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23.1 Introduction

Humans, animals, and plants are in continuous contact with beneficial, harmless, or pathogenic bacteria. Diagnosis of bacterial infections and efficient treatment of infectious diseases are crucial for human health [1]. Eighty years ago, Alexander Fleming’s discovery of penicillin had changed the world of modern medicine by introducing the age of useful antibiotic [2]. A discovery rewarded in 1945 by the Nobel Prize in Medicine. Yet, within 15 years of his findings, Fleming presciently hypothesized that bacteria would likely attain resistance to any antibiotic treatment given the right circumstance. The continued emergence of single and multiple antibiotic-resistant bacterial strains is one of the more important societal issues today. Justifiably, the focus of antibiotic resistance research in the last half century has been on the elucidation of the mechanisms by which microbes can physically alter a drug’s structure, disrupt the interaction between a drug and its cellular target, or alter the behavior and efficiency of its own transport machinery to reduce access to a drug’s cellular target [3].

Despite the significant advances in antibacterial therapy today, death of bacteria in response to antibiotic exposure remains largely unknown. Although it is possible to measure the physiological effects of antibiotics, such as loss of membrane permeability, changes in cell morphology, and molecular effects (e.g., inhibition or cellular pathways) [4], the causes of the ever-increasing prevalence of antibiotic-resistant strains remain obscure. Indeed, this developed resistance has many consequences. In many cases, the infected person with a resistant microorganism is more likely to require hospitalization, to double the duration of their hospital stay, and/or to experience increased risk of death and morbidity. Understanding the mechanisms of antibiotic resistance can advance novel therapeutic approaches and serve as a foundation for the development of new antibiotics.

This chapter discusses the status of bacterial resistance mechanisms and the relationship with oxidative stress and provides an overview of the methods used to assess oxidative stress and mechanisms of antibiotic resistance. A general survey of conventional biological methodologies and the role of proteomics in assessing bacterial resistance and oxidative stress are provided.

23.2 Antibiotics and Bacterial Resistance Mechanisms

Antibiotics (compounds that are by definition “against life”) are typically antibacterial drugs, interfering with processes that are essential to bacterial growth or survival without harm to the eukaryotic host harboring the infecting bacteria [5]. Antibiotics can have bactericidal effect (resulting in cell death) or bacteriostatic effect (stop bacterial growth). In order to understand the mechanism by which bacteria develop antibiotic resistance, it is important to study the different targets and reaction pathways for the main classes of antibacterial drugs and bacterial pathogens.

23.2.1 How Antibiotics Work

It is not clear whether there is a single major mechanism of bacterial cell death from antibiotics or many. Most antibiotics function through inhibition of essential cellular processes, an intervention to which there are likely to be many consequences, and as a result, there would be equally numerous ways to achieve bacterial killing [4].

There are three proven targets for the main antibacterial drugs:

  1. 1.

    Bacterial cell wall biosynthesis: Most bacteria produce a cell wall that is composed partly of a macromolecule called peptidoglycan, itself made up of amino sugars and short peptides. Penicillin, one of the first antibiotics to be used widely (and other β-Lactam antibiotics), targets cell wall synthesis by inhibiting the formation of peptidoglycan cross-links in the bacterial cell wall, a process also known as transpeptidation. The result is a very fragile cell wall that bursts, killing the bacterium.

  2. 2.

    Bacterial protein synthesis: Drugs that target and inhibit protein synthesis can be divided in two classes: 50S ribosome inhibitors and the 30S inhibitors. Indeed the ribosome (serving as the primary site of biological protein synthesis (translation)) is composed of two ribonucleprotein subunits, the 50S and 30S, which assemble (during the initiation phase) following the formation of a complex between an mRNA transcript, N-formylmethionine-charged aminoacyl tRNA, several initiation factors, and a free 30S subunit [6]. Tetracycline, for example, can cross the membranes of bacteria and accumulate in high concentrations in the cytoplasm. Tetracycline then binds to a single site on the 30S ribosomal subunit and blocks that key RNA interaction, which shuts off the lengthening protein chain.

