Virulence Factors Related To Membranes
Lipopolysaccharide as a virulence factor widely interacting with hosts and also target for vaccines
LPS, an important classical structural component of the outer membrane of most Gram-negative bacteria, is a known potent agonist that elicits robust innate inflammatory immunity, its distal end may be capped with O antigen, a long polysaccharide that can range from a few to hundreds of sugars in length, which is critical for bacterial physiology and pathogenesis. At the early stage, scientists were interested in developing vaccines to prevent infection by focusing on LPS, which were later proven highly difficult due to the various serotypes and inefficacious outcomes.,,
OMVs are important part of virulence platforms
OMVs are bacterial components that can be released spontaneously to the environment during growth by many Gram-negative bacteria. Bacterial-derived OMVs have been characterized as a novel secretion mechanism that can deliver a variety of bacterial proteins and lipids into host cells without direct contact with host cells.,, OMVs can package and enrich a wide variety of proteins and nucleic acids, including lipoproteins, periplasmic proteins , plasmid containing chromosomal DNA fragments, phage DNA, virulence factors .
Table 1 The major pathogenesis factors of P. aeruginosa and therapeutics
Chromosomally Encoded Resistance Mechanisms
Similar to imported resistance mechanisms, there are a variety of resistance mechanisms encoded on the P. aeruginosa chromosome. These mechanisms include several aminoglycoside-inactivating enzymes and a class D oxacillinase, OXA-50 . As mentioned above, characterized mechanisms of fluoroquinolone resistance among P. aeruginosa isolates have been restricted to chromosomal genes, including target mutations and active efflux .2). Similar to the case for other gram-negative bacteria, DNA gyrase is the primary target for the fluoroquinolones in P. aeruginosa . Therefore, the first target-specific mutations are typically observed within the quinolone resistance determining region of gyrA . The highest levels of resistance are observed in strains that have mutations in the QRDR of both gyrA and the topoisomerase IV gene parC . Although mutational changes within the other two genes, gyrB and parE, have been described, the prevalence of these mutations appears to be much lower . Recent studies involving screening of the Harvard PA14 library of P. aeruginosa mutants have identified a number of other chromosomal genes that may be involved in antibacterial resistance or in increasing the frequency of mutation to resistance .
Discordance Between Oprd Expression And Susceptibility To Imipenem
Although the relationship between OprD deficiency and imipenem resistance has been well established in the literature, it should not come as a surprise that P. aeruginosa does not always follow expected rules. The genetic versatility of this pathogen and its ability to coregulate multiple resistance mechanisms make P. aeruginosa a constantly moving target and one of our greatest therapeutic challenges. Studies from our laboratory have identified intriguing strains that exhibit discordance between oprD expression and susceptibility to carbapenems.
The first example is an isogenic mutant, P. aeruginosa 410L, that was selected with levofloxacin from P. aeruginosa Tokai#1 . As background, strain Tokai#1 is an isogenic mutant of P. aeruginosa PAO1 that lacks susceptibility to both levofloxacin and imipenem . The MIC for imipenem against Tokai#1 is 8 g/ml, compared to 1 g/ml against the parental PAO1 strain, and this decreased susceptibility correlates with a fivefold decrease in the level of oprD expression . Expression of oprD in mutant strain 410L decreased further, to a level 3-fold below that of Tokai#1 and 17-fold below that of the wild-type parent, PAO1. However, despite the further decrease in oprD expression and the inability to detect OprD in outer membrane preparations, mutant 410L lost its resistance to imipenem and reverted back to a level of susceptibility similar to that of the original PAO1 parent.
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Preventing Emergence Of Chromosomally Encoded Resistance
This review has highlighted the impressive ability of P. aeruginosa to develop antibacterial resistance through mutational changes in the function and/or production of chromosomally encoded resistance mechanisms. Furthermore, the most difficult challenge with this pathogen is the ability of P. aeruginosa to become resistant during treatment of an infection. Unfortunately, the prospect for bringing new, and specifically novel, antipseudomonal drugs to clinical use in the near future is not promising. Therefore, the challenge facing us today is to slow the emergence of resistance through optimizing therapy with currently available drugs.
How Is It Spread
Pseudomonas aeruginosa lives in the environment and can be spread to people in healthcare settings when they are exposed to water or soil that is contaminated with these germs. Resistant strains of the germ can also spread in healthcare settings from one person to another through contaminated hands, equipment, or surfaces.
