Phage Delivery Research Articles

Bacteriophage entrapment strategies for the treatment of chronic wound infections: a comprehensive review.

The growing threat of antimicrobial resistance has made the quest for antibiotic alternatives or synergists one of the most pressing priorities of the 21st century. The emergence of multidrug-resistance in most of the common wound pathogens has amplified the risk of antibiotic-resistant wound infections. Bacteriophages, with their self-replicating ability and targeted specificity, can act as suitable antibiotic alternatives. Nevertheless, targeted delivery of phages to infection sites remains a crucial issue, specifically in the case of topical infections. Hence, different phage delivery systems have been studied in recent years. However, there have been no recent reviews of phage delivery systems focusing exclusively on phage application on wounds. This review provides a compendium of all the major delivery systems that have been used to deliver phages to wound infection sites. Special focus has also been awarded to phage-embedded hydrogels with a discussion on the different aspects to be considered during their preparation.

Efficacy of Bacteriophages in Wound Healing: An Updated Review.

We have attempted to summarize the efficacy of bacteriophage therapy, highlighting the recent advances and phage delivery methods in different clinical trials and animal studies for wound-associated infections. Bacteriophage therapy is the lyse of bacteria by bacteriophages at the site of invasion. As bacteria become more resistant to antibiotics, discovering an alternative is more important than ever, and bacteriophage therapy has yielded promising outcomes. A clear knowledge of the bacteriophage, microbiota, and human host and their interaction is necessary to implement bacteriophage treatment on a large scale. Much technological advancement and regulatory guidelines increased the credibility of phage therapy (PT). Still, the challenges include the development of efficient bacteriophage screening methods, phage therapy strategies for biofilms, and the quality and safety of phage preparations. However, much consideration is to be taken in designing a novel therapeutic approach for antibiotic-resistant infections by using phages, phage lytic proteins, bioengineered phages, or antibiotics in combination.

Optimized Dosing and Delivery of Bacteriophage Therapy for Wound Infections.

Lytic bacteriophages, viruses that lyse (kill) bacteria, hold great promise for treating infections, including wound infections caused by antimicrobial-resistant Pseudomonas aeruginosa. However, the optimal dosing and delivery strategies for phage therapy remain unclear. In a mouse wound infection model, we investigated the impact of dose, frequency, and administration route on the efficacy of phage therapy. We find that topical but not intravenous delivery is effective in this model. High-doses of phage reduces bacterial burden more effectively than low-doses, and repeated dosing achieves the highest eradication rates. Building on these insights, we developed "HydroPhage", a hyaluronan-based hydrogel system that uses dynamic covalent crosslinking to deliver high-titre phages over one week. HydroPhage eradicates infections five times more effectively than intravenous injection. We conclude that hydrogel-based sustained phage delivery enhances the efficacy of phage therapy and offers a practical, well-tolerated option for topical application.

Synergizing Bacteriophage Therapy and Computational Neuroscience: A Ray of Hope in the Fight against Antibiotic Resistance

Antibiotic resistance presents an urgent and pervasive global health crisis that jeopardises human well-being. This dilemma primarily arises from the inappropriate and excessive use of antibiotics across diverse sectors, leading to the emergence of bacteria that have developed resistance to multiple medications. It is a matter of utmost concern due to its direct link to increased mortality rates and the disconcerting possibility of reverting to an era where antibiotics lose their effectiveness. This student experience paper delves into the potential of bacteriophage therapy as a remedy for antibiotic-resistant infections, a pressing issue emphasized by the National Health Service (NHS) in England. Building upon the recent first-hand experience of the first author while shadowing a General Medical Practitioner in Birmingham, UK, the study delves into critical facets of phage therapy. This exploration encompasses phage selection, formulation, delivery, and safety considerations. It also highlights the convergence with computational neuroscience, emphasizing the pivotal role of computational models in comprehending bacterial infections, antibiotic resistance, and neurological responses. This interdisciplinary approach underscores the necessity for collaborative efforts among microbiologists, healthcare professionals, and computational neuroscientists in addressing global healthcare challenges. Additionally, this experience sheds light on the practice of using antibiotics without a prescription, often for the treatment of infections that do not necessitate antibiotic intervention. This observation underscores the urgent requirement for stricter regulatory controls and an extensive educational program, both at the national and global levels.

