**4.2.** *Klebsiella pneumoniae*

**4. Virulence of major pathogens and VAP severity**

**Microorganisms Frequency (%)**

(4) Viruses <1 each

(5) Fungi <1 each

*K. pneumoniae*, *P. aeruginosa*, and *S. aureus*.

**Table 1.** Frequency of etiologic agents of VAP.

**4.1.** *Acinetobacter baumannii*

*Enterococcus* spp. *Moraxella* spp.

110 Contemporary Topics of Pneumonia

*Influenza* virus *Herpes simplex* virus *Cytomegalovirus*

*Aspergillus* spp. *Candida* spp. *Pneumocystis carinii*

Clinical outcomes of VAP depend on a variety of factors, which are inherent to the patient, the hospital assistance, and also the microorganism, including host immune system status, underlying diseases associated, appropriate antibiotic therapy, accurate and rapid clinical and laboratory diagnosis, antimicrobial susceptibility, and virulence of the pathogen. Antimicrobial susceptibility is discussed in Section 5. Here, we present significance of major virulence factors associated with VAP severity of four selected pathogens: *Acinetobacter baumannii*,

There are more than 20 *Acinetobacter* species, with *A. baumannii* being the most commonly isolated in clinical settings, in which it represents important emerging nosocomial pathogen. *A. baumannii* is a Gram-negative bacterium, strictly aerobic, nonfermentative coccobacillus, nonmotile, nonpigmented, and catalase-positive. It is ubiquitous in nature and has been recovered from soil, water, and animals and found as part of the normal skin, throat, and rectal flora of human. Although a frequent colonizer, *A. baumannii* can be the cause of severe and sometimes lethal infections, frequently of nosocomial origin, principally VAP. A survey in U.S. hospitals showed that the majority of the isolates (57.6%) were from the respiratory tract, and *Acinetobacter* species ranked fifth as the causative organism of VAP (6.6%) [28–30]. In recent years, it has been designated a "red alert" human pathogen and has caused considerable concern in the medical community. This pathogen can adhere to surfaces, and it specifically targets moist tissues such as mucous membranes or skin that has been exposed due to accident or injury, and can cause a wide variety of infections. Most of the cases involve the respiratory tract, but bacteremia, meningitis, and wound infection may also occur. A recent systematic review and meta-analysis showed that some invasive procedures frequently used *K. pneumoniae* is generally considered an opportunistic pathogen that affects mainly immunocompromised individuals. It can be found normally in the intestine, oral cavity, and skin, as well as in hospital settings and medical devices [33]. *K. pneumoniae* is able to form biofilms in catheters and endotracheal tubes, which represent major sources of infection in patients with invasive devices [34].

Infections by *K. pneumoniae* that involves biofilm formation tend to be persistent or chronic, since the biofilm protects the pathogen of the host immune response and also of the antibiotic action [35]. An additional risk factor for chronic infections caused by nosocomial strains includes resistance to multiple antibiotics, making difficult for the choice of suitable antibiotics for the treatment [36].

*K. pneumoniae* has about 78 capsular serotypes (or K antigens) [37]. Some of them present an increase in the production of the capsule and present very viscous colonies, which are called hypermucoviscous. Such isolates have also been considered to be hypervirulent because capsule is the most important virulence factor of *K. pneumoniae* [38].

A practical way to check if an isolate is hypermucoviscous is by using the string test. If there is the formation of a viscous chain greater than 5 mm after touching bacterial growth on agar and try to stretch it with a platinum loop. The degree of mucoviscosity correlates with the establishment of invasive infections. Hypervirulent *K. pneumoniae* is highly invasive and can affect previously healthy persons, causing fatal infections, including severe pneumonia among them [38]. The bacterium with the phenotype of hypermucoviscosity is capable of spreading from one organ to other organs (metastatic spread) [39].

