Development of an Anti-Acinetobacter baumannii Biofilm Phage Cocktail: Genomic Adaptation to the Host.

The need for alternatives to antibiotic therapy due to the emergence of multidrug resistant bacteria (MDR), such as the nosocomial pathogen Acinetobacter baumannii, has led to the recovery of phage therapy. In addition, phages can be combined in cocktails to increase the host range. In this study, the evolutionary mechanism of adaptation was utilized in order to develop a phage adapted to A. baumannii, named phage Ab105-2phiΔCI404ad, from a mutant lytic phage, Ab105-2phiΔCI, previously developed by our group. The whole genome sequence of phage Ab105-2phiΔCI404ad was determined, showing that four genomic rearrangements events occurred in the tail morphogenesis module affecting the ORFs encoding the host receptor binding sites. As a consequence of the genomic rearrangements, 10 ORFs were lost and four new ORFs were obtained, all encoding tail proteins; two inverted regions were also derived from these events. The adaptation process increased the host range of the adapted phage by almost 3-fold. In addition, a depolymerase-expressing phenotype, indicated by formation of a halo, which was not observed in the ancestral phage, was obtained in 81% of the infected strains. A phage cocktail was formed by combining this phage with the A. baumannii phage vB_AbaP_B3, known to express a depolymerase. Both the individual phages and the phage cocktail showed strong antimicrobial activity against 5 clinical strains and 1 reference strain of A. baumannii tested. However, in all cases resistance to the bacterial strains was also observed. The antibiofilm activity of the individual phages and the cocktail was assayed. The phage cocktail displayed strong antibiofilm activity.

Maternal and Child Health Pipeline Training Programs: A Description of Training Across 6 Funded Programs.

PURPOSE: The HRSA-funded maternal and child health pipeline training programs (MCHPTPs) are a response to the critical need to diversify the MCH workforce, as a strategy to reduce health disparities in MCH populations. These MCHPTPs support students from undergraduate to graduate education and ultimately into the MCH workforce. DESCRIPTION: The models and components of training across the six MCHPTPs funded in 2016-2021 are summarized, to examine the design and delivery of undergraduate pipeline training and the insights gained across programs. ASSESSMENT: Strategies that emerged across training programs were organized into three themes: recruitment, support for student persistence (in education), and pipeline-to-workforce intentionality. Support for student persistence included financial support, mentoring, creating opportunity for students to develop a sense of belonging, and the use of research as a tool to promote learning and competitiveness for graduate education. Finally, the link to Maternal and Child Health Bureau (MCHB) long-term training and other MCHB opportunities for professional development contributed significant nuance to the pipeline-to-workforce objectives of these programs. CONCLUSIONS: The MCHPTPs not only increase the diversity of the MCH workforce, they also actively prepare the next generation of MCH leaders. The intentional connection of undergraduates to the infrastructure and continuum of MCH training, underscores the comprehensive impact of this funding.

Candida glabrata's recurrent infections: biofilm formation during Amphotericin B treatment.

UNLABELLED: Candida species are responsible for recurrent human infections, mostly in immunocompromised patients, due to their high vulnerability. Candida glabrata has a major role in systemic candidiasis and Amphotericin B (AmB), a polyene only used in hospitals, is frequently used to treat this disease. Lately, however, clinical evidences of Candida recurrent infections during these treatments are being described, probably due to biofilm (re)formation during this therapy. Thus, this work aims at inferring if C. glabrata biofilms are still being formed during AmB treatment. For that, C. glabrata biofilms were formed in the presence of AmB and analysed by dry weight. Matrix composition was analysed quantifying carbohydrates and, specifically, ß-1,3 glucans. Results demonstrated that, although in a lesser extent, C. glabrata is able to develop biofilms in the presence of AmB, with a thick extracellular matrix, with an increase on carbohydrates, especially ß-1,3 glucans. Therefore, it is confirmed that complex biofilms of C. glabrata can be formed during an AmB treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows new insights regarding recurrent candidiasis. The authors demonstrated that Amphotericin B did not totally prevent the development of biofilms during Candida glabrata's infection treatment and that the change in the biofilm matrices may have a high responsibility for the fail in the treatment of systemic candidiasis.