Phage Delivery Strategies for Biocontrolling Human, Animal, and Plant Bacterial Infections: State of the Art.

This review aims at presenting the main strategies that are currently available for the delivery of bacteriophages to combat bacterial infections in humans, animals, and plants. It can be seen that the main routes for phage delivery are topical, oral, systemic, and airways for humans. In animals, the topical and oral routes are the most used. To combat infections in plant species, spraying the plant's phyllosphere or drenching the soil are the most commonly used methods. In both phage therapy and biocontrol using phages, very promising results have been obtained so far. However, more experiments are needed to establish forms of treatment and phage doses, among other parameters. Furthermore, in general, there is a lack of specific standards for the use of phages to combat bacterial infections.

Alginate-Encapsulated Mycobacteriophage: A Potential Approach for the Management of Intestinal Mycobacterial Disease.

Encapsulated medication is a common method of administering therapeutic treatments. As researchers explore alternative therapies, it is likely that encapsulation will remain a feature of these novel treatments, particularly when routes of delivery are considered. For instance, alginate-encapsulation is often favoured where gastric digestion poses an obstacle. When exposed to cations (namely Ca2+), alginate readily forms gels that are resilient to acidic conditions and readily dissociate in response to mid-range pH. This action can be extremely valuable for the encapsulation of phages. The efficient delivery of phages to the intestine is important when considering mycobacteriophage (MP) therapy (or MP prophylaxis) for disseminated mycobacterial infections and chronic gastroenteritis conditions. This study presents the design and in vitro validation of an alginate-encapsulated MP capable of releasing phages in a pH-dependent manner. Ultimately, it is shown that encapsulated phages pretreated with simulated gastric fluid (SGF) are capable of releasing viable phages into simulated intestinal fluid (SIF) and thereby reducing the mycobacterial numbers in spiked SIF by 90%. These findings suggest that alginate encapsulation may be a viable option for therapeutic and prophylactic approaches to the management of intestinal mycobacterial disease, such as Johne's disease.

Engineering pH-sensitive erodible chitosan hydrogel composite containing bacteriophage: An interplay between hydrogel and bacteriophage against Staphylococcus aureus

Encapsulation of phages represents a key approach for improving phage stability and controlling phage delivery dosage. The hydrogel made from positively charged quaternized chitosan (QCS) and multivalent crosslinker, aldehyde-modified poly(xylitol sebacate)-co-poly(ethylene glycol) (APP) was introduced for the first time for drug (phage 44AHJD) delivery. The freeze-thawing (FT) treatment enhanced the porous structure and the stress resistance of native hydrogel with increased compression stress (stiffness) from 10 to 20 kPa. The stiffness of the phage-loaded hydrogel (FTP) was suitable for the proper release of phage particles and polymer chains, both working synergistically against bacterial growth. The FTP followed the Korsmeyer-Peppas model's anomalous diffusion of phage particles at different temperatures (30–45 °C) and pH (6.6–8.5) conditions. FTP was sensitive to pH, which released more phage particles at pH-neutral conditions, while the release under acidic and alkaline conditions was more based on gel degradation. The high biocompatibility of FTP hydrogel at its working concentration of 30 mg mL−1 was demonstrated through a hemolysis ratio of <2 %. Sixty percent of the total encapsulated phages and 6 mg mL−1 of hydrogel debris were released after 10 h of hydrogel submerge treatment, which can fight the growing bacteria and the emergence of phage-resistant bacteria.

Bacteriophage Delivery Systems for Food Applications: Opportunities and Perspectives.

Currently, one-third of all food produced worldwide is wasted or lost, and bacterial contamination is one of the main reasons. Moreover, foodborne diseases are a severe problem, causing more than 420,000 deaths and nearly 600 million illnesses yearly, demanding more attention to food safety. Thus, new solutions need to be explored to tackle these problems. A possible solution for bacterial contamination is using bacteriophages (phages), which are harmless to humans; these natural viruses can be used to prevent or reduce food contamination by foodborne pathogens. In this regard, several studies showed the effectiveness of phages against bacteria. However, when used in their free form, phages can lose infectivity, decreasing the application in foods. To overcome this problem, new delivery systems are being studied to incorporate phages and ensure prolonged activity and controlled release in food systems. This review focuses on the existent and new phage delivery systems applied in the food industry to promote food safety. Initially, an overview of phages, their main advantages, and challenges is presented, followed by the different delivery systems, focused in methodologies, and biomaterials that can be used. In the end, examples of phage applications in foods are disclosed and future perspectives are approached.

