Phage treatment of Pseudomonas aeruginosa yields a phage-resistant population with different susceptibility to innate immune responses and mild effects on metabolic profiles.

In this study, we have investigated innate immune activation capacity and metabolic features of a population of P. aeruginosa PAO1 phage-resistant mutants with diverse genetic modification (large genomic deletions and point mutations) arising after exposure to phages targetting lipopolysaccharide (LPS) or Type-4 pili (T4P). Deletions led to the loss of genes involved in LPS synthesis, cell envelope permeability, efflux systems, biofilm production, oxidative stress tolerance, and DNA repair. Loss of LPS O antigen resulted in bacterial sensitivity to serum complement and stimulation of inflammatory cascades but did not cause increased phagocytosis, while T4P phage-resistant mutants were more effectively phagocytized than LPS-defective mutants. Changes in the utilization of different carbon, nitrogen, sulphur, and phosphorus sources were identified, especially in mutants where the two phage DNA persisted in the bacterial population (pseudolysogeny). However, the metabolic changes did not directly correlate with single-gene mutations or the large gene deletions, suggesting they reflect adaptive changes to the gene modifications that arise during the selection of resistant mutants. In contrast, phage-resistant mutants were susceptible to humoral innate immune responses, suggesting that phage resistance may be a beneficial outcome of phage therapy.

Chitosan-based matrix as a carrier for bacteriophages.

Wound healing is a dynamic and complex process where infection prevention is essential. Chitosan, thanks to its bactericidal activity against gram-positive and gram-negative bacteria, as well as anti-inflammatory and hemostatic properties, is an excellent candidate to design dressings for difficult-to-heal wound treatment. The great advantage of this biopolymer is its capacity to be chemically modified, which allows for the production of various functional forms, depending on the needs and subsequent use. Moreover, chitosan can be an excellent polymer matrix for bacteriophage (phage) packing as a novel alternative/supportive antibacterial therapy approach. This study is focused on the preparation and characteristics of chitosan-based material in the form of a film with the addition of Pseudomonas lytic phages (KTN4, KT28, and LUZ19), which would exhibit antibacterial activity as a potential dressing that accelerates the wound healing. We investigated the method of producing a polymer based on microcrystalline chitosan (MKCh) to serve as the matrix for phage deposition. We described some important parameters such as average molar mass, swelling capacity, surface morphology, phage release profile, and antibacterial activity tested in the Pseudomonas aeruginosa bacterial model. The chitosan polysaccharide turned out to interact with phage particles immobilizing them within a material matrix. Nevertheless, with the high hydrophilicity and swelling features of the prepared material, the external solution of bacterial culture was absorbed and phages went in direct contact with bacteria causing their lysis in the polymer matrix. KEY POINTS: • A novel chitosan-based matrix with the addition of active phages was prepared • Phage interactions with the chitosan matrix were determined as electrostatic • Phages in the matrix work through direct contact with the bacterial cells.

Phages and phage-borne enzymes as new antibacterial agents.

BACKGROUND: Persistent and resistant infections caused by bacteria are increasing in numbers and pose a treatment challenge to the medical community and public health. However, solutions with new agents that will enable effective treatment are lacking or delayed by complex development and authorizations. Bacteriophages are known as a possible solution for invasive infections for decades but were seldom used in the Western world. OBJECTIVES: To provide an overview of the current status and emerging use of bacteriophage therapy and phage-based products, as well as touch on the socioeconomic and regulatory issues surrounding their development. SOURCES: Peer-reviewed articles and authors' first-hand experience. CONTENT: Although phage therapy is making a comeback since its early discovery, there are many hurdles to its current use. The lack of appropriate standardized bacterial susceptibility testing; lack of a simple business model and authorization for the need of many phages to treat a single species infection; and the lack of knowledge on predictable outcome measures are just a few examples. In this review, we explore the possible routes for phage use, either based on local specialty centres or by industry; the current status of phage therapy, which is mainly based on single-centre or single-bacterial cohorts, and emerging clinical trials; local country-level frameworks for phage utilization even without full authorization; and the use of phage-derived products as alternatives to antibiotics. We also explore what may be the current indications based on the possible availability of phages. IMPLICATIONS: Although phages are emerging as a potential treatment for non-resolving and life-threatening infections, the models for their use and production still need to be defined by the medical community, regulatory bodies, and industry. Bacteriophages may have a great potential for infection treatment but many aspects still need to be defined before their routine use in the clinic.