Effect of magnetic hyperthermia on the structure of biofilm and cellular viability of a food spoilage bacterium.

This work evaluated the effect of magnetic hyperthermia (MH) on planktonic cells and biofilms of a major food spoilage bacterium Pseudomonas fluorescens and its performance compared to a conventional direct heating (DH) technique. The results showed that MH had a greater and faster bactericidal effect, promoting a significant reduction in cell viability (≥3 Log CFU) in planktonic and biofilm cells, and leading to a complete eradication of planktonic cells at 55 °C (after only ~8 min). Accordingly, when comparing the same final temperatures, MH was more harmful to the integrity of cell membranes than DH, as observed in confocal laser scanning microscope images. Additionally, scanning electron microscope images revealed that exposure to MH had promoted modifications of the bacterial cell surface as well as of the structure of the biofilm. These results present the possibility of using MH out of the biomedical field as a potential disinfection method in food-related environments.

Infective endocarditis in intravenous drug abusers: an update.

Infective endocarditis despite advances in diagnosis remains a common cause of hospitalization, with high morbidity and mortality rates. Through literature review it is possible to conclude that polymicrobial endocarditis occurs mainly in intravenous drug abusers with predominance in the right side of the heart, often with tricuspid valve involvement. This fact can be associated with the type of drug used by the patients; therefore, knowledge of the patient's history is critical for adjustment of the therapy. It is also important to emphasize that the most common combinations of organisms in polymicrobial infective endocarditis are: Staphylococcus aureus, Streptococcus pneumonia and Pseudomonas aeruginosa, as well as mixed cultures of Candida spp. and bacteria. A better understanding of the epidemiology and associated risk factors are required in order to develop an efficient therapy, although PE studies are difficult to perform due to the rarity of cases and lack of prospective cohorts.

Selection and characterization of a multivalent Salmonella phage and its production in a nonpathogenic Escherichia coli strain.

We report the selection and amplification of the broad-host-range Salmonella phage phi PVP-SE1 in an alternative nonpathogenic host. The lytic spectrum and the phage DNA restriction profile were not modified upon replication in Escherichia coli Bl21, suggesting the possibility of producing this phage in a nonpathogenic host, contributing to the safety and easier approval of a product based on this Salmonella biocontrol agent.

Salmonella Enteritidis bacteriophage candidates for phage therapy of poultry.

AIMS: Salmonella is a worldwide foodborne pathogen causing acute enteric infections in humans. In the recent years, the use of bacteriophages has been suggested as a possible tool to combat this zoonotic pathogen in poultry farms. This work aims to isolate and perform comparative studies of a group of phages active against a collection of specific Salmonella Enteritidis strains from Portugal and England. Also, suitable phage candidates for therapy of poultry will be selected. METHODS AND RESULTS: The Salm. Enteritidis strains studied were shown to have a significantly high occurrence of defective (cryptic) prophages; however, no live phages were found in the strains. Bacteriophages isolated from different environments lysed all except one of the tested Salm. Enteritidis strains. The bacteriophages studied were divided into different groups according to their genetic homology, RFLP profiles and phenotypic features, and most of them showed no DNA homology with the bacterial hosts. The bacteriophage lytic efficacy proved to be highly dependent on the propagation host strain. CONCLUSIONS: Despite the evidences shown in this work that the Salm. Enteritidis strains used did not produce viable phages, we have confirmed that some phages, when grown on particular hosts, behaved as complexes of phages. This is most likely because of the presence of inactive phage-related genomes (or their parts) in the bacterial strains which are capable of being reactivated or which can recombine with lytic phages. Furthermore, changes of the bacterial hosts used for maintenance of phages must be avoided as these can drastically modify the parameters of the phage preparations, including host range and lytic activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This work shows that the optimal host and growth conditions must be carefully studied and selected for the production of each bacteriophage candidate for animal therapy.