Encapsulation and delivery of phage as a novel method for gut flora manipulation in situ: A review.

Intestinal flora regulation is an effective method to intervene and treat diseases associated with microbiome imbalance. In addition to conventional probiotic supplement, phage delivery has recently exhibited great prospect in modifying gut flora composition and regulating certain gene expression of gut bacteria. However, the protein structure of phage is vulnerable to external factors during storage and delivery, which leads to the loss of infection ability and flora regulation function. Encapsulation strategy provides an effective solution for improving phage stability and precisely controlling delivery dosage. Different functional materials including enzyme-responsive and pH-responsive polymers have been used to construct encapsulation carriers to protect phages from harsh conditions and release them in the colon. Meanwhile, diverse carriers showed different characteristics in structure and function, which influenced their protective effect and delivery efficiency. This review systematically summarizes recent research progress on the phage encapsulation and delivery, with an emphasis on function properties of carrier systems in the protection effect and colon-targeted delivery. The present review may provide a theoretical reference for the encapsulation and delivery of phage as microbiota modulator, so as to expedite the development of functional material and delivery carrier, as well as the advances in practical application of intestinal flora regulation.

Advancements in bacteriophage therapies and delivery for bacterial infection.

Having co-evolved with bacteria over hundreds of millions of years, bacteriophage are effective killers of specific bacterial hosts. Therefore, phage therapies for infection are a promising treatment avenue, can provide a solution for antibiotic resistant bacterial infections, and have specified targeting of infectious bacteria while allowing the natural microbiome to survive which systemic antibiotics often wipe out. Many phages have well studied genomes that can be modified to change target, widen target range, or change mode of action of killing bacterial hosts. Phage delivery can also be designed to increase efficacy of treatment, including encapsulation and delivery via biopolymers. Increased research into phage potential for therapies can allow new avenues to develop to treat a larger range of infections.

Evaluation of dendritic cell-targeting T7 phages as a vehicle to deliver avian influenza virus H5 DNA vaccine in SPF chickens.

There is a growing demand for effective technologies for the delivery of antigen to antigen-presenting cells (APCs) and their immune-activation for the success of DNA vaccines. Therefore, dendritic cell (DC)-targeting T7 phages were used as a vehicle to deliver DNA vaccine. In this study, a eukaryotic expression plasmid pEGFP-C1-HA2-AS containing the HA2 gene derived from the avian H5N1 virus and an anchor sequence (AS) gene required for the T7 phage packaging process was developed. To verify the feasibility of phage delivery, the plasmid encapsulated in DC-targeting phage capsid through the recognition of AS was evaluated both in vitro and in vivo. The pEGFP-C1-HA2-AS plasmid could evade digestion by DNase I by becoming encapsulated into the phage particles and efficiently expressed the HA2 antigen in DCs with the benefit of DC-targeting phages. For chickens immunized with the DC-targeting phage 74 delivered DNA vaccine, the levels of IgY and IgA antibodies, the concentration of IFN-γ and IL-12 cytokines in serum, the proliferation of lymphocytes, and the percentage of CD4+/CD8+ T lymphocytes isolated from peripheral blood were significantly higher than chickens which were immunized with DNA vaccine that was delivered by non-DC-targeting phage or placebo (p<0.05). Phage 74 delivered one-fiftieth the amount of pEGFP-C1-HA2-AS plasmid compared to Lipofectin, however, a comparable humoral and cellular immune response was achieved. Although, the HA2 DNA vaccine delivered by the DC-targeting phage induced enhanced immune responses, the protection rate of virus challenge was not evaluated. This study provides a strategy for development of a novel avian influenza DNA vaccine and demonstrates the potential of DC-targeting phage as a DNA vaccine delivery vehicle.

Comparison of bacterial suppression by phage cocktails, dual-receptor generalists, and coevolutionarily trained phages.