  3. 3.

    Bacterial DNA replication and repair: Bacterial chromosomal topology is maintained by the activities of topoisomerase I, topoisomerase IV, and DNA gyrase (topoisomerase II) [7]. These reactions are exploited by the synthetic quinolone class of antimicrobials which target DNA–topoisomerase complexes. The quinolone class of antimicrobials interferes with the maintenance of chromosomal topology by targeting topoisomerase II and topoisomerase IV, trapping these enzymes at the DNA cleavage stage and preventing strand rejoining [6]. The process leads to the complete inhibition of cell division and results to bacteriostatic effects and ultimately cell death.

While the antibiotic drug–target interactions and their respective direct effects are well known, as discussed above, the bacterial responses to antibiotic drug treatments that contribute to cell death are complex and not as well understood. It was reported that the three major classes of bactericidal drugs, regardless of drug–target interaction, all utilize a common mechanism of inactivation whereby they stimulate the production of lethal doses of hydroxyl radicals via the Fenton reaction [8]. The generation of these highly destructive hydroxyl radicals is the result of the iron misregulation by the superoxide-mediated oxidation of iron–sulfur clusters, a process that promotes a breakdown of iron regulatory dynamics [3]. This oxidative stress contributes to bactericidal antibiotic-mediated cell death. Kohanski and colleagues have studied the antibiotic-induced stress response networks to determine how the primary effect of a given bactericidal drug triggers aspects of cell death that are common to all bactericidal drugs. They showed that an aminoglycoside-antibiotic (which is known to be a protein synthesis inhibitor) also induces oxidative stress and cell death. These studies indicate that oxidative stress is involved in the antibiotic resistance effects in pathogenic bacteria and that exposure of bacteria to antibiotics may alter the antioxidant defense system and redox mechanisms in cells. The discovery of the existence of a common oxidative damage cellular death pathway can be helpful for the development of more effective antibacterial therapies. ROS (reactive oxygen species), such as superoxide (O2 •−), and hydroxyl radicals (HO•), as well as RNS (reactive nitrogen species) such as nitric oxide (NO) and peroxynitrite (ONOO−), are highly toxic, as a result of their actions as oxidizing and nitrating agents, and can have damaging effects on bacterial physiology. There is still much to be learned about how oxidative stress related changes in bacterial physiology, affect antibiotic-mediated cell death and the emergence of resistance [6]. Here, we provide an overview of the current state of the art in this field and the methods that can be used to study such effects.

23.2.2 How Bacteria Fight Antibiotics Effects

Bacteria can develop resistance to virtually any antimicrobial agent at varietal stages [9, 10]. The evaluation of a new antimicrobial agent typically involves the study of organisms that are either naturally resistant or susceptible to that agent, thus defining a broad spectrum of activity for that agent [11]. The following section discusses the origin, evolution, and current understanding of antibiotic resistance and the processes that make antibiotic resistance inevitable.