The capsule consists of polysaccharides and is generally constituted by repeating units of three to six sugars [38]. The main functions assigned to it include: (1) protection of *K. pneumoniae* against opsonization and phagocytosis [40]; (2) interference with dendritic cells (DCs) maturation and, consequently, in the production of pro-Th1 cytokines mediated by DCs [41]; (3) anti-inflammatory effect by the inhibition of IL-8 expression [42–44]; and (4) reduction of the amount of antimicrobial peptides reaching the bacterial surface, thus, promoting resistance to them [45].

In addition to capsule, iron acquisition is a virulence property that also contributes to the persistence of the microorganism in the patient body and, consequently, to VAP, since iron is essential for bacterial growth. Pathogenic members of the Enterobacteriaceae family usually display a variety of iron uptake systems, of which at least 12 have been described in *K. pneumoniae.* Isolates associated with pulmonary infections also produce yersiniabactin and salmochelin, which are not sequestered by the host protein lipocalin 2 of the innate immune defense [46, 47]. Additionally, hypervirulent *K. pneumoniae* produces a higher amount and more active siderophore molecules than classical *K. pneumoniae*, which increases its pathogenic potential [48].

A, coagulase, leukocidin, and γ-toxin [55]. Protein A is an important virulence factor in the pathogenesis of experimental staphylococcal pneumonia in mice [56]. Moreover, protein A mediates: (1) invasion across airway epithelial cells through activation of RhoA GTPase signaling and proteolytic activity; (2) binding to tumor necrosis factor receptor 1 (TNFR1) on lung epithelial cells, and (3) activation of a specific intracellular signaling causing the recruitment of neutrophils. These activities increase inflammation of the airway epithelium and,

Microbiology of Ventilator-Associated Pneumonia http://dx.doi.org/10.5772/intechopen.69430 113

Cysteine proteases, in particular staphopain A (ScpA), cleave the pulmonary surfactant protein-A (SP-A), a major surfactant component with immune functions that is important during *S. aureus* infections [55]. Additionally, *S. aureus* releases enzymes with significant roles as virulence factors, including proteases, nucleases, lipases, hyaluronidase, and staphylokinase

Interestingly, *S. aureus* display a great ability to subvert innate and adaptive immune responses to favor its replication [59]. In some situations, such as in immunocompromising conditions, there is a higher susceptibility to acquire *S. aureus* infection, mainly by hospitalized patients. In this context, *S. aureus* and especially the epidemic methicillin-resistant *S. aureus* strains cause severe necrotizing pneumonia by producing Panton-Valentine leukocidin (PVL) that has been reported to cause rapidly progressive necrosis of the lung tissue in young immunocompetent patients. The severity of disease, survival, and clinical outcome of VAP patients can also be associated with the presence of the Panton-Valentine leukocidin genes in MRSA [60]. The role of PVL in the pathogenesis of MRSA infection is not clear, but recently, it was demonstrated that the PVL have strong affinity for host extracellular matrix proteins being, therefore, implicated as a *S. aureus* adherence molecule. Moreover, PVL as a cytotoxin targets human polymorphonuclear neutrophils, and monocytes or macrophages, or both, leading to their apoptosis or necrosis as result of the Bax-independent apoptosis occurring by means of a novel pathway that presumably involved PVL-mediated pore formation in the mitochondria membranes.

**5. Antimicrobial susceptibility and management of patients**

multiple agents with broader spectrum of activity [25].

Choosing an initial antibiotic for suspected VAP is a difficult task. A scheme of empiric antibiotic therapy must take into account that *S. aureus*, *P. aeruginosa*, *Acinetobacter* spp., and Enterobacteriaceae members together represent more than 80% of VAP cases worldwide and several strains are defined as MDR pathogens [26]. To provide suitable antibiotic exposure regarding the possibility of infection by MDR pathogens, the empiric therapy should contain

However, antibiotic choices should be based on local prevalence and the antimicrobial susceptibility profile of the usual pathogens, since data from guidelines or other hospitals can be ineffective [61]. For empiric MRSA coverage, vancomycin or linezolid are strongly recommended. On the other hand, if it is indicated as MSSA coverage, the following antibiotics should be used: piperacillin-tazobactam, cefepime, levofloxacin, imipenem, or meropenem. Suspected etiology for MRSA or MSSA should be based on the presence of risk factors [61].

thus, contribute to tissue damage [57].

that facilitate the invasion of the infected tissue [58].