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Biofilms Formation And Cyclic Di
As in most Gram negative bacteria, P. aeruginosabiofilm formation is regulated by one single molecule: cyclic di-GMP. At low cyclic di-GMP concentration, P. aeruginosa has a free-swimming mode of life. But when cyclic di-GMP levels increase, P. aeruginosa start to establish sessile communities on surfaces. The intracellular concentration of cyclic di-GMP increases within seconds when P. aeruginosa touches a surface . This activates the production of adhesive pili, that serve as “anchors” to stabilize the attachment of P. aeruginosa on the surface. At later stages, bacteria will start attaching irreversibly by producing a strongly adhesive matrix. At the same time, cyclic di-GMP represses the synthesis of the flagellar machinery, preventing P. aeruginosa from swimming. When suppressed, the biofilms are less adherent and easier to treat. The biofilm matrix of P. aeruginosa is composed of nucleic acids, amino acids, carbohydrates, and various ions. It mechanically and chemically protects P. aeruginosa from aggression by the immune system and some toxic compounds. P. aeruginosa biofilm’s matrix is composed of 2 types of sugars named PSL and PEL:
Resistance Challenges For Treatment Of P Aeruginosa
P. aeruginosa presents a serious therapeutic challenge for treatment of both community-acquired and nosocomial infections, and selection of the appropriate antibiotic to initiate therapy is essential to optimizing the clinical outcome . Unfortunately, selection of the most appropriate antibiotic is complicated by the ability of P. aeruginosa to develop resistance to multiple classes of antibacterial agents, even during the course of treating an infection. Epidemiological outcome studies have shown that infections caused by drug-resistant P. aeruginosa are associated with significant increases in morbidity, mortality, need for surgical intervention, length of hospital stay and chronic care, and overall cost of treating the infection . Even more problematic is the development of resistance during the course of therapy, a complication which has been shown to double the length of hospitalization and overall cost of patient care . P. aeruginosa can develop resistance to antibacterials either through the acquisition of resistance genes on mobile genetic elements or through mutational processes that alter the expression and/or function of chromosomally encoded mechanisms. Both strategies for developing drug resistance can severely limit the therapeutic options for treatment of serious infections.
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Description Of The Study Population
One hundred fifty-three patients were included, for whom the characteristics are indicated in Table . They had a median age of 64years . Median SAPS II was 58 , and ICU mortality was 34%. Median length of stay was 21days , and the duration of mechanical ventilation was 16days .
Table 1 Clinical characteristics of the population and comparison of the patients with P. aeruginosa without mutation vs with mutation
The use of ceftazidime was significantly associated with the occurrence of an over-expression of inducible cephalosporinase 23.1% of the patients who received ceftazidime developed an over-expression of cephalosporinase, compared to 7.9% of the patients who did not receive ceftazidime . The use of meropenem was significantly associated with the occurrence of impermeability .
Perspectives And Future Directions
The global overuse and misuse of antibiotics during the last 80 years has led to a profound increase in antimicrobial resistance. Between 2000 and 2010, global antibiotic consumption increased by nearly 70% and antibiotic resistant infections have accordingly become more pervasive, according to global epidemiological antimicrobial resistance surveillance networks . AMR is a complex, One Health issue, involving human, animal and environmental factors. The solution to AMR is therefore also likely to be a complex one, involving multiple strategies maintaining AMR surveillance, containing AMR transmission, reducing selection pressure, developing novel antimicrobials or reverting antibiotic resistant microbes back to the susceptible phenotype with the use of antibiotic adjuvants . Although progress in the development of naturally derived and peptide-based antimicrobials has been made . The conservation of existing antibiotics through careful stewardship is paramount to help mitigate the gap between the demand for new drugs and the diminishing supply pipeline. Antibiotic adjuvants will also play an important role in extending the shelf life of our existing antimicrobial therapeutic agents.
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Acquiring The Antibiotic Resistance Throughout Bacterial Life Cycle
Bacteria can acquire antibiotic resistance through mutations or horizontal gene transfer., Mutations of OprD in P. aeruginosa confers resistance to carbapenems and mutations of DNA gyrase causes resistance to quinolone antibiotics. Importantly, mutations in the -lactamase gene ampC causes a significant increase in resistance to cephalosporins. There are already a host of enzymes in this bacterium may counter antibiotics while it continues to gain new resistance factors, which is debatably the biggest challenge for drug industry and scientific research. As bacteria can conveniently obtain antibiotic resistance genes from the same or different bacterial species through horizontal gene transfer, despite challenging targeting this mechanism may be a niche to search for better treatments. A typical example is that P. aeruginosa may obtain aminoglycoside and -lactam resistance genes through horizontal gene transfer from the environment or other microbes at an unpredictable, fast pace,, it may be highly difficult for scientists and clinicians to design new tools in impeding this natural robust mechanism in this bacterium.