In bacteria, the front line of this resistance system is the cell envelope. In Gram-negative bacteria this includes the outer membrane, which is composed of an asymmetric lipopolysaccharide–phospholipid bilayer, and provides an effective physical barrier to the entry of molecules (including many antibiotics) into the cell. Outer membrane-spanning porins that facilitate the entry of small molecules into cells also passively excludes many antibiotics. In Gram-positive bacteria the absence of an outer membrane results in increased sensitivity to many antibiotics. Nonetheless, many Gram-positive bacteria, such as Mycobacteria species, can fight the cytotoxic effects of antibiotics using physiological defenses [12]. In addition to the protective cell envelope, there are different other mechanisms of acquired antimicrobial resistance. These include possible changes in the drug target (e.g., reduction of receptor affinity and the substitution of an alternative pathway), the production of a detoxifying enzyme, or decreased antibiotic uptake (through diminished permeability or an active efflux system) [11]. Genetic modifications can also be used to increase bacteria resistance against antibiotics [13]. These modifications are performed via plasmid conjugation, phage-based transduction, or lateral gene transfer [14, 15], activation of latent mobile genetic elements, and the mutagenesis of its own DNA [12, 13]. The presence of antibiotic resistance elements in pathogenic bacteria is, mostly the result of the horizontal gene transfer, a process by which bacteria acquire resistance genes form environmental bacteria [16, 17]. A principal mechanism for the fast spread of antibiotic-resistance genes through bacterial populations is that such genes get collected on plasmids that are independently replicated within and passed between bacterial cells and species [5]. This fast acquisition of resistance is facilitated by the environmental antibiotic pressure.

Under antibiotic stress, a few spontaneous drug-resistant mutants can enhance the survival capacity of the overall population in that stressful environment. This protective effect is resultant from the production and sharing of the metabolite indole (produced by antibiotic-resistant mutants), a signaling molecule, that could turn on drug-efflux pumps and activate oxidative stress protective mechanisms [18]. In any event, the survival of a bacterium amidst oxidative stress depends on the evolution of a series of defense mechanisms, which include:

  1. 1.

    Detoxifying enzymes (enzymatic antibiotic inactivation) [19] and free radical-scavenging substrates.

  2. 2.

    DNA and protein repair systems.

  3. 3.

    Competition by substrates favoring bacterial survival.

In many cases, these defenses may be coordinately regulated [20].

23.3 The Role of Oxidative Stress, Antibiotic Function, and Emerging Bacterial Resistance Against It

In addition to the general antibiotic effects mentioned above, which include inhibition of cell wall assembly, protein synthesis, and DNA replication, a string of several other mechanisms have been correlated to cell death meditated by antibiotics. Prominently, oxidative stress has been suggested as a possible pathway involved in antibiotic effects and also in the development of antibiotic resistance in bacteria [21]. Studies suggested oxidative stress as a secondary mechanism to the primary modes of action of antibiotics [22].

Oxidative stress is a redox disequilibrium state, in which the generation of ROS overwhelms the antioxidant defense mechanisms [23]. According to the hierarchical oxidative stress model, a minor level of oxidative stress merely induces protective effects. However, at high level, excessive ROS may cause severe damage to cells, including necrosis and apoptosis [24]. Therefore, quantitative study of ROS release in cells is of particular interest in assessing the relationship between antibiotics and oxidative stress. Examples of techniques that have been used for this purpose are summarized in Table 23.1 and discussed below. Figure 23.1 provides an overview of the various parameters and methods involved in such studies.

Table 23.1 Methods and strategies used to assess oxidative stress in antibiotic-treated bacteria
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Fig. 23.1
figure 1

Antibiotics effects on bacteria and assessment methods

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Methods for monitoring ROS are a critical first step in unraveling the role of these species and their contribution to bacterial resistance and antibiotics susceptibility. Continuous monitoring of these species in biological systems is a significant challenge due to their high reactivity and short lifetime. Moreover, study of their kinetic characteristics is difficult due to the many interrelated redox reactions and low concentrations that change dynamically over time. Commonly used techniques involve indirect absorbance or fluorescence measurements [25, 26]. However, fluorescence probes are relatively nonspecific [27] due to light sensitivity, photobleaching, and poor selectivity. Many biological studies infer ROS levels from indirect measurements of products of protein nitrosylation (3-nitrotyrosine) or lipid peroxidation (4-hydroxynonenal). There are few methods that can measure ROS directly. Electron paramagnetic resonance (EPR) [28, 29] can be used to assess production of free radicals directly but the method has relatively low sensitivity and measurements can be challenging in complex biological environments. Other methods include chemiluminescence (CL), electrospray ionization-mass spectrometry (ESI-MS), gel electrophoresis, polymerase chain reaction (PCR), gene expression, and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS).