Isolation and Characterization of the Acadevirus Members BigMira and MidiMira Infecting a Highly Pathogenic Proteus mirabilis Strain.

Proteus mirabilis is an opportunistic pathogen and is responsible for more than 40% of all cases of catheter-associated urinary tract infections (CAUTIs). Healthcare-associated infections have been aggravated by the constant emergence of antibiotic-resistant bacterial strains. Because of this, the use of phages to combat bacterial infections gained renewed interest. In this study, we describe the biological and genomic features of two P. mirabilis phages, named BigMira and MidiMira. These phages belong to the Acadevirus genus (family Autographiviridae). BigMira and MidiMira are highly similar, differing only in four missense mutations in their phage tail fiber. These mutations are sufficient to impact the phages' depolymerase activity. Subsequently, the comparative genomic analysis of ten clinical P. mirabilis strains revealed differences in their antibiotic resistance profiles and lipopolysaccharide locus, with the latter potentially explaining the host range data of the phages. The massive presence of antimicrobial resistance genes, especially in the phages' isolation strain P. mirabilis MCS, highlights the challenges in treating infections caused by multidrug-resistant bacteria. The findings reinforce BigMira and MidiMira phages as candidates for phage therapy purposes.

Klebsiella phage KP34gp57 capsular depolymerase structure and function: from a serendipitous finding to the design of active mini-enzymes against K. pneumoniae.

IMPORTANCE: In this work, we determined the structure of Klebsiella phage KP34p57 capsular depolymerase and dissected the role of individual domains in trimerization and functional activity. The crystal structure serendipitously revealed that the enzyme can exist in a monomeric state once deprived of its C-terminal domain. Based on the crystal structure and site-directed mutagenesis, we localized the key catalytic residues in an intra-subunit deep groove. Consistently, we show that C-terminally trimmed KP34p57 variants are monomeric, stable, and fully active. The elaboration of monomeric, fully active phage depolymerases is innovative in the field, as no previous example exists. Indeed, mini phage depolymerases can be combined in chimeric enzymes to extend their activity ranges, allowing their use against multiple serotypes.

The impact of agarose immobilization on the activity of lytic Pseudomonas aeruginosa phages combined with chemicals.

The implementation of non-traditional antibacterials is currently one of the most intensively explored areas of modern medical and biological sciences. One of the most promising alternative strategies to combat bacterial infections is the application of lytic phages combined with established and new antibacterials. The presented study investigates the potential of agarose-based biocomposites containing lytic Pseudomonas phages (KT28, KTN4, and LUZ19), cupric ions (Cu2+), strawberry furanone (HDMF), and gentamicin (GE) as antibacterials and anti-virulent compounds for novel wound dressings. Phages (KT28, KTN4, LUZ19, and triple-phage cocktail) alone and in combination with a triple-chemical mixture (Cu + GE + HDMF) when applied as the liquid formulation caused a significant bacterial count reduction and biofilm production inhibition of clinical P. aeruginosa strains. The immobilization in the agarose scaffold significantly impaired the bioavailability and diffusion of phage particles, depending on virion morphology and targeted receptor specificity. The antibacterial potential of chemicals was also reduced by the agarose scaffold. Moreover, the Cu + GE + HDMF mixture impaired the lytic activity of phages depending on viral particles' susceptibility to cupric ion toxicity. Therefore, three administration types were tested and the optimal turned out to be the one separating antibacterials both physically and temporally. Taken together, the additive effect of phages combined with chemicals makes biocomposite a good solution for designing new wound dressings. Nevertheless, the phage utilization should involve an application of aqueous cocktails directly onto the wound, followed by chemicals immobilized in hydrogel dressings which allow for taking advantage of the antibacterial and anti-virulent effects of all components. KEY POINTS: • The immobilization in the agarose impairs the bioavailability of phage particles and the Cu + GE + HDMF mixture. • The cupric ions are toxic to phages and are sequestrated on phage particles and agarose matrix. • The elaborated TIME-SHIFT administration effectively separates antibacterials both physically and temporally.

High-resolution reconstruction of a Jumbo-bacteriophage infecting capsulated bacteria using hyperbranched tail fibers.