Method for bacteriophage isolation against target Campylobacter strains.

AIMS: Poultry meat is considered a major source of Campylobacter. This micro-aerobic bacterium is commonly responsible for foodborne illness. This work focuses on the isolation of Campylobacter coli lytic bacteriophages (phages) against target C. coli strains. METHODS AND RESULTS: A method involving the enrichment of free-range chicken samples in a broth containing the target C. coli strains and salts (CaCl(2) and MgSO(4)) was used for phage isolation. This method allowed the isolation of 43 phages that were active against 83% of the C. coli strains used in the isolation procedure. Approximately 65% of the phages were also effective against Campylobacter jejuni strains. CONCLUSIONS: The use of target pathogens in the phage isolation step improves the likelihood of detecting and isolating phages for the control of these specific strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This technique will be valuable in the context of phage therapy for enriching for phages that are active against specifically identified strains of bacteria, for example from a food poisoning outbreak or epidemic strains resistant to multiple antibiotics. In these situations, using the conventional methods for searching for bacteriophages active for these particular strains can be a time-consuming, if not an unsuccessful process. Using the isolation method described in this manuscript, the particular strains can be added to the enrichment broth increasing the probability of finding phages against them. Therefore, it will shorten the time needed for seeking phages able to lyse target strains, which in most of the cases, because of the rapid increase in antimicrobial-resistant bacteria, is of crucial importance.

Crystal violet staining to quantify Candida adhesion to epithelial cells.

In vitro studies of adhesion capability are essential to characterise the virulence of Candida species. However, the assessment of adhesion by traditional methods is time-consuming. The aim of the present study is the development of a simple methodology using crystal violet staining to quantify in vitro adhesion of different Candida species to epithelial cells. The experiments are performed using Candida albicans (ATCC 90028), C. glabrata (ATCC 2001), C. parapsilosis (ATCC 22019) and C. tropicalis (ATCC 750). A human urinary bladder epithelial cell line (TCC-SUP) is used. Yeast and epithelial cells were stained with crystal violet, epithelial cells were then destained using intermediate washing, and the dye in the yeast cells was extracted with acetic acid. The method was validated for the different Candida reference species by comparison with traditional microscope observation and enumeration. The method was then used to assess Candida adhesion to epithelial cells and also to silicone. For all Candida spp. high correlation values (r2= 0.9724-0.9997) between the number of adherent yeasts (microscope enumeration) and absorbance values were obtained for an inoculum concentration >10(6) cells/mL. The proposed technique was easy to perform and reproducible, enabling the determination of adhesion ability of Candida species to an epithelial cell line.

[Induction and migration of cryptic/defective Salmonella enterica prophages as a consequence of infection with lytic phages is an additional factor in stability of a coevolutionary vector].

The influence of infection of natural isolates of Salmonella enterica with lytic (nonlysogenic) phages on the expression of resident cryptic or defective prophages in host bacteria was studied. The induction of defective/cryptic phages after infection with nonlysogenic phages and packaging of bacterial chromosomal fragments in capsids of defective phages is demonstrated. This may lead to migration and wide distribution of both the genomes of defective phages per se and various fragments of the bacterial chromosome (including pathogenic islands) in new bacterial strains with concomitant change of their properties, the acquired new features of pathogenicity among them.

The role of polysaccharide intercellular adhesin (PIA) in Staphylococcus epidermidis adhesion to host tissues and subsequent antibiotic tolerance.