Electrochemistry is another method that can provide real-time measurements of ROS in complex biological environments [30]. Electrochemical sensors are relatively inexpensive and easy-to-use. Most work has been done with individual microelectrodes by Amatore’s group to study cell secretion [31–34]. Practical problems that impede the broad implementation of electrochemical microsensors in the study of oxidative stress are mainly related to the difficulties of calibration and operation in complex biological samples due to interferences and instability of the radicals. Additionally and unfortunately, commercial microelectrochemical probes and high throughput electrochemical instrumentation are of limited availability and are not widely accessible to life scientists. Sensors that could simultaneously and continuously assess the evolution of multiple ROS with high selectivity can provide quantitative measurements of free radicals in real time with high spatial and temporal resolution directly in bacterial cultures. Examples of applications of this technology were demonstrated in several biological environments [35, 36]. However, the use of these sensors to study bacterial pathogens and antibiotic susceptibility are limited [37, 38].

Several studies have used chemiluminescence (CL) to probe the presence of ROS in bacteria. Albesa et al. used CL measurements to assess the involvement of oxidative stress, particularly the role of superoxide in the action of antibiotics against different bacteria including Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Enterococcus faecalis [39]. Using CL, Albesa found that diverse antibiotics can increase superoxide release in various strains but only those strains that are sensitive to antibiotics show oxidative stress response [39]. In addition to superoxide, several studies have demonstrated the role of hydroxyl radicals (HO•) as an essential contributor to the oxidative stress response, and their involvement in the mode of action of antibiotics. Kohanski et al. studied E. coli treated with norfloxacin, ampicillin, and kanamycin and Staphylococcus aureus treated with norfloxacin and chloramphenicol and demonstrated release of hydroxyl radicals via the Fenton reaction [8]. A hydroxyl radical-specific dye, hydroxyphenyl fluorescein (HPF), was used in this study to assess the formation of hydroxyl radicals. In the Fenton reaction, the ferrous iron is driving formation of hydroxyl radicals; therefore iron chelators will inhibit hydroxyl radical generation allowing direct quantification of oxidative effects due to the presence of these radicals. Similar results were also observed by Grant et al. in norfloxacin-treated E. coli after addition of HPF [40]. Dwyer et al. hypothesized that generation of hydroxyl radicals might also involve superoxide-mediated oxidation of the iron–sulfur clusters, and showed that the generation of superoxide radicals disrupted iron regulatory dynamics, inducing iron misregulation in cells [3]. Yeom et al. also showed that antibiotics could accelerate cell death by promoting the Fenton reaction leading to an oxidative stress response in ampicillin-treated Pseudomonas aeruginosa [22]. Results of this study demonstrated that the antibiotic action is affected by modulation of reduced nicotinamide-adenine dinucleotide (NADH) levels and iron chelation. In addition to direct or indirect measurements of ROS, the involvement of ROS in cell death and antibiotic resistance can also be assessed by using ROS scavengers. Theoretically, an ROS scavenger will neutralize excessive ROS and increase the percentage of surviving bacteria after antibiotics exposure. Therefore, the addition of antioxidants to bacteria exposed to an oxidative stress environment would protect cells from the damaging effects of ROS. Goswami et al. investigated the protective effect of antioxidants in Escherichia coli exposed to ciprofloxacin. Both glutathione and ascorbic acid antioxidants have shown substantial protective effects, which demonstrated the involvement of ROS in the antibiotics-mediated cell death [41]. Reduced killing effects were observed when thiourea, a hydroxyl radical scavenger, was added to an antibiotic-treated cell culture. These results validate the hypothesis that cell death is mediated by hydroxyl radicals. Wang et al. reported similar effects after adding thiourea and/or 2,2′-bipyridyl to oxolinic acid, moxifloxacin, and quinolone-treated E. coli [42]. Grant et al. used thiourea to remove hydroxyl radicals in Mycobacterium smegmatis and M. tuberculosis treated with ciprofloxacin, isoniazid, rifampin, streptomycin, and clofazimine, respectively, in order to establish the relationship between dissolved oxygen and ROS [40].

