**1. Introduction**

Conflict and war can have obvious and profound effects on the transmission and severity of infectious diseases. Although poverty is often listed as the most important external influence on disease rates, the impacts of war and conflict are also important and can lead not only to damaged public health infrastructure, displaced populations and disrupted habitats of zoonotic reservoirs and vectors, but also to the economic perturbations that increase poverty rates within affected populations [1]. Sedentary rural populations are particularly vulnerable to the disruptions of conflict due to their limited ability to adapt after the destruction or confiscation of their crops and arable land [2]. After initial losses, limited access to seeds, tools or draft animals also leads to a vicious cycle of decreasing production, resulting in increased starvation and decreased health status. This is complicated even further by dysfunctional distribution caused by lack of transportation and fuel, damaged roadways, road blocks and local violence. Malnutrition is the inevitable result of these disruptions to the food security of affected populations. Children under the age of five are the most vulnerable to nutritional deficits and thus they frequently suffer disproportionately from the effects of violence and conflict. Pregnant and breast-feeding women are also vulnerable to the effects of disrupted food supplies due to their increased nutritional needs. Increases in malnutrition in turn lead to compromised immune systems, leaving individuals susceptible to many conflict-associated infectious diseases.

Populations that are impacted by violence are often forced or choose to leave their traditional family homes to migrate to a safer environment. Public health workers have known for many years that displaced populations are particularly vulnerable to epidemics of infectious disease [3]. This increased vulnerability can be due to a variety of factors including compromised immune systems as a result of malnutrition, decreased sanitation, insufficient housing or protection from disease vectors, exposure to regional diseases to which immigrants have little or no immunity, and other factors. Thus conflict and poverty are related, and can work

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

synergistically to increase disease risks in affected populations, especially those that are displaced.

Conflict can also increase disease risk in populations of warfighters (referred to as "soldiers" here even though it is acknowledged that not all warfighters are soldiers). Soldiers often deploy to regions that are endemic for diseases to which they have no previous exposure. As a result of their lack of exposures, the soldiers are often immunologically naïve to the endemic diseases of the areas to which they deploy, rendering them vulnerable to outbreaks of exotic diseases. Although modern militaries often have sophisticated immunization programs, there are no effective vaccines for many diseases of military importance, including malaria, dengue and leishmaniasis. In such situations, the soldiers must rely on other means of protection including strictly enforced hygiene, anti-vector measures, chemoprophylaxis and personal protection such as mosquito netting and repellents. Disease risk to soldiers is especially high during the initial stages of a conflict when the soldiers may live in rudimentary shelters that provide inadequate protection from vectors, vermin and the elements. Food safety, good water quality, and adequate general hygiene are particularly difficult to maintain during this phase of the conflict. As the conflict ages, the soldiers often acquire better housing and protection from the environmental hazards; however, the protracted stresses associated with war fighting, accompanied by more mundane factors such as boredom and separation from families, can also contribute to immune system dysfunctions and increased disease transmission.

Paradoxically, the impact that conflict-associated diseases have on militaries can improve the success of programs to lower the risk of acquiring those diseases. This observation does not imply in any way that wars are beneficial to public health, but only that the efforts that militaries put toward protecting their troops can improve treatment and prevention of those same diseases in other environments free of conflict or war. For instance, the impact of malaria during WWI and the need to acquire more dependable supplies of the anti-malarial quinine stimulated efforts by the U.S. military to develop cheaper synthetic substitutes like meparene and chloroquine. Similarly, the military's use of insecticidal chemicals such as DDT and BHC served as models for subsequent successful civilian programs to control the disease vectors of malaria and dengue. Also, effective vaccines are often developed initially in response to military need. Finally, one of the most effective repellents used as a personal protective measure against mosquitoes and other vectors, N,N-diethy-meta-toluamide or DEET, was developed by the U.S. Army in large part as a result of its experiences with malaria and other vector-borne diseases during WWII [4]. Such developments are direct results of the attention to the disease during military operations in endemic areas, as well as the medical and scientific expertise and relatively large sums of money available to develop disease interventions for the population of soldiers.

Many of these general conclusions about the impact of conflict on disease rates have particular significance to the disease leishmaniasis. This chapter provides a review of conflict leishma‐ niasis, especially as related to two events: (a) the civil war in Sudan and, (b) recent conflicts in the Middle East involving the militaries of the U.S. and its allies.

#### **1.1. General background on the disease and its vector**

synergistically to increase disease risks in affected populations, especially those that are

Conflict can also increase disease risk in populations of warfighters (referred to as "soldiers" here even though it is acknowledged that not all warfighters are soldiers). Soldiers often deploy to regions that are endemic for diseases to which they have no previous exposure. As a result of their lack of exposures, the soldiers are often immunologically naïve to the endemic diseases of the areas to which they deploy, rendering them vulnerable to outbreaks of exotic diseases. Although modern militaries often have sophisticated immunization programs, there are no effective vaccines for many diseases of military importance, including malaria, dengue and leishmaniasis. In such situations, the soldiers must rely on other means of protection including strictly enforced hygiene, anti-vector measures, chemoprophylaxis and personal protection such as mosquito netting and repellents. Disease risk to soldiers is especially high during the initial stages of a conflict when the soldiers may live in rudimentary shelters that provide inadequate protection from vectors, vermin and the elements. Food safety, good water quality, and adequate general hygiene are particularly difficult to maintain during this phase of the conflict. As the conflict ages, the soldiers often acquire better housing and protection from the environmental hazards; however, the protracted stresses associated with war fighting, accompanied by more mundane factors such as boredom and separation from families, can

also contribute to immune system dysfunctions and increased disease transmission.

Paradoxically, the impact that conflict-associated diseases have on militaries can improve the success of programs to lower the risk of acquiring those diseases. This observation does not imply in any way that wars are beneficial to public health, but only that the efforts that militaries put toward protecting their troops can improve treatment and prevention of those same diseases in other environments free of conflict or war. For instance, the impact of malaria during WWI and the need to acquire more dependable supplies of the anti-malarial quinine stimulated efforts by the U.S. military to develop cheaper synthetic substitutes like meparene and chloroquine. Similarly, the military's use of insecticidal chemicals such as DDT and BHC served as models for subsequent successful civilian programs to control the disease vectors of malaria and dengue. Also, effective vaccines are often developed initially in response to military need. Finally, one of the most effective repellents used as a personal protective measure against mosquitoes and other vectors, N,N-diethy-meta-toluamide or DEET, was developed by the U.S. Army in large part as a result of its experiences with malaria and other vector-borne diseases during WWII [4]. Such developments are direct results of the attention to the disease during military operations in endemic areas, as well as the medical and scientific expertise and relatively large sums of money available to develop disease interventions for the

Many of these general conclusions about the impact of conflict on disease rates have particular significance to the disease leishmaniasis. This chapter provides a review of conflict leishma‐ niasis, especially as related to two events: (a) the civil war in Sudan and, (b) recent conflicts in

the Middle East involving the militaries of the U.S. and its allies.

displaced.

148 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

population of soldiers.

Leishmaniasis is a result of a parasitic infection by any of several species of the protozoan *Leishmania.* The parasite genus is somewhat catholic in distribution, infecting up to 12 million people on all continents except Australia and Antarctica [5]. Parasites have been identified in Australia but, to date, have not infected humans, only kangaroos and wallabies [6]. The disease manifests in a variety of pathologies depending on the species of parasite and perhaps other factors, with effects ranging from mild scarring to severe disfiguration to death [7]. Localized cutaneous leishmaniasis usually affects body parts that are typically uncovered such as the face and hands. An initial papule lesion often develops into a circumscribed ulcer that may elicit little pain or itching. However, secondary infections are common. Many smaller lesions of the cutaneous form will self-heal, providing incomplete immunity for the person to further infections with the same parasite species; however, multiple lesions may occur. Diffuse cutaneous leishmaniasis may occur when lesions disseminate and this form of the disease often resembles lepromatous leprosy.

A more severe form of the disease usually associated with infections in the New World is mucocutaneous leishmaniasis. This infection often develops in the nasal septum and can result in severe mutilation of the lip, gums, tonsils, pharynx and palate. Damage may be severe enough to cause death through malnutrition and acute respiratory pneumonia.

The most severe form of the disease is visceral leishmaniasis, or kala-azar. This form is usually fatal if left untreated and most deaths from this disease are due to the visceral form. Victims progress through fevers, malaise, and weight loss that are associated with anemia, hepatome‐ galy and spleenomegaly. Secondary bacterial infections are also common and may lead to tuberculosis, pneumonia or diarrhea; these conditions contribute to the high mortality associated with this form of leishmaniasis.

The *Leishmania* parasites spend part of their development cycles in a variety of reservoirs depending on parasite species. For example, for *L. infantum,* a cause of visceral leishmaniasis, researchers believe the reservoirs are domestic dogs, jackals, foxes and perhaps certain rodents. Conversely, humans are considered to be the reservoir for another parasite associated with visceral leishmaniasis in China, *L. donovani*. [5, 8]. More complete reviews of the various forms of the disease are found in other chapters of this book.

Leishmaniasis is a vector-borne disease and the primary means of transmission from reservoir to host is via the phebotomine sand fly, though there have been reports of transmission by blood transfusion [9]. Common names associated with this insect can be misleading because they are known as 'straw mosquitoes' in some parts of their range even though they are not mosquitoes. Also, non-vector insects, such as certain midges, are sometimes known as sand flies so it is important to understand the difference between this vector and other insects. The phlebotomine sand fly is a small, delicate insect which typically flies in short hops close to the surface of the ground. Knowledge about the larval development sites is incomplete so attempts to control the disease through larval control have been frustrating. The adult sand flies are usually less than 3.5 mm in length and are covered with dense "hairs". When at rest they hold their wings in a characteristic "V" shape over their backs. Only the female sand fly takes a blood meal as the blood is essential to complete the development of egg batches. Like the male, her primary source of nutrition is carbohydrates from plant juices. The acquisition of the *Leishma‐ nia* parasite, then, is incidental to taking blood meals. The sand flies usually feed at night or in the early evening. In the New World, they are mostly encountered near forested environments, often being found near particular types of trees or tree buttresses. However, in the Old World, they are mostly associated with rodent burrows in dry or desert environments. Unlike mosquitoes, the larvae do not develop in water, though they do require a moist, warm environment in which to grow. This explains their association with rodent burrows as this environment provides the organic matter needed for nutrition of the growing larvae as well as a degree of protection from extremes in heat and moisture.