Highly Complex And Multiple Mechanisms In Host Immune Responses To The Bacterium
The opportunistic pathogen P. aeruginosa exists almost everywhere and in any environmental conditions. In immunosuppressed people, there is extreme susceptibility to P. aeruginosa infection, developing either acute or chronic phenotypes. As the first line of host defense systems, the innate immune system plays a vital role in battling with P. aeruginosa via multiple mechanisms, such as phagocytosis and inflammatory responses. Several types of host systems, such as pattern recognition receptors , plasma membrane signals, intracellular enzymes, and secreted cytokines/chemokines participate in inflammatory response against the bacterial infections. Although a well-balanced inflammatory response is required for restraining P. aeruginosa invasion, overzealous inflammation is associated with rapid disease progression, tissue injury, and even death. Some host molecules including cytosolic protein annexin A2 , autophagy-related protein 7 , NLRC4, as well as non-coding RNAs , are also implicated in P. aeruginosa-induced inflammation and/or other aspects of host defense mechanisms,,, and understanding of the mechanisms of inflammatory response is just beginning to be unfolded.
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Quorum Sensing Is The Most
Roles and acting mechanisms of QS
QS describes a method that is widely utilized by bacteria for cellcell mass communication. Both Gram-negative and -positive bacteria detect the local population density by sensing chemical signals and coordinate gene expression and group-beneficial behaviors., Bacteria produce autoinducer or quoromone as diffusion signaling molecules and release into the environment for communication. Once the population reaching a threshold, the autoinducers activate their cognate receptors to directly or indirectly induce gene expression. Over the past two decades, QS has been extensively studied as a potential target for antivirulence agents, which may be harnessed to counteract bacterial virulence via a noncytotoxic mechanism as alternatives of traditional antibiotics.,
Another essential function for QS in P. aeruginosa is to regulate the production of multiple virulence factors, such as extracellular proteases, iron chelators, efflux pump expression, biofilm development, swarming motility, and the response to host immunity. As a model organism, P. aeruginosa serves as one of the most suitable bacteria to study the fundamental mechanisms of QS signaling regulating virulence and search for chemical agents to block the QS system.,
Interaction between quorum-sensing systems and environments
Quorum Sensing Biofilm And Motility Attenuation
Quorum sensing regulates a wide range of genes involved in virulence and bacterial adaptation . For instance, QS is required for the surfing and swarming motility phenotypes associated with increased resistance to antimicrobials. The surfing phenotype is regulated via three QS systems in P. aeruginosa Las, Rhl and Pqs . In addition, QS has been found to influence tolerance to antibiotics in P. aeruginosa biofilms. QS provides structural rigidity through the regulation of Pel polysaccharides and eDNA release necessary for the extracellular polysaccharide matrix. In addition, the production of rhamnolipids, surfactants important for the establishment and maintenance of biofilms, is controlled under QS . Therefore, QS has been recognized as a significant potential target for developing anti-resistance therapies. Strategies to combat antimicrobial resistance by targeting adaptive resistance mechanisms have significant potential for reversing antibiotic resistance in P. aeruginosa. Adaptive resistance is often mediated through complex global regulatory systems, such as the QS system, and regulate an extensive set of genes involved in resistance. Targeting these regulatory systems may prevent the activation of expression of these resistance genes that would normally be expressed under the environmental conditions of infection.
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Multiple Mutations And Multidrug Resistance
No single mutation compromises every antipseudomonal drug. Nevertheless, up-regulated efflux can simultaneously compromise fluoroquinolones and most -lactams, leaving only the aminoglycosides and imipenem . A combination of up-regulated efflux, loss of OprD, and impermeability to aminoglycosides compromises every drug class except the polymyxins. Each of the necessary mutations arises in 1 cell per 107 to 109 cells, and, although simultaneous emergence is mathematically and biologically improbable, sequential emergence is all too likely because infections that are resistant to the first antibiotic administered are likely to be treated with a second antibiotic, and so on. Mutations that up-regulate efflux may act additively with those that effect permeability, -lactamase expression, or topoisomerase susceptibility so as to exacerbate resistance .