Another strategy to assess ROS release in antibiotic-treated bacteria is to use mutant strains that have an altered antioxidant defense mechanism. The antioxidant defensive system in bacteria comprises specific antioxidative enzymes including superoxide dismutase (SOD), catalase, and peroxidase. Through manipulation of E. coli strains, knockout strain can be generated to create an artificial imbalance state that allows selective study of oxidant/antioxidant mechanisms. Goswami et al. studied the effect of mutations in oxidative stress defense genes in SOD and alkyl hydroperoxide reductase on the sensitivity of E. coli to antibiotics and found that both superoxide and H2O2 may be involved in the antibacterial action of ciproflaxin [41]. Additional information about oxidative stress in bacteria treated by antibiotics can be obtained by phenotypic and gene expression analysis. Dwyer et al. performed several phenotypic and genetic analyses on gyrase-inhibited E. coli. and demonstrated that both superoxide and hydroxyl radical oxidative species are generated following inactivation by gyrase inhibitors. This oxidative response can amplify the inhibitor effect by oxidatively damaging DNA, proteins, and lipids [3]. The antimicrobial gyrase-catalyzed effects included DNA strand breakage and damage of the replication machinery that may ultimately result in cell death.

Investigation of oxidative stress-related processes and the role of ROS in bacteria exposed to antibiotics provide valuable information regarding the mechanism of bactericidal antibiotic-mediated cell death and the involvement of ROS in this process. These studies can potentially reveal novel aspects related to the mode of action of antibiotics against bacteria, information that can be useful in the future for the development of new antimicrobial drugs. Moreover, since ROS can cause damage to cell membrane and most importantly, proteins, it is equally important to assess changes in the cell proteome to establish the effect of the antibiotic-induced oxidative stress on the cell’s proteome. Proteomic approaches such as mass spectrometry (MS) and gel electrophoresis, e.g., sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) can be used to study these effects. Ongoing efforts in this direction are summarized in the following section.

23.4 Proteomic Investigation of Oxidative Stress in Bacteria

Under conditions that can cause oxidative stress, bacterial cells are exposed to excessive ROS that can oxidize membrane fatty acids, initiating lipid peroxidation [43], oxidize proteins [44], and cause DNA damage [45, 46]. The immediate effects on proteins include tyrosine hydroxylation, methionine or cysteine oxidation, and formation of carbonyl group on side chain amino acids [47]. As a result, modified proteins could be used as potential markers for oxidative stress. Several advanced instrumentations are available for the analysis of the proteome (e.g., proteins, peptides, glycans, protein interactions, and post-translational modifications). These include ESI-MS, MALDI-MS, and chromatography-coupled and tandem techniques such as HPLC-ESI-MS, LC-MS, and LC-MS/MS [47, 48]. In general, these methods are used in conjunction with specific biochemical techniques such as SDS-PAGE, PCR, and cell labeling. As compared to genomic analysis, proteomics studies of bacterial cells are more challenging and difficult due to many variable physicochemical parameters, wide dynamic ranges, and relative protein abundances that differ among different cells. Proteomic tools can reveal information of bacterial surface exposed and cell envelope proteins [49] as well as bacterial secretome. Studies of cell surface proteins could reveal the interaction of cells with the environment and could predict an oxidative stress response. Changes in the secretome of various bacteria can show differences in secreted markers that are related to toxicity and protein alterations. While such studies are relevant for the investigation of bacterial pathogens and their interaction with the environment, few reports have been published on the use of proteomics to assess the relationship between oxidative stress and bacterial pathogenicity.