There are no vaccines or chemoprophylactic drugs to prevent leishmaniasis, so any focus on the prevention of the disease is usually associated with preventing the bite of an infective sand fly. Such efforts are obviously impeded when the public health infrastructure is disrupted, including during times of conflict.


**4. Role of** *P. aeruginosa* **secreted biomolecules in biofilm formation and virulence**

Biofilm formation is the most preferred stage of many bacterial pathogens. Biofilm formation is a multi-step process to start with i) initial attachment of bacteria to the surface (adhesion) and to each other (aggregation), ii) growth regulations and microcolony formation and production of extracellular polymeric substances (EPS) and other exogenous molecules, iii) maturation of biofilms includes structural stability and iv) dispersal of bacterial cells from the mature biofilm into the environment and reestablishment at a new site [34].

Bacteria in its biofilm state are known to withstand antibacterial agents by many ten's and 100's-fold in comparison to its sessile/planktonic state [35]. Biofilm main composition includes up to 90% bacterially self-secreted biopolymers also known as extracellular polymeric substances (EPS) and other exogenous molecules and 10% bacterial cells [36]. These molecules in combined has been termed as house of bacteria and it shelter bacterial cells from numerous challenges includes antibiotics, antiseptics, detergents, shear mechanical stress, etc. [36]. Exogenous molecules synthesized by *P. aeruginosa* is primarily structured by a complex Quorum Sensing (QS) mechanism [37, 38]. In simple terms, QS is an intracellular communication phenomenon in which bacterial species able to detect and respond to its own cell population and ecological cues by regulating genes that facilitates them in survival and colonization in both biotic and abiotic environment. In *P. aeruginosa* QS is hierarchical and its driven through four known signaling system. At the top or first stage is driven by *las* system that activates the biosynthesis of autoinducing molecules N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and N-butanoyl-L-homoserine lactone (C4-HSL). Binding of LasR-HSL molecules triggers the transcription of second QS system: *rhlR*, *rhlI*, *lasI. LasR system further regulates the third and fourth:* 2-heptyl-3-hydroxy-4-quinolone *(HHQ ) and* pseudomonas quinolone signal (PQS) [39]. These four QS circuits are interconnected and depends on each other regarding *P. aeruginosa* biosynthesis of various secreted and surface attached molecules. These includes extracellular biopolymers (Extracellular DNA, polysaccharides, proteins/ enzymes), biosurfactant (rhamnolipids), metabolites (phenazine/pyocyanin), iron chelator (siderophore: pyoverdine, pyochelin), and bacterium cell surface anchored flagella and pili for swarming and twitching motilities [37–40]. These biomolecules and cell appendages independently or in coordination with each other plays dominant role in *P. aeruginosa* growth, fitness, biofilm formation, virulence, pathogenicity in host during infection, antibacterial resistance, and persistence. In this chapter we emphasize only on the diverse role of *P. aeruginosa* secreted extracellular biomolecules. **Figure 1** summarizes the diverse function of *P. aeruginosa* secreted extracellular biomolecules.

### **4.1 Extracellular DNA production, role in** *P. aeruginosa* **biofilm formation and stability**

The role of extracellular (eDNA) in *P. aeruginosa* biofilm was first highlighted by Whitchurch et al. (2002) [41]. Their study revealed that eDNA is predominant in *P. aeruginosa* matrix component and its essential for *P. aeruginosa* biofilm formation [41]. Followed which numerous discoveries were done highlighting several roles of eDNA in *P. aeruginosa* and in other bacterial pathogens as well as in fungi [42–46]. Structural analysis study revealed that eDNA is similar to bacterial chromosomal DNA in its primary structure and it is not surprising because when chromosomal DNA release from bacterial cells (either via membrane vesicles or cell lysis) into

**59**

eDNA [50].

**Figure 1.**

*Pseudomonas aeruginosa* Secreted Biomolecules and Their Diverse Functions in Biofilm…

its immediate environment is termed as eDNA [47]. eDNA in *P. aeruginosa* cell population is released primarily through QS mechanism [48]. QS system (las and rhl -acyl homoserine lactone and pqs-*Pseudomonas* quinolone signaling), as well as flagella and type IV pili (*fliMpilA)* facilities prophage induction in *P. aeruginosa* cell population and consequently trigger cell lysis and eDNA release [48]. Virulence factor pyocyanin/phenazine biosynthesis also shown to trigger cell lysis (via oxidative stress mediated by hydrogen peroxide) and eDNA release in *P. aeruginosa* [49]. Outer membrane vesicles in *P. aeruginosa* cell also demonstrated to actively release

*immune response in host during infection, evading antibiotics, and other antibacterial agents.*

*Highlighting the major role of biomolecules secreted by* P. aeruginosa*. These biomolecules are essential for establishment of biofilm, bacterial growth, fitness, and survival, induce virulence/pathogenicity and triggering*

Studies have confirmed that eDNA plays a key role in different stages of biofilm formation including initial bacterial to surface attachment (adhesion), bacteriato-bacteria interaction (aggregation), colonization and biofilm formation by connecting cells to cells like nanowires [41–45]. Presence of eDNA on *P. aeruginosa* cell surface have shown to dictates physical surface properties of bacterial cell such as increase in cell surface hydrophobicity and consequently enables physico-chemical interactions forces such as Van der Waals interactions, Acid–Base interactions, hydrophobic interactions that aids in bacterial interactions and biofilm formation [51, 52]. eDNA have proven to induce electrostatic interactions with divalent cations like calcium (Ca2+) and triggers bacterial aggregation [53]. eDNA has been established being an essential factor in structural integrity of *P. aeruginosa* biofilms and many studies have shown that cleaving of DNA using DNase I (enzyme that cleaves DNA through hydrolysis of phosphate di-ester bonds that links nucleotides in DNA) disrupts *P. aeruginosa* adhesion and biofilm formation [41, 44, 45, 54]. Other general roles of eDNA includes nutrient (e.g. good source of carbon, nitrogen, phosphorus) for starving bacteria and facilitate growth, horizontal gene transfer among bacteria cell (antibiotic resistance genes, virulence factor genes, etc), protects biofilms from shear stress by increasing biofilm viscosity. eDNA

*DOI: http://dx.doi.org/10.5772/intechopen.96866*

*Pseudomonas aeruginosa* Secreted Biomolecules and Their Diverse Functions in Biofilm… *DOI: http://dx.doi.org/10.5772/intechopen.96866*

#### **Figure 1.**

*Pseudomonas aeruginosa* - Biofilm Formation, Infections and Treatments

environment and reestablishment at a new site [34].

*P. aeruginosa* secreted extracellular biomolecules.

**virulence**

**4. Role of** *P. aeruginosa* **secreted biomolecules in biofilm formation and**

Biofilm formation is the most preferred stage of many bacterial pathogens. Biofilm formation is a multi-step process to start with i) initial attachment of bacteria to the surface (adhesion) and to each other (aggregation), ii) growth regulations and microcolony formation and production of extracellular polymeric substances (EPS) and other exogenous molecules, iii) maturation of biofilms includes structural stability and iv) dispersal of bacterial cells from the mature biofilm into the

Bacteria in its biofilm state are known to withstand antibacterial agents by many ten's and 100's-fold in comparison to its sessile/planktonic state [35]. Biofilm main composition includes up to 90% bacterially self-secreted biopolymers also known as extracellular polymeric substances (EPS) and other exogenous molecules and 10% bacterial cells [36]. These molecules in combined has been termed as house of bacteria and it shelter bacterial cells from numerous challenges includes antibiotics, antiseptics, detergents, shear mechanical stress, etc. [36]. Exogenous molecules synthesized by *P. aeruginosa* is primarily structured by a complex Quorum Sensing (QS) mechanism [37, 38]. In simple terms, QS is an intracellular communication phenomenon in which bacterial species able to detect and respond to its own cell population and ecological cues by regulating genes that facilitates them in survival and colonization in both biotic and abiotic environment. In *P. aeruginosa* QS is hierarchical and its driven through four known signaling system. At the top or first stage is driven by *las* system that activates the biosynthesis of autoinducing molecules N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and N-butanoyl-L-homoserine lactone (C4-HSL). Binding of LasR-HSL molecules triggers the transcription of second QS system: *rhlR*, *rhlI*, *lasI. LasR system further regulates the third and fourth:* 2-heptyl-

3-hydroxy-4-quinolone *(HHQ ) and* pseudomonas quinolone signal (PQS) [39]. These four QS circuits are interconnected and depends on each other regarding *P. aeruginosa* biosynthesis of various secreted and surface attached molecules. These includes extracellular biopolymers (Extracellular DNA, polysaccharides, proteins/ enzymes), biosurfactant (rhamnolipids), metabolites (phenazine/pyocyanin), iron chelator (siderophore: pyoverdine, pyochelin), and bacterium cell surface anchored flagella and pili for swarming and twitching motilities [37–40]. These biomolecules and cell appendages independently or in coordination with each other plays dominant role in *P. aeruginosa* growth, fitness, biofilm formation, virulence, pathogenicity in host during infection, antibacterial resistance, and persistence. In this chapter we emphasize only on the diverse role of *P. aeruginosa* secreted extracellular biomolecules. **Figure 1** summarizes the diverse function of

**4.1 Extracellular DNA production, role in** *P. aeruginosa* **biofilm formation and**

The role of extracellular (eDNA) in *P. aeruginosa* biofilm was first highlighted by Whitchurch et al. (2002) [41]. Their study revealed that eDNA is predominant in *P. aeruginosa* matrix component and its essential for *P. aeruginosa* biofilm formation [41]. Followed which numerous discoveries were done highlighting several roles of eDNA in *P. aeruginosa* and in other bacterial pathogens as well as in fungi [42–46]. Structural analysis study revealed that eDNA is similar to bacterial chromosomal DNA in its primary structure and it is not surprising because when chromosomal DNA release from bacterial cells (either via membrane vesicles or cell lysis) into

**58**

**stability**

*Highlighting the major role of biomolecules secreted by* P. aeruginosa*. These biomolecules are essential for establishment of biofilm, bacterial growth, fitness, and survival, induce virulence/pathogenicity and triggering immune response in host during infection, evading antibiotics, and other antibacterial agents.*

its immediate environment is termed as eDNA [47]. eDNA in *P. aeruginosa* cell population is released primarily through QS mechanism [48]. QS system (las and rhl -acyl homoserine lactone and pqs-*Pseudomonas* quinolone signaling), as well as flagella and type IV pili (*fliMpilA)* facilities prophage induction in *P. aeruginosa* cell population and consequently trigger cell lysis and eDNA release [48]. Virulence factor pyocyanin/phenazine biosynthesis also shown to trigger cell lysis (via oxidative stress mediated by hydrogen peroxide) and eDNA release in *P. aeruginosa* [49]. Outer membrane vesicles in *P. aeruginosa* cell also demonstrated to actively release eDNA [50].