#### **4.3.** *Pseudomonas aeruginosa*

VAP caused by *P. aeruginosa* has been associated with higher case fatality rates than that by other bacteria. This pathogen is a noninvasivefermenting Gram-negative, aerobic, rod-shaped polar-flagella, with unipolar motility. *P. aeruginosa* is considered emerging as an important nosocomial pathogen worldwide and is responsible for an extensive spectrum of infections in humans associated with significant morbidity and mortality. It is an opportunistic pathogen that is normally found in plants, soils, and in a variety of aquatic environments. The adaptability and high antibiotic resistance allow it to survive in a wide range of other natural and artificial settings, including surfaces in medical facilities. In addition, *P. aeruginosa* is recognized for its ability to form biofilms and directly increase the VAP-induced lung injury. In the United States, *P. aeruginosa* is among the most common hospital pathogens and is the second most common pathogen isolated from patients with VAP and has been associated with prolonged hospitalization, increased cost, and mortality [49–52].

Cell surface virulence factors of *P. aeruginosa* play an important role in colonization of the lower respiratory tract. These factors include *flagellum*, *pili* or fimbriae, lipopolysaccharide (LPS), as well as type III secretion system (T3SS), which is a major determinant of virulence. The T3SS expression is frequently associated with acute invasive infections and has been linked to increased mortality in infected patients, and it is shared among many pathogenic Gram-negative bacteria as a means of injecting toxins directly into host cells [49, 53].

Additionally, several proteases are produced by *P. aeruginosa*. These proteases have established roles in distinct infectious process, such as hydrolysis of immunoglobulin, fibrin, fibrinogen, and also disruption of epithelial tight junctions. Main *P. aeruginosa* proteases include pyocyanin, which induces damage to the respiratory tract, such as epithelial necrosis and reduced ciliary movement; pyoverdin, its main secreted siderophore; protease IV, a serine protease responsible to degradation of complement proteins, fibrinogen, immunoglobulin G, and plasminogen; elastase and metalloproteinases that degrade elastin, collagen types III and IV, surfactant, immunoglobulins, complement factors, and cytokines; and exotoxin A, one of the most potent toxins with cytopathic activity, among others, such as *quorum-sensing*, a very sophisticated gene regulatory mechanism that allows bacteria to coordinate activity through the production of small diffusible molecules. These functions include the formation of biofilms, motility, secretion of virulent factors, and exopolysaccharide production [49, 54].

#### **4.4.** *Staphylococcus aureus*

*S. aureus* strains produce several virulence factors that contribute to the pathogenesis and severity of lower respiratory infections. Some of them can hinder host defenses, such as protein A, coagulase, leukocidin, and γ-toxin [55]. Protein A is an important virulence factor in the pathogenesis of experimental staphylococcal pneumonia in mice [56]. Moreover, protein A mediates: (1) invasion across airway epithelial cells through activation of RhoA GTPase signaling and proteolytic activity; (2) binding to tumor necrosis factor receptor 1 (TNFR1) on lung epithelial cells, and (3) activation of a specific intracellular signaling causing the recruitment of neutrophils. These activities increase inflammation of the airway epithelium and, thus, contribute to tissue damage [57].

a variety of iron uptake systems, of which at least 12 have been described in *K. pneumoniae.* Isolates associated with pulmonary infections also produce yersiniabactin and salmochelin, which are not sequestered by the host protein lipocalin 2 of the innate immune defense [46, 47]. Additionally, hypervirulent *K. pneumoniae* produces a higher amount and more active siderophore molecules than classical *K. pneumoniae*, which increases its pathogenic potential [48].