The aim of this study was to determine the role of polysaccharide intercellular adhesin (PIA) in Staphylococcus epidermidis adhesion to host tissues and subsequent antibiotic tolerance. The adherence of S. epidermidis 1457 and the mutant defective in PIA production (1457-M10) to urinary epithelium and endothelium was estimated by colony counting. Minimum bactericidal concentration and mean reduction of cellular activity (XTT) following antibiotic exposure was determined for planktonic and adhered bacteria. S. epidermidis 1457 adhered to a greater extent to both cells than the mutant strain. The adhered strains had a significantly higher antimicrobial tolerance than their planktonic counterparts. The mutant strain was, in general, the most susceptible to the antibiotics assayed. In conclusion, PIA may influence S. epidermidis adherence to host tissues and their antimicrobial susceptibility. Initial adhesion may be the main step for the acquisition of resistance in S. epidermidis.

Antibiotherapy and pathogenesis of uncomplicated UTI: difficult relationships.

In a time when conventional antibiotics are becoming increasingly less effective for treatment of infections, the relationship between bacteria and antimicrobial resistance is becoming more and more complicated. This paper provides a current review of studies reported in the literature pertaining to the antibiotherapy of human urinary tract infections (UTI), in a way that helps the reader direct a bibliographic search and develop an integrated perspective of the subject. Highlights are given to (bio)pathogenesis of uncomplicated cystitis. Features associated with the antibiotherapy of UTI such as development of resistance are presented in the text systematically. This review discusses recent advances in the understanding of how the predominant uropathogen Escherichia coli interacts with its host and leads to infection; so one can understand some of the reasons behind antibiotherapy failures.

Isolation and characterization of bacteriophages for avian pathogenic E. coli strains.

AIMS: To isolate and characterize bacteriophages, and to evaluate its lytic performance against avian pathogenic Escherichia coli (APEC) strains with high patterns of antibiotic resistance, in order to select phages for a therapeutic product to treat colibacillosis in chickens. METHODS AND RESULTS: Bacteriophages were isolated from poultry sewage and tested against 148 O-serotyped APEC strains. The morphological characterization of the bacteriophages was made by transmission electronic microscopy (TEM) observations and the genetic comparison between bacteriophages DNA was performed by restriction fragment length polymorphism (RFLP) patterns. Results showed that 70.5% of the tested E. coli strains were sensitive to a combination of three of the five isolated phages, that seemed to be virulent and taxonomically belong to the Caudovirales order. Two of them look like 16-19, T4-like phages (Myoviridae) and the third is a T1-like phage and belongs to Syphoviridae family. All of them are genetically different. CONCLUSIONS: It was possible to obtain a combination of three different lytic bacteriophages with broad lytic spectra against the most prevalent O-serotypes of APEC. SIGNIFICANCE AND IMPACT OF THE STUDY: Data reported in this study, presents an in vitro well studied phage product to be used as antimicrobial agent to treat colibacillosis in poultry industry.

The influence of the mode of administration in the dissemination of three coliphages in chickens.

Escherichia coli can cause severe respiratory and systemic infections in chickens, and it is often associated with significant economic losses in the poultry industry. Bacteriophages (phages) have been shown to be potential alternatives to the antibiotics in the treatment of bacterial infections. To accomplish that, phage particles must be able to reach and remain active in the infected organs. The present work aims at evaluating the effect of the route of administration and the dosage in the dissemination of 3 coliphages in the chicken's organs. In vivo trials were conducted by infecting chickens orally, spray, and i.m. with 10(6), 10(7), and 10(8) plaque-forming units/mL suspensions of 3 lytic phages: phi F78E (Myoviridae), phi F258E (Siphoviridae), and phi F61E (Myoviridae). Birds were killed 3, 10, and 24 h after challenge and the phage titer was measured in lungs and air sacs membranes, liver, duodenum, and spleen. When administered by spray, the 3 phages reached the respiratory tract within 3 h. Oral administration also allowed all phages to be recovered in lungs, but only phi F78E was recovered from the duodenum, the liver, and the spleen. These differences can be explained by the possible replication of phi F78E in commensal E. coli strains present in the chicken gut, thus leading to a higher concentration of this phage in the intestines that resulted in systemic circulation of phage with consequent phage in organs. When phages were administered i.m., they were found in all of the collected organs. Despite this better response, i.m. administration is a nonpracticable way of protecting a large number of birds in a poultry unit. In general, the results suggest that oral administration and spray allowed phages to reach and to remain active in the respiratory tract and can, therefore, be considered promising administration routes to treat respiratory E. coli infections in the poultry industry.