The Klebsiella jumbo myophage ϕKp24 displays an unusually complex arrangement of tail fibers interacting with a host cell. In this study, we combine cryo-electron microscopy methods, protein structure prediction methods, molecular simulations, microbiological and machine learning approaches to explore the capsid, tail, and tail fibers of ϕKp24. We determine the structure of the capsid and tail at 4.1 Šand 3.0 Šresolution. We observe the tail fibers are branched and rearranged dramatically upon cell surface attachment. This complex configuration involves fourteen putative tail fibers with depolymerase activity that provide ϕKp24 with the ability to infect a broad panel of capsular polysaccharide (CPS) types of Klebsiella pneumoniae. Our study provides structural and functional insight into how ϕKp24 adapts to the variable surfaces of capsulated bacterial pathogens, which is useful for the development of phage therapy approaches against pan-drug resistant K. pneumoniae strains.

Culture Media Composition Influences the Antibacterial Effect of Silver, Cupric, and Zinc Ions against Pseudomonas aeruginosa.

Different metals, such as silver (Ag), copper (Cu), and zinc (Zn), have been broadly investigated as metals and cations used both in medicine and everyday life due to their broad spectrum of antibacterial activity. Although the antibacterial action of those metals and their ions is well known and studied, the main problem remains in the standardization of experimental procedures to determine the antimicrobial activity as bacteriological media composition might significantly influence the outcome. The presented study aimed to evaluate the appropriability of different culture media (four nutritionally rich and four minimal) in the testing of the antibacterial activity of Ag+, Cu2+, and Zn2+ ions against Pseudomonas aeruginosa. Our investigation revealed the influence of medium ingredients and the presence of phosphates, which significantly reduced the activity of tested metal ions. Moreover, the precipitate formation and decrease in pH in the minimal media were additionally observed. It was assumed that the most favorable medium for metal ion activity testing was Luria-Bertani complex medium and MOPS minimal medium.

Comparative Characteristics and Pathogenic Potential of Escherichia coli Isolates Originating from Poultry Farms, Retail Meat, and Human Urinary Tract Infection.

The pathogenicity of many bacterial strains is determined by the acquisition of virulence genes and depends on many factors. The aim of this study was to analyse the phylogenetic background, virulence patterns, and drug susceptibility of 132 E. coli isolates tested in the context of the ExPEC (Extraintestinal Pathogenic E. coli) pathotype and the correlation of these features with bacterial isolation source: food (retail meat), poultry farms (AFEC-Avian Faecal E. coli), and patients with UTI (urinary tract infection) symptoms. The drug-susceptibility results of tested E. coli isolates obtained indicate that the resistance profile-ampicillin/tetracycline/trimethoprim+sulfamethoxazole/ciprofloxacin (AMP/TE/SXT/CIP)-was most frequently observed. The multidrug resistance (MDR) phenotype was found in 31.8% of isolates from poultry farms, 36.8% of strains isolated from food, and 20% of clinical samples. The greatest similarity of virulence profiles applied to isolates derived from poultry farms and food. Most of the AFEC from poultry farms and food-derived isolates belonged to commensals from phylogroups A and B1, while among the isolates from patients with UTI symptoms, the most common was the B2 phylogroup. The collective analysis showed similarity of the three studied groups of E. coli isolates in terms of the presented patterns of antimicrobial resistance, while the virulence profiles of the isolates studied showed great diversity. The phylogroup analysis showed no similarity between the poultry/food isolates and the UTI isolates, which had significant pathogenic potential.

Exploiting phage-derived carbohydrate depolymerases for combating infectious diseases.

Bacteria are protected against the immune system of their human hosts, as well as against predators such as phages, by expressing diverse surface carbohydrates. Some phages produce specialized depolymerases which can degrade those carbohydrates. Here, we discuss the biological role of depolymerases and how they can be exploited to develop new therapeutic strategies against pathogens.

PhREEPred: Phage Resistance Emergence Prediction Web Tool to Foresee Encapsulated Bacterial Escape from Phage Cocktail Treatment.