Studies have confirmed that eDNA plays a key role in different stages of biofilm formation including initial bacterial to surface attachment (adhesion), bacteriato-bacteria interaction (aggregation), colonization and biofilm formation by connecting cells to cells like nanowires [41–45]. Presence of eDNA on *P. aeruginosa* cell surface have shown to dictates physical surface properties of bacterial cell such as increase in cell surface hydrophobicity and consequently enables physico-chemical interactions forces such as Van der Waals interactions, Acid–Base interactions, hydrophobic interactions that aids in bacterial interactions and biofilm formation [51, 52]. eDNA have proven to induce electrostatic interactions with divalent cations like calcium (Ca2+) and triggers bacterial aggregation [53]. eDNA has been established being an essential factor in structural integrity of *P. aeruginosa* biofilms and many studies have shown that cleaving of DNA using DNase I (enzyme that cleaves DNA through hydrolysis of phosphate di-ester bonds that links nucleotides in DNA) disrupts *P. aeruginosa* adhesion and biofilm formation [41, 44, 45, 54]. Other general roles of eDNA includes nutrient (e.g. good source of carbon, nitrogen, phosphorus) for starving bacteria and facilitate growth, horizontal gene transfer among bacteria cell (antibiotic resistance genes, virulence factor genes, etc), protects biofilms from shear stress by increasing biofilm viscosity. eDNA

directly bindings to cationic antibiotics thus inhibits antimicrobial agents' interaction with bacteria within biofilm, removal of eDNA from biofilms have shown increase of bacterial susceptibility to antimicrobial agents [55]. In *P. aeruginosa* biofilm, eDNA release has shown to lower the pH of the local environment and subsequently these acidification initiates antibiotic resistance phenotype genes (PhoPQ and PmrAB) that fosters alteration of lipid A and the manufacture of spermidine on the *P. aeruginosa* outer membrane and consequently decrease entry/intake of aminoglycoside antibiotics [56].

## **4.2 Multitude task of polysaccharides secreted by** *P. aeruginosa*

Many studies have concluded that polysaccharides as a chief component of many bacterial EPS/biofilm matrix. *P. aeruginosa* biosynthesis alginate, psl, and pel as their three predominant extracellular polysaccharides. Alginate producing isolates of *P. aeruginosa* have been acknowledged as a mucoid phenotype regulates through mutation in the alginate biosynthesis of *algA*-*algD* operon and *mucA* [57]. *AlgD* is the key gene that promotes alginate production followed by combined action of *mucA* and *algU* genes [57]. The physical characterizes of alginate positive *P. aeruginosa* colonizes are highly viscous and gelatinous structure on the edge of the cells [58]. This feature is due to its heavy molecular weight structure of alginate which mainly composed of O-acetylated D-mannuronic acid and its C5′ epimer L-guluronic acid [59]. Alginate productions make *P. aeruginosa* virulent strain and a foremost cause for respiratory infections and mortality in CF patients [60]. Alginate production enhances bacterial adhesion due to its sticky nature and its plays key role in shielding *P. aeruginosa* from host immune defense system by scavenging reactive oxygen species (ROS) and evading neutrophils and macrophages mediated phagocytosis [61, 62]. A study by McCaslin in rat alveolar macrophages, showed that alginate in combination with lipopolysaccharide produced by *P. aeruginosa* plays a synergy role in sparking airway inflammation by impeding alveolar function in removal of apoptotic cells and debris [63]. The anionic (negative charge) feature of alginate undergoes electrostatic interactions with cationic aminoglycosides and thus constrains their dissemination into biofilms [64]. Alginate also induce structural and conformational alteration and aggregation in the antimicrobial peptides by binding with it thereby, hinders its antimicrobial activity against pseudomonas [65].

In absence of alginate biosynthesize, *Psl* or *Pel* genes in *P. aeruginosa* isolates up-regulates and activates over production of psl and pel polysaccharide [58]. These polysaccharides by itself or in combination with each other exhibit non-mucoid bacterial colonies/biofilm and these colonies are termed as rugose small colony variant (RSCV) [58]. Psl biosynthesis in *P. aeruginosa* is induced through a QS (*las*) mediated set of *Psl* genes (*PslA-PslL*) and each or group of *Psl* genes and its corresponding protein/enzyme plays a unique role in synthesizing and integrating Psl polysaccharide [58]. For instance, PslB enzyme is responsible for sugar-nucleotide precursor production, whereas, PslA/PslE/PslJ/PslK/PslL and PslF, PslH, and PslI set of enzymes deals with polymerization of polysaccharide, and integration of the activated sugar subunits into the polysaccharide repeating structure [58]. Psl is a neutrally charged polysaccharide comprised of repeating sugar groups: D-mannose, L-rhamnose, and D-glucose [66, 67]. This polysaccharide plays a crucial role in bacterial cell-to-cell communication by enhancing intracellular c-di-GMP (secondary messenger molecule) and essential for initial *P. aeruginosa* attachment to a surface as tested on various clinical, environmental and common laboratory strains, biofilm biomass and antibiotic tolerance (tested on gentamicin) [68, 69].

**61**

*Pseudomonas aeruginosa* Secreted Biomolecules and Their Diverse Functions in Biofilm…

Pel is a positively charged polysaccharide comprised of amino sugar groups and is biosynthesized is regulated via QS (*rhl* sytem) through activating *pel* operons (*pelA-pelG)* [70, 71]. Pel composed of acetylated 1–4 glycosidic linkages of N-acetylgalactosamine and N-acetylglucosamine [71]. PelA protein is responsible for the deacetylase of the sugar amino group, whereas PelD, PelE, PelF, and PelG enzymes, these set of enzymes accountable for Pel polymerization and passage across the *P. aeruginosa* cytoplasmic membrane [58, 71]. Study also speculated that pel is adapted version of LPS [71]. Pel polysaccharide biosynthesize is a strain dependent, and studies shown that in absence of psl polysaccharides pel genes up regulated to form primary structural framework in non-mucoid *P. aeruginosa* biofilms. This indicates that pel plays important role on later stage of biofilm and not during initial adhesion, aggregation, and colonization [58]. Pel being a cationic biopolymer binds to negatively charged eDNA in *P. aeruginosa* biofilm matrix via ionic bonding/electrostatic interactions henceforth, stabilize biofilm matrix

The biosynthesize and secretion of exogenous proteins/enzymes by *P. aeruginosa* is mediated by QS (*las-rhl*) system [73]. The common proteins virulence factor *P. aeruginosa* secrets includes elastase/LAS A and B, exotoxin A, U, S, T, Y phospholipase C, alkaline protease, type IV protease, phospholipase H and lipolytic enzymes [74]. The primary function of these proteins is to play as a virulence factor and induce bacterial pathogenicity in host. To induce pathogenicity, evade host immune defense and damage epithelial cells, *P. aeruginosa* secrets these proteins predominantly via type II and type III secretion system (out of five protein secretion system) [75, 76]. Type II system constituent of protein secretons that facilitate release of exotoxin A, elastase/LasA and LasB proteases, type IV protease, and phospholipase H, as well as lipolytic enzymes into the host cells. Whereas exotoxins U, S, T, and Y are released into host cells via type III secretion system (T3SS) [76]. T3SS forms needle like membrane structure that are anchored to the bacterial cell surface and facilitates delivery of bacterial protein virulence factors into the host

Some actions of *P. aeruginosa* virulence proteins are discussed below. For example, *P. aeruginosa* toxin A protein have shown to impair protein elongation factor in mammalian cells thereby interferes with host essential protein synthesis [77]. The T3SS proteins (Exo U, S, T, Y) have diverse functions such as hinder DNA synthesis and modulates cell morphology in host, escaping host phagocytosis by impairing host cell actin cytoskeleton polymerization and endothelial barriers, phospholipase activity (cleaving host cell lipid layer and increase cell membrane permeability), modulates host inflammatory response and consequently extending bacteria and its virulence factors into host blood stream, different organs to cause bacteraemia and septicaemia and organ failure [78–80]. Metalloproteases are another group of enzymes such as elastase whose main function is to cleave human elastin and leukocyte elastase and neutrophil elastase and consequently alters host tissue elastic property and stimulate tissue damage. Elastases also proven to degrade human collagen II and IV, impair fibroblast growth and destroy wound healing proteins which are essential for mammalian cell and tissue development and wound repair [81–84]. Other crucial role of *P. aeruginosa* elastases includes cleaves host immunoglobulins (IgA and IgG) that aids bacterium to evade host immune response [85, 86]. Clinical studies in burn and wound patients infected with *P. aeruginosa*, showed protease biosynthesis by this bacterium trigger host

*DOI: http://dx.doi.org/10.5772/intechopen.96866*

**4.3** *P. aeruginosa* **exotoxins proteins role in pathogenicity**

frame [72].

epithelial cells [76].