In vivo toxicity study of phage lysate in chickens.

1. Bacteriophage (phage) crude lysate of Gram-negative bacteria often contains bacterial debris, including lipopolysaccharides found in the outer membrane of the cell wall, which are potentially toxic. 2. In this study, an in vivo evaluation of the toxicity of a suspension of three phages to control pathogenic Escherichia coli strains in poultry was performed. 3. Eighteen commercial layers, 7 weeks old, were intramuscularly injected with phage lysate (8.21 x 10(4) Endotoxin Units/dose). The control group was injected with sterile Luria Bertani (LB) broth. 4. Bird prostration and decrease in body weight gain and water intake per gram of body weight were observed only on the day of the inoculation in the challenged group. Over the following 6 d, no differences were observed in the chickens' activity. 5. These results support the view that phage crude lysate carrying endotoxins are not toxic for chickens.

Efficacy and safety assessment of two enterococci phages in an in vitro biofilm wound model.

Chronic wounds affect thousands of people worldwide, causing pain and discomfort to patients and represent significant economical burdens to health care systems. The treatment of chronic wounds is very difficult and complex, particularly when wounds are colonized by bacterial biofilms which are highly tolerant to antibiotics. Enterococcus faecium and Enterococcus faecalis are within the most frequent bacteria present in chronic wounds. Bacteriophages (phages) have been proposed as an efficient and alternative against antibiotic-resistant infections, as those found in chronic wounds. We have isolated and characterized two novel enterococci phages, the siphovirus vB_EfaS-Zip (Zip) and the podovirus vB_EfaP-Max (Max) to be applied during wound treatment. Both phages demonstrated lytic behavior against E. faecalis and E. faecium. Genome analysis of both phages suggests the absence of genes associated with lysogeny. A phage cocktail containing both phages was tested against biofilms formed in wound simulated conditions at a multiplicity of infection of 1.0 and a 2.5 log CFU.mL-1 reduction in the bacterial load after at 3 h of treatment was observed. Phages were also tested in epithelial cells colonized by these bacterial species and a 3 log CFU.mL-1 reduction was observed using both phages. The high efficacy of these new isolated phages against multi-species biofilms, their stability at different temperatures and pH ranges, short latent periods and non-cytotoxicity to epithelial cells suggest their therapeutic use to control infectious biofilms present in chronic wounds.

Alginate-nanohydroxyapatite hydrogel system: Optimizing the formulation for enhanced bone regeneration.

Ceramic/polymer-based biocomposites have emerged as potential biomaterials to fill, replace, repair or regenerate injured or diseased bone, due to their outstanding features in terms of biocompatibility, bioactivity, injectability, and biodegradability. However, these properties can be dependent on the amount of ceramic component present in the polymer-based composite. Therefore, in the present study, the influence of nanohydroxyapatite content (30 to 70 wt%) on alginate-based hydrogels was studied in order to evaluate the best formulation for maximizing bone tissue regeneration. The composite system was characterized in terms of physic-chemical properties and biological response, with in vitro cytocompatibility assessment with human osteoblastic cells and ex vivo functional evaluation in embryonic chick segmental bone defects. The main morphological characteristics of the alginate network were not affected by the addition of nanohydroxyapatite. However, physic-chemical features, like water-swelling rate, stability at extreme pH values, apatite formation, and Ca2+ release were nanoHA dose-dependent. Within in vitro cytocompatibility assays it was observed that hydrogels with nanoHA 30% content enhanced osteoblastic cells proliferation and expression of osteogenic transcription factors, while those with higher concentrations (50 and 70%) decreased the osteogenic cell response. Ex vivo data underlined the in vitro findings, revealing an enhanced collagenous deposition, trabecular bone formation and matrix mineralization with Alg-nanoHA30 composition, while compositions with higher nanoHA content induced a diminished bone tissue response. The outcomes of this study indicate that nanohydroxyapatite concentration plays a major role in physic-chemical properties and biological response of the composite system and the optimization of the components ratio must be met to maximize bone tissue regeneration.

