emerged as an important nosocomial pathogens second to Staphylococci which is the leading cause of nosocomial infections worldwide [2]. Enterococci are important nosocomial pathogens causing up to 10% of all infections in the hospitalized patients [3]. In these Enterococci infections approximately 60% of infections are caused by *Enterococcus faecalis* and *Enterococcus faecium* causes the remaining [4]. In the last decade both *E. faecalis* and *E. faecium* have emerged as important nosocomial pathogens. Other Enterococcal species causing nosocomial human infections are *E. avium*, *E. gallinarum*, *E. casseliflavus*, *E. durans*, *E. raffinosus* and *E. mundtii*. Majority of clinical isolates (63–81%) are identified as *E. faecalis*, around 13–23% as *E. faecium* and other enterococcal species comprise around 3–4% of the clinical isolates [5].