*Pseudomonas aeruginosa* Secreted Biomolecules and Their Diverse Functions in Biofilm… *DOI: http://dx.doi.org/10.5772/intechopen.96866*

Pel is a positively charged polysaccharide comprised of amino sugar groups and is biosynthesized is regulated via QS (*rhl* sytem) through activating *pel* operons (*pelA-pelG)* [70, 71]. Pel composed of acetylated 1–4 glycosidic linkages of N-acetylgalactosamine and N-acetylglucosamine [71]. PelA protein is responsible for the deacetylase of the sugar amino group, whereas PelD, PelE, PelF, and PelG enzymes, these set of enzymes accountable for Pel polymerization and passage across the *P. aeruginosa* cytoplasmic membrane [58, 71]. Study also speculated that pel is adapted version of LPS [71]. Pel polysaccharide biosynthesize is a strain dependent, and studies shown that in absence of psl polysaccharides pel genes up regulated to form primary structural framework in non-mucoid *P. aeruginosa* biofilms. This indicates that pel plays important role on later stage of biofilm and not during initial adhesion, aggregation, and colonization [58]. Pel being a cationic biopolymer binds to negatively charged eDNA in *P. aeruginosa* biofilm matrix via ionic bonding/electrostatic interactions henceforth, stabilize biofilm matrix frame [72].

#### **4.3** *P. aeruginosa* **exotoxins proteins role in pathogenicity**

The biosynthesize and secretion of exogenous proteins/enzymes by *P. aeruginosa* is mediated by QS (*las-rhl*) system [73]. The common proteins virulence factor *P. aeruginosa* secrets includes elastase/LAS A and B, exotoxin A, U, S, T, Y phospholipase C, alkaline protease, type IV protease, phospholipase H and lipolytic enzymes [74]. The primary function of these proteins is to play as a virulence factor and induce bacterial pathogenicity in host. To induce pathogenicity, evade host immune defense and damage epithelial cells, *P. aeruginosa* secrets these proteins predominantly via type II and type III secretion system (out of five protein secretion system) [75, 76]. Type II system constituent of protein secretons that facilitate release of exotoxin A, elastase/LasA and LasB proteases, type IV protease, and phospholipase H, as well as lipolytic enzymes into the host cells. Whereas exotoxins U, S, T, and Y are released into host cells via type III secretion system (T3SS) [76]. T3SS forms needle like membrane structure that are anchored to the bacterial cell surface and facilitates delivery of bacterial protein virulence factors into the host epithelial cells [76].

Some actions of *P. aeruginosa* virulence proteins are discussed below. For example, *P. aeruginosa* toxin A protein have shown to impair protein elongation factor in mammalian cells thereby interferes with host essential protein synthesis [77]. The T3SS proteins (Exo U, S, T, Y) have diverse functions such as hinder DNA synthesis and modulates cell morphology in host, escaping host phagocytosis by impairing host cell actin cytoskeleton polymerization and endothelial barriers, phospholipase activity (cleaving host cell lipid layer and increase cell membrane permeability), modulates host inflammatory response and consequently extending bacteria and its virulence factors into host blood stream, different organs to cause bacteraemia and septicaemia and organ failure [78–80]. Metalloproteases are another group of enzymes such as elastase whose main function is to cleave human elastin and leukocyte elastase and neutrophil elastase and consequently alters host tissue elastic property and stimulate tissue damage. Elastases also proven to degrade human collagen II and IV, impair fibroblast growth and destroy wound healing proteins which are essential for mammalian cell and tissue development and wound repair [81–84]. Other crucial role of *P. aeruginosa* elastases includes cleaves host immunoglobulins (IgA and IgG) that aids bacterium to evade host immune response [85, 86]. Clinical studies in burn and wound patients infected with *P. aeruginosa*, showed protease biosynthesis by this bacterium trigger host

*Pseudomonas aeruginosa* - Biofilm Formation, Infections and Treatments

**4.2 Multitude task of polysaccharides secreted by** *P. aeruginosa*

entry/intake of aminoglycoside antibiotics [56].

activity against pseudomonas [65].

directly bindings to cationic antibiotics thus inhibits antimicrobial agents' interaction with bacteria within biofilm, removal of eDNA from biofilms have shown increase of bacterial susceptibility to antimicrobial agents [55]. In *P. aeruginosa* biofilm, eDNA release has shown to lower the pH of the local environment and subsequently these acidification initiates antibiotic resistance phenotype genes (PhoPQ and PmrAB) that fosters alteration of lipid A and the manufacture of spermidine on the *P. aeruginosa* outer membrane and consequently decrease

Many studies have concluded that polysaccharides as a chief component of many bacterial EPS/biofilm matrix. *P. aeruginosa* biosynthesis alginate, psl, and pel as their three predominant extracellular polysaccharides. Alginate producing isolates of *P. aeruginosa* have been acknowledged as a mucoid phenotype regulates through mutation in the alginate biosynthesis of *algA*-*algD* operon and *mucA* [57]. *AlgD* is the key gene that promotes alginate production followed by combined action of *mucA* and *algU* genes [57]. The physical characterizes of alginate positive *P. aeruginosa* colonizes are highly viscous and gelatinous structure on the edge of the cells [58]. This feature is due to its heavy molecular weight structure of alginate which mainly composed of O-acetylated D-mannuronic acid and its C5′ epimer L-guluronic acid [59]. Alginate productions make *P. aeruginosa* virulent strain and a foremost cause for respiratory infections and mortality in CF patients [60]. Alginate production enhances bacterial adhesion due to its sticky nature and its plays key role in shielding *P. aeruginosa* from host immune defense system by scavenging reactive oxygen species (ROS) and evading neutrophils and macrophages mediated phagocytosis [61, 62]. A study by McCaslin in rat alveolar macrophages, showed that alginate in combination with lipopolysaccharide produced by *P. aeruginosa* plays a synergy role in sparking airway inflammation by impeding alveolar function in removal of apoptotic cells and debris [63]. The anionic (negative charge) feature of alginate undergoes electrostatic interactions with cationic aminoglycosides and thus constrains their dissemination into biofilms [64]. Alginate also induce structural and conformational alteration and aggregation in the antimicrobial peptides by binding with it thereby, hinders its antimicrobial

In absence of alginate biosynthesize, *Psl* or *Pel* genes in *P. aeruginosa* isolates up-regulates and activates over production of psl and pel polysaccharide [58]. These polysaccharides by itself or in combination with each other exhibit non-mucoid bacterial colonies/biofilm and these colonies are termed as rugose small colony variant (RSCV) [58]. Psl biosynthesis in *P. aeruginosa* is induced through a QS (*las*) mediated set of *Psl* genes (*PslA-PslL*) and each or group of *Psl* genes and its corresponding protein/enzyme plays a unique role in synthesizing and integrating Psl polysaccharide [58]. For instance, PslB enzyme is responsible for sugar-nucleotide precursor production, whereas, PslA/PslE/PslJ/PslK/PslL and PslF, PslH, and PslI set of enzymes deals with polymerization of polysaccharide, and integration of the activated sugar subunits into the polysaccharide repeating structure [58]. Psl is a neutrally charged polysaccharide comprised of repeating sugar groups: D-mannose, L-rhamnose, and D-glucose [66, 67]. This polysaccharide plays a crucial role in bacterial cell-to-cell communication by enhancing intracellular c-di-GMP (secondary messenger molecule) and essential for initial *P. aeruginosa* attachment to a surface as tested on various clinical, environmental and common laboratory strains, biofilm

biomass and antibiotic tolerance (tested on gentamicin) [68, 69].

**60**

cytokinin (interleukins IL6 and IL8) production and induce severe inflammation, septicaemia and elevates mortality level in patients [87–89].

## **4.4 Rhamnolipids** *P. aeruginosa* **biosurfactant**

Rhamnolipids is a glycolipid biosurfactant produced by *P. aeruginosa* mediated through *rhl* QS system involving operons *rhlA*, *rhlB* for biosynthesis and *rhlI* and *rhlR* for regulation [90]. It is made up of sugar group (rhamnose) and a lipid/fatty acid group 3-(hydroxyalkanoyloxy) alkanoic acid and has a both hydrophilic and hydrophobic group like any typical biosurfactant [90, 91]. Rhamnolipids production helps *P. aeruginosa* in uptake and metabolism of hydrophobic molecules such as oils, hexadecane for nutritional source and growth [92]. Rhamnolipids (monorhamnolipids) also adhere to *P. aeruginosa* cell membrane (LPS) and plays key role in influencing *P. aeruginosa* cell surface physical property such as increasing cell surface hydrophobicity which aids in bacterial adhesion to substratum and bacterial cell-to-cell aggregation through hydrophobic interactions [93]. Rhamnolipids also lower the surface tension of *P. aeruginosa* cell surface thus aid them in swarming motility to travel across different location within the substratum [93]. It also proven to influence biofilm architecture by establishing and sustaining fluid channels in biofilms for water and oxygen transport [94]. *P. aeruginosa* employs rhamnolipids to their own advantage to eradicate competing bacteria. Binding of rhamnolipids into competing bacterial cell membrane consequently creates pores and increase cell permeability to induce cell lysis [95]. It is also a known virulence element, by binding to epithelial cell membrane it interrupts epithelial cell membrane integration, disrupts epithelial cell junctions, and triggers death in various mammalian cell types includes leukocytes, macrophages [96]. Rhamnolipids biosynthesis by *P. aeruginosa* in infected patients has been associated with escalation in pathogenicity in cystic fibrosis lung, ventilator-associated pneumonia patients [97].