Phages, as well as phage-derived proteins, especially lysins and depolymerases, are intensively studied to become prospective alternatives or supportive antibacterials used alone or in combination. In the common phage therapy approach, the unwanted emergence of phage-resistant variants from the treated bacterial population can be postponed or reduced by the utilization of an effective phage cocktail. In this work, we present a publicly available web tool PhREEPred (Phage Resistance Emergence Prediction) (https://phartner.shinyapps.io/PhREEPred/), which will allow an informed choice of the composition of phage cocktails by predicting the outcome of phage cocktail or phage/depolymerase combination treatments against encapsulated bacterial pathogens given a mutating population that escapes single phage treatment. PhREEPred simulates solutions of our mathematical model calibrated and tested on the experimental Klebsiella pneumoniae setup and Klebsiella-specific lytic phages: K63 type-specific phage KP34 equipped with a capsule-degrading enzyme (KP34p57), capsule-independent myoviruses KP15 and KP27, and recombinant capsule depolymerase KP34p57. The model can calculate the phage-resistance emergence depending on the bacterial growth rate and initial density, the multiplicity of infection, phage latent period, its infectiveness and the cocktail composition, as well as initial depolymerase concentration and activity rate. This model reproduced the experimental results and showed that (i) the phage cocktail of parallelly infecting phages is less effective than the one composed of sequentially infecting phages; (ii) depolymerase can delay or prevent bacterial resistance by unveiling an alternative receptor for initially inactive phages. In our opinion, this customer-friendly web tool will allow for the primary design of the phage cocktail and phage-depolymerase combination effectiveness against encapsulated pathogens.

The Specific Capsule Depolymerase of Phage PMK34 Sensitizes Acinetobacter baumannii to Serum Killing.

The rising antimicrobial resistance is particularly alarming for Acinetobacter baumannii, calling for the discovery and evaluation of alternatives to treat A. baumannii infections. Some bacteriophages produce a structural protein that depolymerizes capsular exopolysaccharide. Such purified depolymerases are considered as novel antivirulence compounds. We identified and characterized a depolymerase (DpoMK34) from Acinetobacter phage vB_AbaP_PMK34 active against the clinical isolate A. baumannii MK34. In silico analysis reveals a modular protein displaying a conserved N-terminal domain for anchoring to the phage tail, and variable central and C-terminal domains for enzymatic activity and specificity. AlphaFold-Multimer predicts a trimeric protein adopting an elongated structure due to a long α-helix, an enzymatic ß-helix domain and a hypervariable 4 amino acid hotspot in the most ultimate loop of the C-terminal domain. In contrast to the tail fiber of phage T3, this hypervariable hotspot appears unrelated with the primary receptor. The functional characterization of DpoMK34 revealed a mesophilic enzyme active up to 50 °C across a wide pH range (4 to 11) and specific for the capsule of A. baumannii MK34. Enzymatic degradation of the A. baumannii MK34 capsule causes a significant drop in phage adsorption from 95% to 9% after 5 min. Although lacking intrinsic antibacterial activity, DpoMK34 renders A. baumannii MK34 fully susceptible to serum killing in a serum concentration dependent manner. Unlike phage PMK34, DpoMK34 does not easily select for resistant mutants either against PMK34 or itself. In sum, DpoMK34 is a potential antivirulence compound that can be included in a depolymerase cocktail to control difficult to treat A. baumannii infections.

Special Issue: Phage-Bacteria Interplay in Health and Disease.

Bacteriophages are obligatory parasites propagating in bacterial hosts in a lytic or lysogenic/pseudolysogenic cycle [...].

Bacteriophages and antibiotic interactions in clinical practice: what we have learned so far.

Bacteriophages (phages) may be used as an alternative to antibiotic therapy for combating infections caused by multidrug-resistant bacteria. In the last decades, there have been studies concerning the use of phages and antibiotics separately or in combination both in animal models as well as in humans. The phenomenon of phage-antibiotic synergy, in which antibiotics may induce the production of phages by bacterial hosts has been observed. The potential mechanisms of phage and antibiotic synergy was presented in this paper. Studies of a biofilm model showed that a combination of phages with antibiotics may increase removal of bacteria and sequential treatment, consisting of phage administration followed by an antibiotic, was most effective in eliminating biofilms. In vivo studies predominantly show the phenomenon of phage and antibiotic synergy. A few studies also describe antagonism or indifference between phages and antibiotics. Recent papers regarding the application of phages and antibiotics in patients with severe bacterial infections show the effectiveness of simultaneous treatment with both antimicrobials on the clinical outcome.

Protein with negative surface charge distribution, Bnr1, shows characteristics of a DNA-mimic protein and may be involved in the adaptation of Burkholderia cenocepacia.