The evolution and spread of antibiotic-resistant bacteria have renewed interest in phage therapy, the use of bacterial viruses (phages) to combat bacterial infections. The delivery of phages in cocktails where constituent phages target different modalities (e.g., receptors) may improve treatment outcomes by making it more difficult for bacteria to evolve resistance. However, the multipartite nature of cocktails may lead to unintended evolutionary and ecological outcomes. Here, we compare a 2-phage cocktail with a largely unconsidered group of phages: generalists that can infect through multiple, independent receptors. We find that λ phage generalists and cocktails that target the same receptors (LamB and OmpF) suppress Escherichia coli similarly for ~2 days. Yet, a "trained" generalist phage, which previously adapted to its host via 28 days of coevolution, demonstrated superior suppression. To understand why the trained generalist was more effective, we measured the resistance of bacteria against each of our phages. We find that, when bacteria were assailed by two phages in the cocktail, they evolved mutations in manXYZ, a host inner-membrane transporter that λ uses to move its DNA across the periplasmic space and into the cell for infection. This provided cross-resistance against the cocktail and untrained generalist. However, these mutations were ineffective at blocking the trained generalist because, through coevolutionary training, it evolved to bypass manXYZ resistance. The trained generalist's past experiences in training make it exceedingly difficult for bacteria to evolve resistance, further demonstrating the utility of coevolutionary phage training for improving the therapeutic properties of phages.

Biofilm Control in Flow-Through Systems Using Polyvalent Phages Delivered by Peptide-Modified M13 Coliphages with Enhanced Polysaccharide Affinity.

Eradication of biofilms that may harbor pathogens in water distribution systems is an elusive goal due to limited penetration of residual disinfectants. Here, we explore the use of engineered filamentous coliphage M13 for enhanced biofilm affinity and precise delivery of lytic polyvalent phages (i.e., broad-host-range phages lysing multiple host strains after infection). To promote biofilm attachment, we modified the M13 major coat protein (pVIII) by inserting a peptide sequence with high affinity for Pseudomonas aeruginosa (P. aeruginosa) extracellular polysaccharides (commonly present on the surface of biofilms in natural and engineered systems). Additionally, we engineered the M13 tail fiber protein (pIII) to contain a peptide sequence capable of binding a specific polyvalent lytic phage. The modified M13 had 102- and 5-fold higher affinity for P. aeruginosa-dominated mixed-species biofilms than wildtype M13 and unconjugated polyvalent phage, respectively. When applied to a simulated water distribution system, the resulting phage conjugates achieved targeted phage delivery to the biofilm and were more effective than polyvalent phages alone in reducing live bacterial biomass (84 vs 34%) and biofilm surface coverage (81 vs 22%). Biofilm regrowth was also mitigated as high phage concentrations induced residual bacteria to downregulate genes associated with quorum sensing and extracellular polymeric substance secretion. Overall, we demonstrate that engineered M13 can enable more accurate delivery of polyvalent phages to biofilms in flow-through systems for enhanced biofilm control.

Alginate chitosan microbeads and thermos-responsive hyaluronic acid hydrogel for phage delivery

Bacteriophages are potential alternatives to conventional antibiotics in the treatment of multidrug resistant infections. However, optimal treatment administration protocols are not established. The direct application of bacteriophages in local biomaterials-based carriers offers solutions to many of the limitations of current modalities. In this study, thermo-responsive hyaluronic acid-poly(N-isopropylacrylamide) (HA-pNIPAM) hydrogels and alginate-chitosan microbeads have been developed as bioresorbable local bacteriophage delivery systems designed to enable rapid and delayed phage release, respectively. The long-tailed Staphylococcus aureus phage ISP and the short-tailed Pseudomonas aeruginosa phage LUZ19 were selected as structurally diverse phages to load within both biomaterials. HA-pNIPAM hydrogels were synthesized and their thermo-responsive crosslinking was confirmed after rheological assessment. Alginate microbeads where prepared by ionic-gelation and chitosan surface deposition was confirmed by amine sensitive assays. After embedding phages, their antimicrobial potential was retained when stored at 4 °C. Release of both phages from HA-pNIPAM hydrogels was gradual and consistent over 21 days, whereby, continuous release of the short-tailed LUZ19 was observed for 21 days from the microbeads, while release of the long-tailed ISP portrayed burst-like release during early timepoints, with gradually reducing phage release thereafter. These findings suggest that the release kinetics of phage from biomaterials are impacted by both the biomaterial and the phages themselves. These results highlight the potential of HA-pNIPAM hydrogel and alginate-chitosan microbeads for local delivery of ISP and LUZ19 bacteriophages for both rapid burst release and controlled, extended release.