#### **4.5 Pyocyanin a unique virulence factor and its diverse function**

*P. aeruginosa* biosynthesis and secretes a unique secondary metabolite called phenazines. Different types of phenazines are produced by *P. aeruginosa* however, pyocyanin is the most predominant one. Pyocyanin biosynthesis occurs at the later stage in *P. aeruginosa* population density or in biofilm, in laboratory culture it is generally expressed at the late exponential stage via regulation through QS (PQS) system [98]. Pyocyanin production is easily identified by its color, bluish -pure pyocyanin and green color when grown in laboratory in bacterial growth media (e.g. Tryptone Soy broth, Nutrient media, Luria broth, these media are all yellow in color and blue pyocyanin mix with yellow turns green). The two set genes of *phzA1-G1 and phzA2-G2* encrypts initial phenazine molecule (phenazine-1-carboxylic acid, PCA) followed by conversion of PCA to pyocyanin (N-methyl-1 hydroxyphenazine) encoded by genes *phzM* and *phzS* [98]. Pyocyanin production has been associated with the severity of infection and acknowledged as a hyper virulent strain [99]. Analysis of pyocyanin production on variety of clinical and environmental isolates indicates pyocyanin production is very common among all isolates however, the amount of pyocyanin production is depended upon strain phenotype and genotype variations. A study by Fothergill et al. (2007) on strains isolated from different clinical sites (CF, keratitis) and environmental (water) strains indicated that Liverpool epidermic strain (LES) from CF patients (attended Liverpool CF centre in England between years 1995 to 2004) exhibited significantly high pyocyanin production in comparison to keratitis and water isolates [99]. Pyocyanin plays diverse role in establishment of *P. aeruginosa* biofilm formation

**63**

*Pseudomonas aeruginosa* Secreted Biomolecules and Their Diverse Functions in Biofilm…

including inducing oxidative stress in competing bacteria and outcompete their growth (e.g. *S. aureus*) and fungi (e.g. *candida albicans*) [100, 101]. Pyocyanin promote cell signaling by activating transcription factor SoxR and stimulating various genes expression includes efflux pump genes *mexGHI-opmD*, and *PA2274* (monooxygenase, to control oxidative stress response in *P. aeruginosa*) [102]. By regulating target genes pyocyanin also maintain bacterial fitness, pyocyanin/

phenazine deficient mutant (Δ*phz*) showed drastic change in its colony morphology (wrinkled colony), whereas pyocyanin over producing mutant strain (DKN370) remained smooth [103]. Pyocyanin induce oxidative stress and cell death (via H2O2 production) in *P. aeruginosa* population in late exponential phase and triggers eDNA production [49]. An interesting discovery by Das et al. 2012 and 2015 revealed that pyocyanin intercalates with DNA and influence *P. aeruginosa* cell surface hydropho-

Pyocyanin has been in limelight in many decades due to its virulence property. In context to *P. aeruginosa* infection in human, pyocyanin production has been linked to increase in virulence and severity of infection [99]. Different studies reported different concentration of pyocyanin to be found in sputum of CF patients from 0.9 to 16.5 μg/ml and 27.3 μg/ml in bronchitis patients sputum and also significantly higher amount (5.3 μg/g) also found in burn wound exudates [105, 106]. In mammalian cells, it declines intracellular cAMP and ATP levels, provoke neutrophils apoptosis, and modulates host immune system [105–108]. Pyocyanin being a zwitter ion (positive and negative charge group and can penetrate into host cell membrane), and redox (electron donating and accepting property) molecule it oxidized cytosol (mammalian intracellular fluid), produces reactive oxygen species (ROS) by diffusing into host cells and undergoes redox reaction to accept electrons from NADPH and donates to molecular oxygen [109, 110]. ROS production leads to the decline in intracellular glutathione (a master antioxidant in mammalian cells essential for cell health and fitness) level which leads to bronchial epithelial cell

) secretion and

bicity and subsequently promote biofilm formation [51, 104].

death and tissue damage [109, 110]. It also impedes chlorine ion (Cl<sup>−</sup>

**4.6 Siderophore benefits** *P. aeruginosa* **growth and biofilm formation**

Siderophore are small molecules and belongs to the class of "iron-chelating compounds". They are intrinsically secreted by microorganisms primarily for scavenging and uptake of Ferric ion, Fe3+ for their own benefits including nutrition, metabolism, growth, and virulence in mammals [114]. For example, Bacillus spp. *(subtilis and anthracis*) biosynthesis primary siderophore (bacillibactin), enterobactin, vibriobactin, yersinibactin, and pyoverdine by *E.coli*, *Vibrio cholerae*, *Yersinia pestis* and *P. aeruginosa* respectively. Pyoverdine is a fluorescent green color compound and its biosynthesis is encoded by the operons of *pvd*. Pyoverdine forages Fe3+ from host iron-binding molecules (transferrin) and binds strongly to it thus contribute to pathogenicity in host as shown in the immunocompromised mouse model [115, 116]. Pyoverdine also benefits from *P. aeruginosa* virulence factor protease action in degrading human iron-binding protein (ferritin), thus outcompetes host and scavenges iron [117]. Burn mouse model study have shown that pyoverdine

and mucous hypersecretion by [113].

transport in CF patients' lungs (bronchial epithelial cells) and consequently halt mucous clearance in human airways [111]. In burn wound patients infected with *P. aeruginosa*, pyocyanin production shown to provoke premature senescence and apprehend human fibroblast growth by levying oxidative stress [106, 112]. Mouse model study revealed that exposing pyocyanin to mouse lung airways triggers repress of transcription factors protein FoxA2 expression (essential for tissue development) and consequently leads to over production of host cells (cell hyperplasia)

*DOI: http://dx.doi.org/10.5772/intechopen.96866*

*Pseudomonas aeruginosa* Secreted Biomolecules and Their Diverse Functions in Biofilm… *DOI: http://dx.doi.org/10.5772/intechopen.96866*

including inducing oxidative stress in competing bacteria and outcompete their growth (e.g. *S. aureus*) and fungi (e.g. *candida albicans*) [100, 101]. Pyocyanin promote cell signaling by activating transcription factor SoxR and stimulating various genes expression includes efflux pump genes *mexGHI-opmD*, and *PA2274* (monooxygenase, to control oxidative stress response in *P. aeruginosa*) [102]. By regulating target genes pyocyanin also maintain bacterial fitness, pyocyanin/ phenazine deficient mutant (Δ*phz*) showed drastic change in its colony morphology (wrinkled colony), whereas pyocyanin over producing mutant strain (DKN370) remained smooth [103]. Pyocyanin induce oxidative stress and cell death (via H2O2 production) in *P. aeruginosa* population in late exponential phase and triggers eDNA production [49]. An interesting discovery by Das et al. 2012 and 2015 revealed that pyocyanin intercalates with DNA and influence *P. aeruginosa* cell surface hydrophobicity and subsequently promote biofilm formation [51, 104].

Pyocyanin has been in limelight in many decades due to its virulence property. In context to *P. aeruginosa* infection in human, pyocyanin production has been linked to increase in virulence and severity of infection [99]. Different studies reported different concentration of pyocyanin to be found in sputum of CF patients from 0.9 to 16.5 μg/ml and 27.3 μg/ml in bronchitis patients sputum and also significantly higher amount (5.3 μg/g) also found in burn wound exudates [105, 106]. In mammalian cells, it declines intracellular cAMP and ATP levels, provoke neutrophils apoptosis, and modulates host immune system [105–108]. Pyocyanin being a zwitter ion (positive and negative charge group and can penetrate into host cell membrane), and redox (electron donating and accepting property) molecule it oxidized cytosol (mammalian intracellular fluid), produces reactive oxygen species (ROS) by diffusing into host cells and undergoes redox reaction to accept electrons from NADPH and donates to molecular oxygen [109, 110]. ROS production leads to the decline in intracellular glutathione (a master antioxidant in mammalian cells essential for cell health and fitness) level which leads to bronchial epithelial cell death and tissue damage [109, 110]. It also impedes chlorine ion (Cl<sup>−</sup> ) secretion and transport in CF patients' lungs (bronchial epithelial cells) and consequently halt mucous clearance in human airways [111]. In burn wound patients infected with *P. aeruginosa*, pyocyanin production shown to provoke premature senescence and apprehend human fibroblast growth by levying oxidative stress [106, 112]. Mouse model study revealed that exposing pyocyanin to mouse lung airways triggers repress of transcription factors protein FoxA2 expression (essential for tissue development) and consequently leads to over production of host cells (cell hyperplasia) and mucous hypersecretion by [113].

#### **4.6 Siderophore benefits** *P. aeruginosa* **growth and biofilm formation**

Siderophore are small molecules and belongs to the class of "iron-chelating compounds". They are intrinsically secreted by microorganisms primarily for scavenging and uptake of Ferric ion, Fe3+ for their own benefits including nutrition, metabolism, growth, and virulence in mammals [114]. For example, Bacillus spp. *(subtilis and anthracis*) biosynthesis primary siderophore (bacillibactin), enterobactin, vibriobactin, yersinibactin, and pyoverdine by *E.coli*, *Vibrio cholerae*, *Yersinia pestis* and *P. aeruginosa* respectively. Pyoverdine is a fluorescent green color compound and its biosynthesis is encoded by the operons of *pvd*. Pyoverdine forages Fe3+ from host iron-binding molecules (transferrin) and binds strongly to it thus contribute to pathogenicity in host as shown in the immunocompromised mouse model [115, 116]. Pyoverdine also benefits from *P. aeruginosa* virulence factor protease action in degrading human iron-binding protein (ferritin), thus outcompetes host and scavenges iron [117]. Burn mouse model study have shown that pyoverdine

*Pseudomonas aeruginosa* - Biofilm Formation, Infections and Treatments

septicaemia and elevates mortality level in patients [87–89].