Western blotting, two-dimensional gel electrophoresis, gel imaging, and mass spectrometry were used to perform a full proteomic analysis of Paracoccidioides exposed to H2O2, used as an example of ROS, for the purpose of mimicking oxidative stress. Furthermore, intracellular NADPH/NADP+ ratio were determined. One hundred and seventy-nine oxidative stress-responsive proteins/isoforms were identified and grouped. Paracoccidioides yeast response was characterized by up-regulated proteins/isoforms that represented a total of 64.8 % of all proteins/isoforms identified in this study [50]. Dosselli et al. used a proteomic approach to examine the oxidative response of Staphylococcus aureus undergoing photodynamic therapy (PDT), an antimicrobial method of killing bacteria by ROS. Several functional classes of proteins appeared to be selectively affected by PDT treatment. Moreover, cell growth and nutrition uptake were also inhibited by this treatment [51].

In another proteomic study, NAD-specific glutamate dehydrogenase, phosphoglycerate kinase, and Acyl-CoA dehydrogenase in Fusobacterium nucleatum were found to be up-regulated by oxidative stress after 72 h of atmospheric oxygen exposure and four additional exposure cycles [52]. Huang et al. showed that the treatment of Helicobacter pylori with 10 mM H2O2 induced overexpression of the following: cytotoxin-associated protein A (CagA), vacuolating cytotoxin (VacA), adherence-associated protein (AlpA), alkylhydroperoxide reductase (AhpC), catalase (KatA), serine protease (HtrA), aconitate hydratase, and fumarate reductase [53]. Combined results from 2D gel electrophoresis and MALDI-TOF MS analysis showed expression of 60 different proteins in Bacillus anthracis treated with 0.3 mM H2O2; 17 of these proteins are differently expressed over time. Time-dependent changes in generation of metabolic and repair/protection proteins were also studied [54]. Shu et al. reported a proteomic study of the oxidative stress response induced by low-dose H2O2 in Bacillus anthracis targeting activity and efficacy of Dps-like proteins (Dps1, Dps2, and Dps3) encoded by Bacillus cereus. Electrophoretic mobility shift assay (EMSA) and real-time reverse transcription-PCR were used to determine transcription level of the dps genes. The deletion of dps1 and dps2 caused a dramatic decrease in the survival rate. Since Dps1 and Dps2 were induced by oxidative stress, the authors concluded that bacteria developed certain defensive strategy when facing oxidative stress, while Dps3 only responded to general stress [55]. Sardar et al. investigated the changes in the proteome of Leishmania donovani promastigote during oxidative and nitrosative stresses using real-time PCR, MALDI-TOF/TOF mass spectrometry, Western blot, and iTRAQ labeling. There were nine proteins that were involved in redox homeostasis and were up-regulated while three others were down-regulated. For proteins involved in β-oxidation, TCA cycle, mitochondrial respiration, and oxidative phosphorylation, up-regulation was observed. The heat shock proteins (HSPs) and chaperone also were up-regulated. Antioxidant levels in L. donovani promastigotes decrease when the cells were treated with menadione, SNAP, and the combination of these two reagents. Possibly, antioxidants were consumed to maintain the normal physiological condition [56]. Through proteomic analysis, damage by ROS and/or RNS can be comprehensively investigated and fully revealed. Furthermore, a recent proteomic study indicates that pathogenic bacteria exhibit a complex response to ROS that includes the rapid adaption of metabolic pathways in response to oxidative-stress challenges [57]. A better understanding about the adaptation and the protection mechanisms of bacteria against oxidative stress are slowly being obtained using the above-mentioned methods.

