**6. Laboratory diagnosis**

It has been reported that more than 50% of MRSA are also resistant to macrolides, lincosamides, fluoroquinolones, and aminoglycosides. This high level of resistance not only impedes successful therapy but also allows the microorganism to persist in the hospital, expanding its reservoir. So, vancomycin is the first-line treatment to VAP patients caused by MRSA. Nevertheless, some studies have described *S. aureus* strains with decreased susceptibility to vancomycin (vancomycin intermediate-resistant *S. aureus*, VISA). The acquired-resistance of MRSA to vancomycin is related to acquire mutations that appear in MRSA during vancomycin therapy [62, 63]. More recently, studies describing MRSA strains with high-level vancomycin resistance (vancomycin-resistant *S. aureus*, VRSA) were described. The mechanism of resistance is associated to the presence of transposon Tn1546, acquired from vancomycinresistant *Enterococcus faecalis*, which is known to alter cell wall structure and metabolism, but

the resistance mechanisms in VISA and VRSA isolates are less well defined [62].

MDR pathogens, only one anti-pseudomonas drug should be prescribed [61].

acquired β-lactam resistance [52, 64, 65].

114 Contemporary Topics of Pneumonia

Antibiotic options for Gram-negative coverage are more varied and must contain two antipseudomonal antibiotics from different classes in the presence of risk factors for MDR pathogens for the initial treatment of suspected VAP. If the patient does not present risk factors for

The frequency of infections caused by *P. aeruginosa* has increased in combination with the morbidity and mortality among hospitalized patients, all of which are exacerbated by antimicrobial resistance. Studies have demonstrated that resistance to carbapenems, aminoglycosides, and fluoroquinolones has increased gradually over the past few years, as well as episodes caused by MDR strains. Many *P. aeruginosa* isolates display an intrinsic reduced susceptibility to several antibacterial agents, as well as a tendency to develop resistance during therapy, especially in carbapenem-resistant strains. The most common mechanism of imipenem resistance in *P. aeruginosa* is a combination of chromosomal AmpC production and porin alterations. It also produces extended-spectrum β-lactamases (ESBLs) and can harbor other antibiotic resistance enzymes such as *K. pneumoniae* carbapenemases (KPC) and imipenem metallo-β-lactamases. β-Lactamase production, especially ESBLs, remains the main factor to

*K. pneumoniae* may present two major types of antibiotic resistance: (1) expression of ESBLs, which make them resistant to cephalosporins and monobactams and (2) the expression of carbapenemases that make *K. pneumoniae* resistant to almost all available β-lactams, including carbapenems. The first reported of carbapenemase by *K. pneumoniae* was in the USA, in 1996, which was designated KPC. Currently four classes of carbapenemases (classes A–D) have already been described and KPCs are classified into class A. To date, 16 KPC class A variants have already been identified. In addition to KPCs, *K. pneumoniae* strains may carry other forms of carbapenemases, such as class B metallo-β-lactamases (such as New Delhi's metalloβ-lactamase NDM-1 enzymes) and OXA class. In addition to β-lactamases, mutations in outer membrane proteins (OMPs) may also make the bacterium more resistant to β-lactams, par-

*A. baumannii* is also considered an emerging cause of nosocomial outbreaks, especially by MDR strains in ICUs. The most significant mechanism of carbapenem resistance in *A. baumannii* is the production of carbapenemases, which can be either intrinsic or acquired. Carbapenems

ticularly if it was in combination with the expression of a carbapenemase [66].

The diagnosis of VAP is usually based on clinical, radiographic, and microbiological criteria. Microbiological diagnosis is important in the management of VAP, since early diagnosis can influence clinical outcomes. The usual methods for microbiological diagnosis are based on quantitative or semiquantitative culture, but the results can take 48 h or more to be available. The Gram stain method has been used as screening of infection and to guide initial antibiotic therapy. However, utility of microscopy examination of respiratory secretions is still controversial.

Molecular methods can also be used to obtain results more quickly and initiate rational antibiotic therapy of patients with VAP. Many method formats are available for the detection of target genes for microbial identification and also for the detection of antimicrobial resistance genes.