**4.4 Rhamnolipids** *P. aeruginosa* **biosurfactant**

cytokinin (interleukins IL6 and IL8) production and induce severe inflammation,

Rhamnolipids is a glycolipid biosurfactant produced by *P. aeruginosa* mediated through *rhl* QS system involving operons *rhlA*, *rhlB* for biosynthesis and *rhlI* and *rhlR* for regulation [90]. It is made up of sugar group (rhamnose) and a lipid/fatty acid group 3-(hydroxyalkanoyloxy) alkanoic acid and has a both hydrophilic and hydrophobic group like any typical biosurfactant [90, 91]. Rhamnolipids production helps *P. aeruginosa* in uptake and metabolism of hydrophobic molecules such as oils, hexadecane for nutritional source and growth [92]. Rhamnolipids (monorhamnolipids) also adhere to *P. aeruginosa* cell membrane (LPS) and plays key role in influencing *P. aeruginosa* cell surface physical property such as increasing cell surface hydrophobicity which aids in bacterial adhesion to substratum and bacterial cell-to-cell aggregation through hydrophobic interactions [93]. Rhamnolipids also lower the surface tension of *P. aeruginosa* cell surface thus aid them in swarming motility to travel across different location within the substratum [93]. It also proven to influence biofilm architecture by establishing and sustaining fluid channels in biofilms for water and oxygen transport [94]. *P. aeruginosa* employs rhamnolipids to their own advantage to eradicate competing bacteria. Binding of rhamnolipids into competing bacterial cell membrane consequently creates pores and increase cell permeability to induce cell lysis [95]. It is also a known virulence element, by binding to epithelial cell membrane it interrupts epithelial cell membrane integration, disrupts epithelial cell junctions, and triggers death in various mammalian cell

types includes leukocytes, macrophages [96]. Rhamnolipids biosynthesis by *P. aeruginosa* in infected patients has been associated with escalation in pathogenic-

*P. aeruginosa* biosynthesis and secretes a unique secondary metabolite called phenazines. Different types of phenazines are produced by *P. aeruginosa* however, pyocyanin is the most predominant one. Pyocyanin biosynthesis occurs at the later stage in *P. aeruginosa* population density or in biofilm, in laboratory culture it is generally expressed at the late exponential stage via regulation through QS (PQS) system [98]. Pyocyanin production is easily identified by its color, bluish -pure pyocyanin and green color when grown in laboratory in bacterial growth media (e.g. Tryptone Soy broth, Nutrient media, Luria broth, these media are all yellow in color and blue pyocyanin mix with yellow turns green). The two set genes of *phzA1-G1 and phzA2-G2* encrypts initial phenazine molecule (phenazine-1-carboxylic acid, PCA) followed by conversion of PCA to pyocyanin (N-methyl-1 hydroxyphenazine) encoded by genes *phzM* and *phzS* [98]. Pyocyanin production has been associated with the severity of infection and acknowledged as a hyper virulent strain [99]. Analysis of pyocyanin production on variety of clinical and environmental isolates indicates pyocyanin production is very common among all isolates however, the amount of pyocyanin production is depended upon strain phenotype and genotype variations. A study by Fothergill et al. (2007) on strains isolated from different clinical sites (CF, keratitis) and environmental (water) strains indicated that Liverpool epidermic strain (LES) from CF patients (attended Liverpool CF centre in England between years 1995 to 2004) exhibited significantly high pyocyanin production in comparison to keratitis and water isolates [99]. Pyocyanin plays diverse role in establishment of *P. aeruginosa* biofilm formation

ity in cystic fibrosis lung, ventilator-associated pneumonia patients [97].

**4.5 Pyocyanin a unique virulence factor and its diverse function**

**62**

contribute to severity in infection and mutants deficient in pyoverdine production showed significantly less virulence [116]. Infection model study in *Caenorhabditis elegans*, showed that pyoverdine penetrates host cells and undermines mitochondrial dynamics and triggers hypoxic response thus hinders ATP generation in host [118]. Other features of pyoverdine including communicating molecule to control biosynthesis of virulence proteins in *P. aeruginosa* including exotoxin A and protease [119]. Iron is essential to sustain bacterial growth thus pyoverdine aids in survival of *P. aeruginosa* in infection site, triggers biofilm formation where, pyoverdine deficient mutant strains forms fragile biofilm [120]. *P. aeruginosa* also produces another siderophore molecule called pyochelin, however pyochelin has lower affinity for Fe3+ than pyoverdine. However, this pyochelin-iron complex in coordination with pyocyanin undergoes oxidative-reductive reaction and contribute to oxidative damage (via hydroxyl radical formation) and inflammation in host [121, 122]. In CF patients pyochelin found to be involved in inflammation and tissue damage [123].


**Abstract**

The desire of more and more segments of the population for self-improvement and self-expression through amateur sports has acquired a global scale. The evaluation and analysis of the amateur athletes' achievements in the chosen physical activity become of practical importance for most amateur athletes and open up the possibility of developing innovative interventions to stimulate the engagement in physical activity of as much population as possible. For this purpose, we have developed an elementary methodology for monitoring the achieved level of strength and overall endurance of students, designed to monitor and motivate amateur athletes of various types of physical activity. The purpose of this research project was to conduct the targeted analysis of the array of physiological indicators of those engaged in physical activity, and also to justify and test technologies for collecting and interpreting objective indicators of performing elementary physical test exercises and calculating their dependence on objective indicators of the cardiovascular system. Subsequently, we planned to develop a simple methodology for collecting, evaluating, and interpreting test information. We suggest using this technique in the daily training sessions of amateur athletes of various qualifications.

**Keywords:** motor activity, physical activity, age-related changes, heart diseases, cardiovascular and respiratory systems, lifestyle, physical exercises, health promotion, physical training, physical fitness, functional state, working capacity, adaptive capabilities, cardiovascular system, regular physical activity, mobile services, endurance development, fitness level, healthy lifestyle


*Article* **Neutron Diffraction Study of the Martensitic Transformation of Ni2.07Mn0.93Ga Heusler Alloy**

**Lara Righi 1,2**

1 Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, Parco Area delle Scienze 17/A, 43124 Parma, Italy<EMAIL_ADDRESS>
2 IMEM-CNR, Parco Area delle Scienze 37/A, 43124 Parma, Italy

**Abstract:** The martensitic transition featuring the ternary Heusler alloy Ni2.09Mn0.91Ga was investigated by neutron diffraction. Differential scanning calorimetry indicated that structural transition starts at 230 K on cooling with a significant increase in the martensitic transformation onset compared to the classical Ni2MnGa. The low-temperature martensite presents the 5M type of modulated structure, and the structural analysis was performed by the application of the superspace approach. As already observed in Mn-rich modulated martensites, the periodical distortion corresponds to an incommensurate wave-like shift of the atomic layers. The symmetry of the modulated martensite at 220 K is orthorhombic with unit cell constants *a* = 4.2172(3) Å, *b* = 5.5482(2) Å, and *c* = 4.1899(2) Å; space group Immm(00γ)s00; and modulation vector *q* = *γc*\* = 0.4226(5)*c*\*. Considering the different neutron scattering lengths of the elements involved in this alloy, it was possible to ascertain that the chemical composition was Ni2.07Mn0.93Ga, close to the nominal formula. In order to characterize the martensitic transformation upon increasing the temperature, a series of neutron diffraction patterns was collected at different temperatures. The structural analysis indicated that the progressive change of the martensitic lattice is characterized by the exponential change of the *c*/*a* parameter approaching the limit value *c*/*a* = 1 of the cubic austenite.

**Keywords:** Heusler alloys; martensitic transformation; neutron diffraction

#### **1. Introduction**

Ni-Mn-*X* with *X* = Ga, In, Sn, Sb Heusler alloys are considered the prototypical multifunctional material encompassing a huge magnetocaloric effect (MCE) [1], magnetic field giant induced strains (MFIS), magnetic shape memory effect, and interesting exchange bias phenomena [2–4]. Most of the stunning physical properties of this class of intermetallics are based on the martensitic phase transformation (MT) taking place by temperature change [5,6], application of mechanical loads [7,8], or induced by the application of an external magnetic field [3,4]. In Heusler alloys, the parent phase exhibits the L21 superstructure, and the martensitic transition is accompanied by a lattice distortion yielding tetragonal, orthorhombic, or monoclinic structures (see Figure 1) depending on the chemical composition [6,7]. In the case of Ni-Mn based alloys, it is frequently observed that the martensitic phases are featured by modulated structural distortions generally associated to five-layered or seven-layered supercells defined 5M or 7M, respectively [7,9]. In Ni2MnGa, the martensitic phase stable below 200 K is characterized by an incommensurate modulated 5M-type crystal structure that was successfully solved within the crystallographic approach called superspace [9,10]. Indeed, the MCE is related to the drastic entropy change that can be induced by a magnetic field change during the martensitic transformation or eventually at the corresponding Curie temperature [11].

**Citation:** Righi, L. Neutron Diffraction Study of the Martensitic Transformation of Ni2.07Mn0.93Ga Heusler Alloy. *Metals* **2021**, *11*, 1749. https://doi.org/10.3390/ met11111749

Academic Editor: Enrique Jimenez-Melero

Received: 29 September 2021 Accepted: 27 October 2021 Published: 31 October 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Figure 1.** Fundamental crystal structures of austenitic (**a**) and martensitic (**b**) phases wherein the colored spheres define different crystallographic sites (light green = Ga, cyan = Ni, pink = Mn). (**c**) Reciprocal relation between the two lattices. The a and m subscripts of the crystallographic axes stand for austenite and martensite respectively.

In this context, the compositional combination Ni2+*x*Mn1−*x*Ga has received increasing attention [12]. Large magnetocaloric effects in single crystals of Ni2+*x*Mn1−*x*Ga [13] alloy are observed upon application of magnetic fields suitable for diffuse technological applications. A recently published study is focused on the intermartensitic transformation occurring for the variant Ni2.15Mn0.85Ga [14]. The authors described a structural transition occurring at 215 K from the low-temperature tetragonal L10 phase to monoclinic modulated 7Mtype martensite. Furthermore, the martensitic 7M phase is stable at room temperature, and the transition from martensite to austenite is reported to take place beyond 325 K. A further increment of Ni to reach Ni2.18Mn0.82Ga composition stabilizes the tetragonal L10 martensite at room temperature.

Moreover, in such a Ni-rich alloy both the MT and Curie temperature merge in a magnetostructural first-order transition [15,16] with a beneficial effect on the MCE outcome. Besides compositions showing 0.15 ≤ *x* ≤ 0.18, the compositional regimes based on *x* < 0.1 include valuable magnetic and MCE properties. Single crystals with Ni2.04Mn0.96Ga composition were investigated by T. Liang [17] for the giant strains induced by external magnetic field. Strain-temperature curves evidenced that the martensitic phase undergoes below 240 K on cooling. Moreover, it was demonstrated that off-stoichiometric Ni2.08Mn1.04Ga0.88 alloy shows interesting magnetocaloric properties in polycrystalline ribbons [14]. Notably, in a Ni2+*x*Mn1−*x*Ga system, the compositions showing a moderate excess of Ni are still not completely explored, and structural studies of the martensitic transitions have not yet been reported. In this work, the structural investigation of the nominal Ni2.09Mn0.98Ga Heusler alloy was undertaken by neutron diffraction experiments. It was found that, below 230 K, the martensitic phase displays an incommensurate 5M modulated crystal structure. The structural refinement based on the neutron diffraction data was achieved by employing the model obtained for the Ni2MnGa counterpart. Furthermore, the study of the structural transformation versus temperature points out that the progressive change of the martensitic lattice is closely related to geometrical constraints in agreement with the invariant plane strain requirements.