Accumulation of sequential mutations may be facilitated by hypermutators, which either lack the ability to perform DNA proofreading or mismatch repair, or which use DNA polymerases with a reduced copying fidelity. Because resistance is most likely to emerge in hypermutators, antibiotics may select for hypermutators, thereby increasing the probability that further resistance will emerge. Hypermutators were found in sputum samples obtained from 11 of 30 patients who had cystic fibrosis and chronic P. aeruginosa infections, compared with 0 of 75 samples from patients without cystic fibrosis who had acute infections .
Characterization Of Oprd Promoter Elements
Although the relationship between OprD and resistance to the carbapenems has been recognized for 2 decades, characterization of the oprD promoter and understanding of the regulation of OprD remain limited compared to our understanding of AmpC- and efflux-mediated resistance mechanisms. Figure Figure44 depicts the key promoter elements for oprD, based upon 5 rapid amplification of cDNA ends of the oprD promoter of P. aeruginosa PAO1 grown in Mueller-Hinton broth . In these studies, two start sites for oprD transcription were identified, with transcription initiating at similar frequencies from an adenine 71 bases and a thymine 23 bases upstream of the translational start codon for oprD. Putative 10 and 35 elements are also presented in Fig. Fig.44.
Characterization of oprD promoter elements. Transcription of oprD in P. aeruginosa PAO1 initiates with equal frequencies from two start sites, located 23 bases and 71 bases upstream of the structural gene . The putative 10 and 35 promoter elements for SS1 are highlighted in red, and the putative 10 and 35 promoter elements for SS2 are highlighted in blue.
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New Antibiotic Formulations And Compounds
With an alarming rise in pathogens with resistance to existing drugs, a number of new antibiotics have recently entered the antibiotic development pipeline however, the hope for patients and clinicians is rapidly dwindling once a new antibiotic resistance strain emerges. Hence, we should invest unconventionally research efforts in searching for new treatments of MDR bacteria. Recently developed antimicrobials are discussed below.
The biological activity of substituted guanidines was known in the mid-1930s when a series of guanidines and metformin compounds were found to possess bactericidal and disinfectant properties. Subsequently, many guanidine derivatives were studied for therapeutic purposes. Chin et al., recently reported a polyguanidine polycarbonates with strong antimicrobial activities through a distinctive mechanism that does not intensify drug resistance in multi-drug resistance superbugs including methicillin-resistant S. aureus, P. aeruginosa, A. baumannii, K. pneumoniae, suggesting that the polyguanidine compounds may be potential antibacterial candidates.
How To Optimize Anti
Clinicians should be aware that in addition to adequate antimicrobial coverage, other factors including optimal dosing, interval of drug administration, and duration of therapy are key factors influencing clinical outcomes.
For example, in a recent multinational study performed in ICU patients, 16% of the patients did not achieve free antibiotic concentrations sufficiently greater than the MIC required to ensure a positive clinical outcome . Another recent study performed in patients with VAP due to Gram-negative bacteria showed that a serum exposure of anti-pseudomonal cephalosporins greater than 53% fT> MIC was significantly associated with a favorable outcome or presumed eradication. Therefore, these and other studies support the importance of considering adequate exposure-response profiles when optimizing drug therapy in these patient groups.
In our opinion, the best way to optimize beta-lactam antibiotic dosing may be the use of prolonged or continuous infusion with the use of a loading dose to ensure early attainment of target concentration exceeding the MIC . Moreover, although it is not available in most clinical laboratory, we also suggest the use of therapeutic drug monitoring .
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Inflammasomes Drive Inflammation And Pyroptosis
The inflammasome is a multiprotein complex, which is attributed to the production and release of inflammatory cytokines, IL-1 and IL-18., Recent work reveals that inflammasome is involved in pyroptosis dependent on the cleavage of Gasdermin D, which contributed to the formation of plasma membrane pores, and in turn promoting the release of inflammatory cytokines and pyroptosis., Typically, inflammasome consists of cytosolic PRRs, ASC , and caspase 1. Inflammasome PRRs are responsible for detecting exogenous PAMPs like TLRs, which are also essential for monitoring P. aeruginosa invasion and activate host inflammatory response for promoting the clearance of P. aeruginosa., Remarkably, TLRs are involved in the priming of inflammasome activation by promoting the transcription of inflammasome-related genes. NLRC4 and AIM2 are well characterized among numerous inflammasome PRRs linked to infection of P. aeruginosa.