Adaptation of opportunistic pathogens to their host environment requires reprogramming of a vast array of genes to facilitate survival in the host. Burkholderia cenocepacia, a Gram-negative bacterium with a large genome of ∼8 Mb that colonizes environmental niches, is exquisitely adaptable to the hypoxic environment of the cystic fibrosis lung and survives in macrophages. We previously identified an immunoreactive acidic protein encoded on replicon 3, BCAS0292. Deletion of the BCAS0292 gene significantly altered the abundance of 979 proteins by 1.5-fold or more; 19 proteins became undetectable while 545 proteins showed ≥1.5-fold reduced abundance, suggesting the BCAS0292 protein is a global regulator. Moreover, the ∆BCAS0292 mutant showed a range of pleiotropic effects: virulence and host-cell attachment were reduced, antibiotic susceptibility was altered, and biofilm formation enhanced. Its growth and survival were impaired in 6% oxygen. In silico prediction of its three-dimensional structure revealed BCAS0292 presents a dimeric ß-structure with a negative surface charge. The ΔBCAS0292 mutant displayed altered DNA supercoiling, implicated in global regulation of gene expression. Three proteins were identified in pull-downs with FLAG-tagged BCAS0292, including the Histone H1-like protein, HctB, which is recognized as a global transcriptional regulator. We propose that BCAS0292 protein, which we have named Burkholderia negatively surface-charged regulatory protein 1 (Bnr1), acts as a DNA-mimic and binds to DNA-binding proteins, altering DNA topology and regulating the expression of multiple genes, thereby enabling the adaptation of B. cenocepacia to highly diverse environments.

The Antibacterial Effect of PEGylated Carbosilane Dendrimers on P. aeruginosa Alone and in Combination with Phage-Derived Endolysin.

The search for new microbicide compounds is of an urgent need, especially against difficult-to-eradicate biofilm-forming bacteria. One attractive option is the application of cationic multivalent dendrimers as antibacterials and also as carriers of active molecules. These compounds require an adequate hydrophilic/hydrophobic structural balance to maximize the effect. Herein, we evaluated the antimicrobial activity of cationic carbosilane (CBS) dendrimers unmodified or modified with polyethylene glycol (PEG) units, against planktonic and biofilm-forming P. aeruginosa culture. Our study revealed that the presence of PEG destabilized the hydrophilic/hydrophobic balance but reduced the antibacterial activity measured by microbiological cultivation methods, laser interferometry and fluorescence microscopy. On the other hand, the activity can be improved by the combination of the CBS dendrimers with endolysin, a bacteriophage-encoded peptidoglycan hydrolase. This enzyme applied in the absence of the cationic CBS dendrimers is ineffective against Gram-negative bacteria because of the protective outer membrane shield. However, the endolysin-CBS dendrimer mixture enables the penetration through the membrane and then deterioration of the peptidoglycan layer, providing a synergic antimicrobial effect.

Outer Membrane Vesicles (OMVs) of Pseudomonas aeruginosa Provide Passive Resistance but Not Sensitization to LPS-Specific Phages.

Outer membrane vesicles (OMVs) released from gram-negative bacteria are key elements in bacterial physiology, pathogenesis, and defence. In this study, we investigated the role of Pseudomonas aeruginosa OMVs in the anti-phage defence as well as in the potential sensitization to LPS-specific phages. Using transmission electron microscopy, virion infectivity, and neutralization assays, we have shown that both phages efficiently absorb on free vesicles and are unable to infect P. aeruginosa host. Nevertheless, the accompanying decrease in PFU titre (neutralization) was only observed for myovirus KT28 but not podovirus LUZ7. Next, we verified whether OMVs derived from wild-type PAO1 strain can sensitize the LPS-deficient mutant (Δwbpl PAO1) resistant to tested phages. The flow cytometry experiments proved a quite effective and comparable association of OMVs to Δwbpl PAO1 and wild-type PAO1; however, the growth kinetic curves and one-step growth assay revealed no sensitization event of the OMV-associated phage-resistant P. aeruginosa deletant to LPS-specific phages. Our findings for the first time identify naturally formed OMVs as important players in passive resistance (protection) of P. aeruginosa population to phages, but we disproved the hypothesis of transferring phage receptors to make resistant strains susceptible to LPS-dependent phages.

Genome-driven elucidation of phage-host interplay and impact of phage resistance evolution on bacterial fitness.