23.5 Future Perspectives and Emerging Trends

This chapter reviewed the main mechanisms of antibiotic action, as well as mechanisms allowing bacteria to develop antibiotic resistance. We also introduced the role of oxidative stress in antibiotic-induced cell death as well as the how pathogenic bacteria have developed antibiotic resistance to oxidative stress. Various methods commonly used to assess oxidative stress were summarized along with their advantages and limitations, and their contribution to the study of antibiotic resistance. Since ROS are difficult to measure, the major challenge in this field remains the identification of a molecular connection between antibiotics and oxidative stress in bacteria [22]. Development of quantitative analytical methods to allow real-time quantitative measurements of ROS and antioxidant status could facilitate fundamental future investigations in this field. Improving the understanding of the intracellular communication and molecular mechanism used by bacteria is important in future research developments to be able to rationally design effective clinical interventions to respond to the growing threat of resistant bacterial infections [18].

The acquisition of multidrug resistance is a serious problem for the modern medicine. Resistance to antibiotics is facilitated by the presence of antibiotic-resistance genes on transferable genetic elements and also by the use of antibiotics in a way that allows them to act as selective agents. The use of antimicrobials for the promotion of animal growth and its link to the increased resistance has been a topic of heated debate [58]. As in humans, subtherapeutic doses in animals can select for resistant strains; if the bacteria cross from animal hosts to human hosts, then reservoirs of resistance may markedly reduce the effective lifetime of human antibiotics [5].

As a possible solution to this growing problem of resistance to conventional antibiotics, some authors suggested the use of antimicrobial peptides to partially substitute low effective antibiotics [59–61]. The interest in antimicrobial peptides began with the work of Fleming in 1922, with his discovery of antimicrobial activities in different secretions (saliva, nasal mucus, and tears…), blood, leukocytes, and lymphatic tissues called lysozyme [62]. Antimicrobial peptides are an abundant and diverse group of molecules. Their amino acid composition, amphipathicity, cationic charge, and size allow them to attach to and insert into membrane bilayers to form pores by “barrel-stave,” “carpet,” or “toroidal-pore” mechanisms [59]. One of the challenges to the use of these peptides as antimicrobial human therapy is their potential for toxicity. All clinical trials to date have used topical applications to address surface infections, rather than the more effective systemic administration (parenteral and oral). Another disadvantage of natural peptides is the potential sensitivity to proteases, creating potentially unfavorable pharmacokinetics. And, finally, the high cost of manufacturing peptides has limited both the testing and development of large numbers of variants and clinical targets to which these molecules can be applied [60].

Another emerging field expected to open new avenues in the fight against bacterial infection is nanotechnology. Nanotechnology is emerging as a new interdisciplinary field of chemistry, physics, and material science with broad applicability to biology and medicine [63]. A wide number of engineered nanoparticles (NPs) have shown excellent antibacterial activity on several Gram-positive and Gram-negative bacteria. The scientific debate concerning the mechanism of the antibacterial effect of NPs is still open. High surface area to volume ratios and unique chemico-physical properties of various nanomaterials are believed to contribute to the observed antimicrobial activities [64–73]. Among the different antimicrobial agents, silver has been the most extensively studied and used since ancient times to fight infections and prevent food spoilage. The antibacterial, antifungal, and antiviral properties of silver ions, silver compounds, and silver NPs have been extensively studied [67, 74–77]. In addition to silver, nitric oxide-releasing NPs (NO-NPs) are effective candidates in the inhibition of growth of many resistant bacteria (e.g., methicillin-resistant bacteria, Gram-negative bacteria resistant to commonly used antibiotics) [78]. The authors of this study suggested that as NO provides multiple mechanisms of bactericidal and immunological activity, the risk of pathogen resistance to NO-NPs is limited. Although many scientists are presenting nanoparticles (or “Nanoantibiotics”) as a new promising paradigm for treating multiresistant bacteria [63, 79, 80], other works indicate that the development of NP resistance is also possible [81]. While NPs have demonstrated potential as effective antimicrobial agents against multidrug-resistant bacteria, future fundamental studies are needed to evaluate the specific toxicity mechanisms and assess the risks of a possible development of resistance, before these can be fully implemented in real world applications.