#### **2. Materials and Methods**

The sample with nominal composition Ni2.09Mn0.91Ga was obtained from high purity elements by arc melting and subsequent annealing in a quartz ampoule under Ar atmosphere and quenched in cold water. The annealing treatment was performed at 983 K for 72 h. Afterwards, the small ingots were ground in a mortar and successively annealed at 1073 K for 2 h in order to remove the defects induced by the grinding process. Powder X-ray diffraction measurement, performed with an X'TRA Thermo diffractometer (Thermo Fisher Scientific, Losanna, Switzerland) equipped with a CuKα source and a solid-state detector, indicated the presence of single-phase austenite (see Figure S1 in the Supplementary Information).

Differential scanning calorimetry (DSC) was carried out under nitrogen in the temperature range from room temperature to 200 K and then upon heating from 200 K to 350 K. The cooling and heating scans were performed with a rate of 10 K/min

Neutron diffraction experiments were performed on the D2B beamline (ILL, Grenoble, France) at ILL (Institute Laue-Longevin, Grenoble, France) with a wavelength of 1.594 Å in the temperature range from 220 K to 350 K, and diffraction patterns were collected at selected temperatures. The low-temperature diffraction pattern (220 K) used to determine the crystal structure of the martensitic phase was recorded at a low scanning rate in order to assure the required statistics and a good peak/background ratio. The structural refinements were based on the Rietveld method [18] with the JANA2006 suite (Institute of Physics Department of Structure Analysis, Praha, Czech Republic).

#### **3. Results and Discussion**

The critical temperature of the martensitic transition for the prepared Ni-rich composition was determined by thermal analysis. The DSC measurement, illustrated in Figure 2a, manifested a pronounced transformation at 231 K on cooling, and the reverse martensitic transformation is observed at 244 K. The minute substitution of Mn with Ni in the stoichiometric Ni2MnGa alloy induces an increase of 35 K at the MT onset. Indeed, the critical temperatures recorded by DSC scans are in agreement with those reported for a similar Ni-rich composition [17].

**Figure 2.** (**a**) DSC scan of the Ni2.07Mn0.93Ga sample (Ms = start of martensite, Mf = finish of martensite, As = start of austenite, Af = finish of austenite). (**b**) Neutron diffraction patterns in the 24–37◦ 2*θ* range collected at different temperatures.

A neutron diffraction experiment on the polycrystalline alloy with the nominal formula Ni2.09Mn0.91Ga was undertaken to establish the martensitic structure and the structural evolution of the martensitic transformation. In Figure 2b, a limited region of the neutron diffraction patterns collected at different increasing temperatures is shown. In accordance with the indication provided by DSC, the martensitic transition takes place beyond 240 K.

The phase diagram accounting for the *T*c and MT in the Ni2+*x*Mn1−*x*Ga system and reported by V.V. Kovaylo et al. [12] indicates that for a moderate excess of Ni, the paramagnetic state occurs above 350 K. Therefore, a neutron diffraction collection was performed at 350 K above *T*c in order to suppress the magnetic contribution to the scattering and determine the chemical composition of the austenitic phase. Neutron diffraction data are particularly suited for the study of the chemical order in the Heusler lattice. The neutron scattering lengths are not Z-dependent as in the case of X-ray diffraction [19], and this allows the faithful determination of occupancy factors for each crystallographic site from powder diffraction experiments. Neutron diffraction studies [20] dealing with the characterization of Heusler alloys have demonstrated that a Rietveld refinement is a suitable tool for the reliable determination of the stoichiometric composition of the pre-

pared alloys. In the case of this Ni-rich alloy, the structural refinement was based on the cubic structure showing the Fm-3m space group typical for the L21 superstructure. The crystal data obtained by the structural refinement are listed in the Tables S1 and S2 reported in SI. The structural model was conceived by considering an excess of Ni in the site of Mn; this assumption is in agreement with previous studies [15–17] that accounted for the predominant tendency of the Ni excess to occupy the Mn position. The chemical ratio of Ni and Mn located in the same site was constrained to maintaining the global occupancy of the site equivalent to 1. The best fitting (see Figure S2) was obtained with a cubic structure with the unit cell constant *a* = 5.83194(4) Å and occupancy factors of 0.93(5) and 0.07(5) for Mn and Ni, respectively, in the 2*a* site, yielding a chemical composition of the austenitic phase corresponding to Ni2.07Mn0.93Ga showing a slight deviation from the nominal formula. Since the martensitic transition is a diffusion-less process, the actual atomic distribution in the ordered lattice is preserved in the martensitic phase as well. The neutron diffraction pattern collected at 220 K shows a close analogy with that reported for the stoichiometric Ni2MnGa compound [21]. As remarked before, below 200 K, the martensitic phase of Ni2MnGa possesses an incommensurate modulated structure that was successfully solved with the application of the superspace approach [22]. The superspace approach has been recurrently employed to analyze the 5M and 7M modulated structures of martensites in Ni-Mn-based Heusler alloys [9,10,23–25]. In agreement with the crystallographic representation of these complex structures [21], the diffraction peaks are divided into two groups: the main reflections are related to the basic body-centered martensitic lattice depicted in Figure 1b, and the weak peaks called satellites are associated with the modulation vector *q* = *αa***\*** + *βb***\*** + *γc***\*** connected to the incommensurate distortion of the fundamental crystal structure. To perform the Rietveld refinement of the neutron diffraction pattern collected at 220 K, the orthorhombic model derived for 5M modulated martensite in Ni2MnGa Heusler compound [9,10] was adopted. The basic martensitic structure (see Figure 1) originated by the Bain distortion of the austenitic L21 lattice is characterized by the body-centered Immm symmetry.

The (3+1)-dimensional structure belongs to the Immm(00γ)s00 superspace group and the modulated atomic positions of Ni Mn and Ga are defined by a wave-like shape expressed by a sinusoidal function:

$$
\mu^{\bar{i}}(\mathbf{x}\_4) = A\_1^{\bar{i}} \sin(2\pi nx\_4) \tag{1}
$$

that is based on the *x*4 superspace coordinate for *j*-th atomic site and the *n* index representing the order of the Fourier series [21]. Further details of the superspace approach are illustrated in the Supplementary Information. Therefore, the refinement of the amplitudes *Ai* 1 allows to find the incommensurate modulated displacing of the atomic positions. Main reflections and satellites are used in the Rietveld refinement and convergence is achieved for the values summarized in Tables S3 and S4. The refined modulation vector *q*= *γc***\*** (*α* and *β* variables assume zero value for symmetry) exhibits a deviation from the commensurate value of 0.4 *c***\*** = 2/5 *c***\***; indeed, in analogy with what was already found for the stoichiometric Ni2MnGa [9], this martensite can be regarded as incommensurate modulated 5M having *q* = 0.4226 *c*\* = 2/5(1 + *δ*)*c*\* where *δ* = 0.023 defines the aperiodicity of the system. Figure 3a represents the wave-like shift of the atomic layers stacked along the *c* axis where the atomic coordinates are extrapolated by assuming an ideal *q* = 2/5*c***\*** commensurate condition. In Figure 3b, the incommensurate deviation from the fundamental positions in the basic lattice is depicted. For all three atomic sites, the amplitudes A1*x* assume rather similar values, indicating that the systematic shift is damped to the 00l planes. Figure 3c reports the Rietveld plot of the structural refinement with agreement factors *R*p = 6.45% and *R*wp = 9.23%.

**Figure 3.** (**a**) View of the incommensurate modulated structure of the Ni2.07Mn0.93Ga martensitic phase. (**b**) Sinusoidal displacement of the atomic positions obtained by the Rietveld refinement of the (3+1)-dimensional model. The *t* parameter is related to the *x*4 coordinate. (**c**) Rietveld plot of the structural refinement for the modulated martensite at 220 K.

The magnetic characterizations involving Ni2+*x*Mn1−*x*Ga alloys with a slight excess of Ni revealed ferromagnetism in the low temperature martensitic phase [12]. Therefore, the structural refinement was carried out considering a ferromagnetic alignment of the spins along the b axis of the orthorhombic martensitic lattice as illustrated in Figure S3. The structural refinement returns a magnetic moment located on the Mn site of 2.86(6) *μ*B for the modulated martensite at 220 K. The decrement of the magnetic moment if compared to that reported for Mn in the classical Ni2MnGa [26] is in agreement with magnetic measurements described in previous publications [12,27].

This effect regards the dilution of the Mn atoms distributed in the Heusler sublattice with Ni atoms that bring a small, localized moment (0.33 *μ*B approximately [26]). The structural evolution of the martensitic phase across the structural transformation is monitored by collecting neutron diffraction patterns at selected temperatures from 220 K to 250 K. The values associated with each temperature were determined by changing the labelling of the crystallographic axes from abc to acb (with reference to the crystal data reported in Table S3; in this new set the martensitic lattice has the dimensions *a*m = 4.2167 Å, *b*m = 4.1870 Å, *c*m = 5.5484 Å, and *α* = *β* = *γ* = 90◦) in order to assign the shorter unit cell constant along the c axis. From 220 K to 240 K, the modulated martensite manifests a slight change of the unit cell parameters as indicated in Figure 4a. The growth of the austenite takes place at around 240 K, and as illustrated in the inset of Figure 4a, this phase linearly increments its relative amount as the temperature rises. Beyond 240 K, the crystal lattice of the martensite displays a progressive change with a marked elongation of the shorter axis. By increasing the temperature, the parameter of the modulation vector shows a decrement of its value toward the 0.4 condition (see Figure 4b) equivalent to the commensurate variant of the 5M martensite [9]. This trend was observed for Mn-rich Ni-Mn-Ga martensitic 5M modulated structures [9,23], and it is a clear indication that the wave-like modulation is dominated by the phonon softening of TA2 modes as remarked by Zheludev et al. [28]. The evolution of the tetragonality parameters is depicted in Figure 4c against the temperature change. The tetragonality is calculated considering a pseudotetragonal lattice with *a* = √2*a*m and *b* = √2*b*m. Indeed, upon heating, the martensite

manifests a progressive rearrangement of the lattice dimensions to mitigate the Bain distortion. This behavior is observed below the MT temperature but is particularly evident with the beginning of austenite growth. This evidence confirms that, in agreement with the crystallographic theory of martensitic transformations [29], as long as the structural transition from martensite to austenite is ongoing, the two coexisting crystal structures are metrically correlated to comply with the invariant plane strain requirements. The structural constraint forcing the martensitic unit cell parameters to assume certain values can be also studied by the application of the co-factor approach between 235 and 255 K.