When considering the interactions between bacteriophages and their host, the issue of phage-resistance emergence is a key element in understanding the ecological impact of phages on the bacterial population. It is also an essential parameter for the implementation of phage therapy to combat antibiotic-resistant pathogens. This study investigates the phenotypic and genetic responses of five Pseudomonas aeruginosa strains (PAO1, A5803, AA43, CHA, and PAK) to the infection by seven phages with distinct evolutionary backgrounds and recognised receptors (LPS/T4P). Emerging phage-insensitivity was generally accompanied by self and cross-resistance mechanisms. Significant differences were observed between the reference PAO1 responses compared to other clinical representatives. LPS-dependent phage infections in clinical strains selected for mutations in the "global regulatory" and "other" genes, rather than in the LPS-synthesis clusters detected in PAO1 clones. Reduced fitness, as proxied by the growth rate, was correlated with large deletion (20-500 kbp) and phage carrier state. Multi-phage resistance was significantly correlated with a reduced growth rate but only in the PAO1 population. In addition, we observed that the presence of prophages decreased the lytic phage maintenance seemingly protecting the host against carrier state and occasional lytic phage propagation, thus preventing a significant reduction in bacterial growth rate.

The Mutation in wbaP cps Gene Cluster Selected by Phage-Borne Depolymerase Abolishes Capsule Production and Diminishes the Virulence of Klebsiella pneumoniae.

Klebsiella pneumoniae is considered one of the most critical multidrug-resistant pathogens and urgently requires new therapeutic strategies. Capsular polysaccharides (CPS), lipopolysaccharides (LPS), and exopolysaccharides (EPS) are the major virulence factors protecting K. pneumoniae against the immune response and thus may be targeted by phage-based therapeutics such as polysaccharides-degrading enzymes. Since the emergence of resistance to antibacterials is generally considered undesirable, in this study, the genetic and phenotypic characteristics of resistance to the phage-borne CPS-degrading depolymerase and its effect on K. pneumoniae virulence were investigated. The K63 serotype targeting depolymerase (KP36gp50) derived from Klebsiella siphovirus KP36 was used as the selective agent during the treatment of K. pneumoniae 486 biofilm. Genome-driven examination combined with the surface polysaccharide structural analysis of resistant mutant showed the point mutation and frameshift in the wbaP gene located within the cps gene cluster, resulting in the loss of the capsule. The sharp decline in the yield of CPS was accompanied by the production of a larger amount of smooth LPS. The modification of the surface polysaccharide layers did not affect bacterial fitness nor the insensitivity to serum complement; however, it made bacteria more prone to phagocytosis combined with the higher adherence and internalization to human lung epithelial cells. In that context, it was showed that the emerging resistance to the antivirulence agent (phage-borne capsule depolymerase) results in beneficial consequences, i.e., the sensitization to the innate immune response.

Multifunctionality of Nanosized Calcium Apatite Dual-Doped with Li+/Eu3+ Ions Related to Cell Culture Studies and Cytotoxicity Evaluation In Vitro.

Li+/Eu3+ dual-doped calcium apatite analogues were fabricated using a microwave stimulated hydrothermal technique. XRPD, FT-IR, micro-Raman spectroscopy, TEM and SAED measurements indicated that obtained apatites are single-phased, crystallize with a hexagonal structure, have similar morphology and nanometric size as well as show red luminescence. Lithium effectively modifies the local symmetry of optical active sites and, thus, affects the emission efficiency. Moreover, the hydrodynamic size and surface charge of the nanoparticles have been extensively studied. The protein adsorption (lysozyme, LSZ; bovine serum albumin, BSA) on the nanoparticle surface depended on the type of cationic dopant (Li+, Eu3+) and anionic group (OH-, Cl-, F-) of the apatite matrix. Interaction with LSZ resulted in a positive zeta potential, and the nanoparticles had the lowest hydrodynamic size in this protein medium. The cytotoxicity assessment was carried out on the human osteosarcoma cell line (U2OS), murine macrophages (J774.E), as well as human red blood cells (RBCs). The studied apatites were not cytotoxic to RBCs and J774.E cells; however, at higher concentrations of nanoparticles, cytotoxicity was observed against the U2OS cell line. No antimicrobial activity was detected against Gram-negative bacteria with one exception for P. aeruginosa treated with Li+-doped fluorapatite.