Real-time quantification of Pseudomonas fluorescens cell removal from glass surfaces due to bacteriophage varphiS1 application.

AIMS: To study the efficacy of the lytic phage varphiS1 in eliminating Pseudomonas fluorescens in the early stage of biofilm formation, using an in situ and real time methodology for cell quantification. METHODS AND RESULTS: Cell adhesion and phage infection studies were carried out in a parallel plate flow chamber under laminar conditions. Cells were allowed to adhere until reaching 1.7-1.8 x 10(6) cells cm(-2) and phage infection was performed with two different phage concentrations (2 x 10(9) PFU ml(-1) and 1 x 10(10) PFU ml(-1)). Phage concentration clearly affects the speed of infection. The less concentrated phage solution promoted a three times slower rate of cell removal but did not affect the overall percentage of cell removal. In fact, after a longer infection period the less concentrated phage solution reached the same 93% cell removal value. CONCLUSIONS: Phages are efficient in the eradication of bacterial cells at the early stage of biofilm formation and their presence at the surface did not allow bacterial recolonization of the surface. SIGNIFICANCE AND IMPACT OF THE STUDY: To date, no published studies have been made concerning in situ and real time quantification of cell removal from surfaces due to phage action.

The effect of lens wear on refractive index of conventional hydrogel and silicone-hydrogel contact lenses: a comparative study.

PURPOSE: The purpose of this work was to evaluate the ability of four silicone-hydrogel contact lenses (galyfilcon A, balafilcon A, lotrafilcon A and lotrafilcon B) to retain their equilibrium water content before and after wear, through measurements of refractive index and compare with that of a conventional disposable hydrogel contact lens (etafilcon A). METHODS: The refractive indices of 115 contact lenses were measured using an automated refractometer (CLR 12-70, Index Instruments, Cambridge, U.K.) before and after a schedule of daily wear by 58 patients for 30 days in the case of silicone-hydrogel lenses and 15 days for the conventional contact lenses. RESULTS: In the silicone-hydrogel contact lenses the changes on the refractive indices were not statistically significant, however after being worn the refractive index of the conventional etalfilcon A hydrogel contact lens increased significantly (p<0.001). CONCLUSION: The results presented here show that after being worn the silicone-hydrogel contact lens, show more capacity to retain or to reach their initial equilibrium water content than conventional hydrogel contact lenses. This suggests that the silicone-hydrogel contact lenses are less susceptible to spoilation over time maintaining its biocompatibility and contributing to the clinical success of lens performance.

Phage therapy as an alternative or complementary strategy to prevent and control biofilm-related infections.

The complex heterogeneous structure of biofilms confers to bacteria an important survival strategy. Biofilms are frequently involved in many chronic infections in consequence of their low susceptibility to antibiotics as well as resistance to host defences. The increasing need of novel and effective treatments to target these complex structures has led to a growing interest on bacteriophages (phages) as a strategy for biofilm control and prevention. Phages can be used alone, as a cocktail to broaden the spectra of activity, or in combination with other antimicrobials to improve their efficacy. Here, we summarize the studies involving the use of phages for the treatment or prevention of bacterial biofilms, highlighting the biofilm features that can be tackled with phages or combined therapy approaches.