**Figure 4.** (**a**) Unit cell parameter change versus temperature. The *a* and *b* parameters are rescaled to the austenitic lattice. The grey region indicates the temperature range with coexistence of austenite and martensite. Inset: reciprocal amount of parent and product phase during the MT. (**b**) Temperature dependence of the *γ* variable of the modulation vector. (**c**) Tetragonality parameters *c*/*a*, *c*/*b*, and co-factor *λ*2 against temperature.

The co-factor *λ*2 was introduced by J.M. Ball and R.D. James [30,31] in order to evaluate the compatibility conditions of martensitic and austenitic lattice. A co-factor parameter *λ*2 equal to 1 indicates zero elastic energy of the austenite/martensite interface, therefore implying a stress-free propagation of the phase boundaries during the MT process [30–34]. One of the implications of the condition *λ*2 = 1 is the narrowing of the thermal hysteresis of the first-order martensitic transition. The temperature span of the direct and reverse martensitic transition is a detrimental aspect for the technological application of the MCE, and theoretical tools such as the co-factor method were adopted to predict a favorable compositional chemistry to control the thermal drift. Further, the co-factor was applied to the Ni-Mn-Ga Heusler alloy to evaluate the lattice compatibility between modulated 7M martensites and the corresponding parent phase [35]. In the case of Ni2.07Mn0.93Ga the co-factor is calculated to provide additional information on the reciprocal correlation of the martensitic unit cell parameters ruled by the invariant plane strain conditions. The matrix selected for the co-factor extrapolation and the crystallographic assumptions used for this approach are summarized in the Supplementary Information. As indicated in Figure 4c, from 240 to 250 K, the λ2 parameter moves from 0.8 to 1.01, indicating a linear dependence with the temperature. Interestingly, the co-factor approaches the ideal value when the austenite exceeds 80% in weight.

#### **4. Conclusions**

The actual structural study brings new insights into the crystal structure of the martensitic phase characterizing Ni2+*x*Mn1−*x*Ga compositions with *x* < 1. The crystal structure of the martensitic phase Ni2.07Mn0.93Ga was solved by using neutron diffraction data. At 220 K this phase displays an incommensurate modulated 5M structure with a modulation vector closely related to the Ni2MnGa stoichiometric compound. The structural analysis of neutron diffraction patterns collected at different temperatures upon heating was used

to study the progressive mitigation of the classical Bain distortion of the martensite. As expected, the parameters *c*/*a* and *c*/*b* tend to assume higher values as the temperature increases, indicating the progressive restoring of the cubic symmetry. The co-factor parameter, calculated in the temperature range of the hysteresis of the transformation, highlights that the maximal lattice compatibility between austenite and martensite is reached when the volume of the parent phase is predominant. Likewise, this approach suggests that the mechanism ruling the diffusion-less transformation from orthorhombic martensite to cubic austenite is apparently not influenced by the structural modulation.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/met11111749/s1, Figure S1: X-ray diffraction pattern of the polycrystalline Ni2.07Mn0.93Ga alloy, Figure S2: Rietveld plot based on neutron diffraction data for the austenitic phase at 350 K. Figure S3: Ferromagnetic orientation of the spins located on the Mn/Ni site of the orthorhombic body centered lattice. Table S1: Crystal data of austenitic phase at 350 K, Table S2: Atomic coordinates, occupancy factors and thermal parameters for the cubic austenite at 350 K. Table S3: Crystal data of the modulated martensitic phase based on the structural refinement of the neutron diffraction data collected at 220K. Table S4: Atomic coordinate, occupancy factors, thermal parameters and modulation parameters A1x for the martensite at 220 K.

**Funding:** This research received no external funding.

**Data Availability Statement:** Data presented in this article are available at request from the corresponding author.

**Acknowledgments:** The author is grateful to R. Magnani for technical support during thermal measurements. The experimental and scientific support provided by ILL (Institute Laue-Langevin, France) during the neutron diffraction experiments performed at D2B beamline It is acknowledged.

**Conflicts of Interest:** The author declares no conflict of interest.

#### **References**


**2. Establishing reference intervals in umbilical cord blood**

blood volume per draw; thus, in neonates and infants, a less‐than‐optimal amount of blood may be available for testing. Approximately 1.5 – 4 mL of blood drawn for admission blood test may cause iatrogenic anemia to neonates especially in extremely low birth weight infants [1, 2]. Umbilical cord blood (UCB) is remained blood in the placenta and attached umbilical cord after the birth of baby. Several studies suggest that UCB could be an alternative source for admission of blood tests in neonates [1, 3, 4]. Especially, very low birth weight infants who typically have greater phlebotomy blood loss on the first day of life than any other day during their hospitalization would benefit most from admission laboratory studies being obtained

30 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

At birth, full‐term newborns show relative polycythemia, macrocytosis, and marked poly‐ chromasia with nucleated red blood cells (RBCs) [7, 8]. The red cell distribution width (RDW) is elevated, showing anisocytosis, compared with adult standards. Full‐term new‐ borns have a high white blood cell (WBC) count with relative transient neutrophilia at birth while soon after birth, neutrophils gradually decrease and lymphocytes become major population in neonate's peripheral blood. This neutrophilia may arise from bone marrow mobilization under stress during labor, and these WBCs show shift‐to‐left neu‐ trophils such as metamyelocytes, myelocytes, and even circulating blasts [9, 10]. The platelet counts are similar to the older children and adults. Neonatal thrombocytopenia is

Thrombocytopenia is one of the most common hematological abnormalities except iatro‐

During the pregnancy, human placenta forms an imperfect barrier, allowing bidirectional passage of soluble antigens and cells between a mother and a baby without any mixing between the two blood supplies [13]. This results in the presence of fetal cells in the maternal circulation, known as fetal microchimerism, and maternal cells in the fetal circulation, known as maternal microchimerism [14]. Maternal microchimerism was first described in 1963 by Rajendra G. Desai who identified maternal leukocytes and platelets in UCB [15]. This bidirec‐ tional trafficking of cells begins at seven to 16 weeks, increases steadily after 24 weeks, and reaches a peak at parturition [16]. At delivery, maternal microchimerism has been reported in 42% of normal pregnancies [13]. For this, microchimerism does not occur in all pregnan‐ cies, altered maternal‐fetal bidirectional passage has been associated with disruption of the feto‐maternal interface, and the biologic role of this bidirectional passage is unclear. This pas‐ sage is implicated in development of the fetal immune system [17]. Substantial numbers of maternal cells cross the placenta to reside in fetal lymph nodes, inducing the immune system, the development of CD4+CD25highFOXP3+ regulatory T cells (Tregs), which suppresses fetal

UCB is a rich source of hematopoietic cells or precursors to blood cells. Since the first UCB stem cell transplantation in 1988 to treat a child with Fanconi's anemia, UCB has been used as an important source of hematopoietic stem cell transplantation [18]. UCB could be collected at birth without any harm to the newborn infant. UCB cells have many theoretical advantages as grafts for stem cell transplantation because of the immaturity of newborn cells and immatu‐ rity of the immune system at birth. These properties should decrease the alloreactive potential

/L in any neonate of a viable gestational age.

from UCB [5, 6].

defined as a platelet count less than 150 × 109

genic anemia in neonates [11, 12].

antimaternal immunity.

The production of health‐associated reference values must be implemented in accordance with a well‐defined protocol. The Clinical Laboratory Standards Institute (CLSI) offers a protocol for determining reference intervals that meet the minimum requirements for reliability and usefulness related to quantitative clinical laboratory tests [21]. The CLSI suggested a protocol outline for obtaining reference values and establishing reference intervals. First, researchers should establish a list of analytical interferences and sources of biological variability from medical and scientific literatures. Then, they must establish selection and partition criteria and an appropriate questionnaire designed to reveal these criteria in the potential reference individuals. An appropriate written consent should be signed by legal guardians of neonates. Researchers have to categorize the potential refer‐ ence individuals based on the results of questionnaire and health assessments and exclude individuals based on the exclusion criteria. For UCB, gestational age, birth weight, mater‐ nal age, maternal health, and maternal history of medical, smoking, and alcohol consump‐ tion are important.

The reference interval is defined as the internal between and including two numbers, an upper and lower reference limit, which are estimated to enclose a specified percentage (usually 95%). For most analytes, the lower and upper reference limits are estimated as the 2.5th and 97.5th percentiles of the distribution of test results for reference populations. To decide on an appro‐ priate number of reference individuals, in consideration of desire confidence limits, the CLSI suggests that a minimum of 120 reference values for 90% confidence limits, 146 observations for 95% confidence limits, and 210 reference values for 99% confidence limits. It is necessary to define whether the sample should be arterial or venous UCB in a manner consistent with the routine practice for patient specimens. Inspection of the reference value data, preparing a histogram, and identifying possible data errors and/or outliers are essential to evaluate the distribution of data. Furthermore, partitioning into subclasses for separate reference intervals should be considered if appropriate according to gestational age, gender of neonates, and maternal age.

For difficulties of sample obtaining, it is not easy to establish reference intervals of parameters for neonates according to the CLSI guideline. Even published reference intervals using neo‐ nates' peripheral blood or UCB are very useful and informative for clinical laboratory tests, physicians should keep in mind that some of published reference intervals did not satisfy the CLSI guideline for sample collection.