VAP caused by *P. aeruginosa* has been associated with higher case fatality rates than that by other bacteria. This pathogen is a noninvasivefermenting Gram-negative, aerobic, rod-shaped polar-flagella, with unipolar motility. *P. aeruginosa* is considered emerging as an important nosocomial pathogen worldwide and is responsible for an extensive spectrum of infections in humans associated with significant morbidity and mortality. It is an opportunistic pathogen that is normally found in plants, soils, and in a variety of aquatic environments. The adaptability and high antibiotic resistance allow it to survive in a wide range of other natural and artificial settings, including surfaces in medical facilities. In addition, *P. aeruginosa* is recognized for its ability to form biofilms and directly increase the VAP-induced lung injury. In the United States, *P. aeruginosa* is among the most common hospital pathogens and is the second most common pathogen isolated from patients with VAP and has been associated with pro-

Cell surface virulence factors of *P. aeruginosa* play an important role in colonization of the lower respiratory tract. These factors include *flagellum*, *pili* or fimbriae, lipopolysaccharide (LPS), as well as type III secretion system (T3SS), which is a major determinant of virulence. The T3SS expression is frequently associated with acute invasive infections and has been linked to increased mortality in infected patients, and it is shared among many pathogenic

Additionally, several proteases are produced by *P. aeruginosa*. These proteases have established roles in distinct infectious process, such as hydrolysis of immunoglobulin, fibrin, fibrinogen, and also disruption of epithelial tight junctions. Main *P. aeruginosa* proteases include pyocyanin, which induces damage to the respiratory tract, such as epithelial necrosis and reduced ciliary movement; pyoverdin, its main secreted siderophore; protease IV, a serine protease responsible to degradation of complement proteins, fibrinogen, immunoglobulin G, and plasminogen; elastase and metalloproteinases that degrade elastin, collagen types III and IV, surfactant, immunoglobulins, complement factors, and cytokines; and exotoxin A, one of the most potent toxins with cytopathic activity, among others, such as *quorum-sensing*, a very sophisticated gene regulatory mechanism that allows bacteria to coordinate activity through the production of small diffusible molecules. These functions include the formation of biofilms, motility, secretion of virulent factors, and exopolysaccharide production [49, 54].

*S. aureus* strains produce several virulence factors that contribute to the pathogenesis and severity of lower respiratory infections. Some of them can hinder host defenses, such as protein

Gram-negative bacteria as a means of injecting toxins directly into host cells [49, 53].

longed hospitalization, increased cost, and mortality [49–52].

**4.3.** *Pseudomonas aeruginosa*

112 Contemporary Topics of Pneumonia

**4.4.** *Staphylococcus aureus*

Cysteine proteases, in particular staphopain A (ScpA), cleave the pulmonary surfactant protein-A (SP-A), a major surfactant component with immune functions that is important during *S. aureus* infections [55]. Additionally, *S. aureus* releases enzymes with significant roles as virulence factors, including proteases, nucleases, lipases, hyaluronidase, and staphylokinase that facilitate the invasion of the infected tissue [58].

Interestingly, *S. aureus* display a great ability to subvert innate and adaptive immune responses to favor its replication [59]. In some situations, such as in immunocompromising conditions, there is a higher susceptibility to acquire *S. aureus* infection, mainly by hospitalized patients. In this context, *S. aureus* and especially the epidemic methicillin-resistant *S. aureus* strains cause severe necrotizing pneumonia by producing Panton-Valentine leukocidin (PVL) that has been reported to cause rapidly progressive necrosis of the lung tissue in young immunocompetent patients. The severity of disease, survival, and clinical outcome of VAP patients can also be associated with the presence of the Panton-Valentine leukocidin genes in MRSA [60]. The role of PVL in the pathogenesis of MRSA infection is not clear, but recently, it was demonstrated that the PVL have strong affinity for host extracellular matrix proteins being, therefore, implicated as a *S. aureus* adherence molecule. Moreover, PVL as a cytotoxin targets human polymorphonuclear neutrophils, and monocytes or macrophages, or both, leading to their apoptosis or necrosis as result of the Bax-independent apoptosis occurring by means of a novel pathway that presumably involved PVL-mediated pore formation in the mitochondria membranes.