Gated Ethidium- and Bleomycin-Loading in Phage T4 That Is Subsequently Purified Leak-Free

Chemotherapy-inhibiting tumor cell evolution to drug-resistance is potentially suppressed by using a drug delivery vehicle (DDV) that has gating. Gating would be used to increase tumor-selectivity of delivery of DDV packaged drug. Tumor-selectivity increase would make possible increase in tumor-delivered drug dose, which would suppress opportunities to evolve drug resistance. Currently used DDVs do not have gating but gating is a natural feature of some bacteriophages (phages). Phage T4, which has recently been found highly persistent in murine blood, is a potential gated DDV. Thus, here, we proceed towards a T4-DDV by developing (1) improved procedure for generating high concentrations and amounts of phage T4, (2) elevated temperature-driven gate-opening and ethidium- and bleomycin-loading, and (3) purification of loaded T4 by rate zonal centrifugation. We test for loading by native agarose gel electrophoresis (AGE) with fluorescence detection. We observe loading in both phage T4 and T4 (tail-free) heads. The loaded particles have an openable, closed gate. Stored, mature T4 phages and phage heads do not release ethidium during at least a month at 4 °C and 6 days at 37 and 42 °C. Tumor-specific T4 phage delivery is projected via both the EPR effect and high T4 persistence.

Nebulization as the mode to administer therapeutic bacteriophages to resolve Acinetobacter pneumonia in rabbits

Multidrug-resistant strains of , a significant hospital pathogen, have developed resistance to virtually all available antibiotics. Carbapenems antibiotics are among the most commonly used antibiotics against Acinetobacter infections, but they can be rendered ineffective by the metallo-beta-lactamase enzyme. In this study, bacteriophage isolated against producing was evaluated for its therapeutic potential in the rabbit pneumonia model. was isolated from the sputum and was speciated as per the standard microbiological techniques. Bacteriophage specific for producing from the sewage water. Pneumonia was introduced in the rabbits as per Esposito Pennington method and efficacy of the bacteriophage in resolving pneumonia was evaluated. For in-vivo experiments, five groups of rabbits were used including infection-free, phage control, bacteria-infected control, and the other two groups infected with and treated either with an antibiotic, colistin (2.5 mg/kg, twice a day intraperitoneally) or Acinetobacter phage (3×10 PFU/mL, given once through nebulizer). The experimental animals were monitored for 72 hours for mortality, and the surviving rabbits were killed for bacteriological and histopathological analysis In the infected group, pneumonia was developed within 48 hours, and 6/10 animals were dead after 72 hours. All the animals in the antibiotic group survived but showed signs of pneumonia, and there was up to 4 log CFU/g±0.24 reduction in the bacterial count. In phage treated group, all the animals survived at the end of 72 hours and all the animals were healthy with no signs of pneumonia. The experiment showed new insights into the application of bacteriophage through nebulization, a non-invasive method of phage delivery to rescue rabbits from pneumonia caused by -producing .

Phage Therapy of Mycobacterium Infections: Compassionate Use of Phages in 20 Patients With Drug-Resistant Mycobacterial Disease.

Nontuberculous Mycobacterium infections, particularly Mycobacterium abscessus, are increasingly common among patients with cystic fibrosis and chronic bronchiectatic lung diseases. Treatment is challenging due to intrinsic antibiotic resistance. Bacteriophage therapy represents a potentially novel approach. Relatively few active lytic phages are available and there is great variation in phage susceptibilities among M. abscessus isolates, requiring personalized phage identification. Mycobacterium isolates from 200 culture-positive patients with symptomatic disease were screened for phage susceptibilities. One or more lytic phages were identified for 55 isolates. Phages were administered intravenously, by aerosolization, or both to 20 patients on a compassionate use basis and patients were monitored for adverse reactions, clinical and microbiologic responses, the emergence of phage resistance, and phage neutralization in serum, sputum, or bronchoalveolar lavage fluid. No adverse reactions attributed to therapy were seen in any patient regardless of the pathogen, phages administered, or the route of delivery. Favorable clinical or microbiological responses were observed in 11 patients. Neutralizing antibodies were identified in serum after initiation of phage delivery intravenously in 8 patients, potentially contributing to lack of treatment response in 4 cases, but were not consistently associated with unfavorable responses in others. Eleven patients were treated with only a single phage, and no phage resistance was observed in any of these. Phage treatment of Mycobacterium infections is challenging due to the limited repertoire of therapeutically useful phages, but favorable clinical outcomes in patients lacking any other treatment options support continued development of adjunctive phage therapy for some mycobacterial infections.

Topical liquid formulation of bacteriophages for metered-dose spray delivery

Bacteriophage (phage) therapy is a promising treatment strategy to combat antibiotic-resistant bacteria. Clinical reports from a century ago, as well as recent reports have revealed safety and efficacy of phage therapy for bacterial wound infections. However, the conventional liquid phage formulation and delivery platforms reported lack of dose control as it easily runs off from the infection site and it is impossible to determine total volume transfer. The aim of this study was to formulate phage liquids for topical delivery using a metered-dose spray. Two types of anti-Pseudomonas phages, PEV1 (myovirus) and PEV31 (podovirus) were formulated in 35% ethanol in water containing non-ionic polymers. The formulations were evaluated for physical properties, ease of spray, dripping upon spraying, drying time, in vitro release profiles, antibacterial activity, and storage stability. The optimized phage-polymer spray formulations were easily sprayable with minimal dripping and fast drying time. Phages were rapidly released from the formulation and inhibited the growth of Pseudomonas aeruginosa. Both PEV1 and PEV31 remained biologically stable in the optimized formulations during storage at 4 °C for eight weeks. This study showed the topical spray formulations containing non-ionic polymers in ethanol/water could be a promising and innovative therapeutic system for delivering phages.

Further Considerations on How to Improve Phage Therapy Experimentation, Practice, and Reporting: Pharmacodynamics Perspectives.

Phage therapy uses bacterial viruses (bacteriophages) to infect and kill targeted pathogens. Approximately one decade ago, I started publishing on how possibly to improve upon phage therapy experimentation, practice, and reporting. Here, I gather and expand upon some of those suggestions. The issues emphasized are (1) that using ratios of antibacterial agents to bacteria is not how dosing is accomplished in the real world, (2) that it can be helpful to not ignore Poisson distributions as a means of either anticipating or characterizing phage therapy success, and (3) how to calculate a concept of ‘inundative phage densities.’ Together, these are issues of phage therapy pharmacodynamics, meaning they are ways of thinking about the potential for phage therapy treatments to be efficacious mostly independent of the details of delivery of phages to targeted bacteria. Much emphasis is placed on working with Poisson distributions to better align phage therapy with other antimicrobial treatments.

Incorporation of Mycobacteriophage Fulbright into Polycaprolactone Electrospun Nanofiber Wound Dressing.

The Genus Mycobacterium includes pathogens known to cause disease in mammals such as tuberculosis (Mycobacterium tuberculosis) and skin infections (M. abscessus). M. smegmatis is a model bacterium that can cause opportunistic infections in human tissues and, rarely, a respiratory disease. Due to the emergence of multidrug-resistant bacteria, phage therapy is potentially an alternative way of treating these bacterial infections. As bacteriophages are specific to their bacterial host, it ensures that the normal flora is unharmed. Fulbright is a mycobacteriophage that infects the host bacteria M. smegmatis. The main goal of this study is to incorporate Mycobacteriophage Fulbright into a polycaprolactone (PCL) nanofiber and test its antimicrobial effect against the host bacteria, M. smegmatis. Stability tests conducted over 7 days showed that the phage titer does not decrease when in contact with PCL, making it a promising vehicle for phage delivery. Antimicrobial assays showed that PCL_Fulbright effectively reduces bacterial concentration after 24 h of contact. In addition, when stored at −20 °C, the phage remains viable for up to eleven months in the fiber. Fulbright addition on the nanofibrous mats resulted in an increase in water uptake and decrease in the mechanical properties (strength and Young’s modulus) of the membranes, indicating that the presence of phage Fulbright can greatly enhance the physical and mechanical properties of the PCL. Cytotoxicity assays showed that PCL_Fulbright is not cytotoxic to Balbc/3T3 mouse embryo fibroblast cell lines; thus, phage-incorporated PCL is a promising alternative to antibiotics in treating skin infections.