The Safety of Bacteriophages in Treatment of Diseases Caused by Multidrug-Resistant Bacteria.

Given the urgency due to the rapid emergence of multidrug-resistant (MDR) bacteria, bacteriophages (phages), which are viruses that specifically target and kill bacteria, are rising as a potential alternative to antibiotics. In recent years, researchers have begun to elucidate the safety aspects of phage therapy with the aim of ensuring safe and effective clinical applications. While phage therapy has generally been demonstrated to be safe and tolerable among animals and humans, the current research on phage safety monitoring lacks sufficient and consistent data. This emphasizes the critical need for a standardized phage safety assessment to ensure a more reliable evaluation of its safety profile. Therefore, this review aims to bridge the knowledge gap concerning phage safety for treating MDR bacterial infections by covering various aspects involving phage applications, including phage preparation, administration, and the implications for human health and the environment.

Bacteriophages and Their Host Range in Multidrug-Resistant Bacterial Disease Treatment.

The rapid emergence of multidrug-resistant (MDR) bacteria in recent times has prompted the search for new and more potent antibiotics. Bacteriophages (commonly known as phages) are viruses that target and infect their bacterial hosts. As such, they are also a potential alternative to antibiotics. These phages can be broadly categorized into monovalent (with a narrow host range spectrum and specific to a single bacterial genus) and polyvalent (with a broad host range and specific to more than two genera). However, there is still much ambiguity in the use of these terms, with researchers often describing their phages differently. There is considerable research on the use of both narrow- and broad-host range phages in the treatment of infections and diseases caused by MDR bacteria, including tuberculosis, cystic fibrosis, and carbapenem-resistant Enterobacterales (CRE) infectious diseases. From this, it is clear that the host range of these phages plays a vital role in determining the effectiveness of any phage therapy, and this factor is usually analyzed based on the advantages and limitations of different host ranges. There have also been efforts to expand phage host ranges via phage cocktail development, phage engineering and combination therapies, in line with current technological advancements. This literature review aims to provide a more in-depth understanding of the role of phage host ranges in the effectiveness of treating MDR-bacterial diseases, by exploring the following: phage biology, the importance of phages in MDR bacteria diseases treatment, the importance of phage host range and its advantages and limitations, current findings and recent developments, and finally, possible future directions for wide host range phages.

Identification and antibiotic pattern analysis of bacillary dysentery causing bacteria isolated from stool samples of infected patients.

Bacillary dysentery is a type of dysentery and a severe form of shigellosis. This dysentery is usually restricted to Shigella infection, but Salmonella enterica and enteroinvasive Escherichia coli strains are also known as this infection's causative agents. The emergence of drug-resistant, bacillary dysentery-causing pathogens is a global burden, especially for developing countries with poor hygienic environments. This study aimed to isolate, identify, and determine the drug-resistant pattern of bacillary dysentery-causing pathogens from the stool samples of the Kushtia region in Bangladesh. Hence, biochemical tests, serotyping, molecular identification, and antibiotic profiling were performed to characterize the pathogens. Among one hundred fifty (150) stool samples, 18 enteric bacterial pathogens were isolated and identified, where 12 were Shigella strains, 5 were S. enterica sub spp. enterica strains and one was the E.coli strain. Among 12 Shigella isolates, 8 were Shigella flexneri 2a serotypes, and 4 were Shigella sonnei Phage-II serotypes. Except for three Salmonella strains, all isolated strains were drug-resistant (83%), whereas 50% were multidrug-resistant (MDR), an alarming issue for public health. In antibiotic-wise analysis, the isolated pathogens showed the highest resistance against nalidixic acid (77.78%), followed by tetracycline (38.89%), kanamycin (38.89%), amoxicillin (27.78%), streptomycin (27.78%), cefepime (22.22%), ceftriaxone (22.22%), ampicillin (16.67%), ciprofloxacin (16.67%), and chloramphenicol (16.67%). The existence of MDR organisms that cause bacillary dysentery in the Kushtia area would warn the public to be more health conscious, and physicians would administer medications cautiously. The gradual growth of MDR pathogenic microorganisms needs immediate attention, and the discovery of effective medications must take precedence. Supplementary information: The online version contains supplementary material available at 10.1007/s11756-022-01299-x.

Direct Priming of CD8+ T Cells Persists in the Face of Cowpox Virus Inhibitors of Antigen Presentation.

Vaccinia virus (VACV) was the vaccine used to eradicate smallpox and is being repurposed as a vaccine vector. CD8+ T cells are key anti-viral mediators, but require priming to become effector or memory cells. Priming requires an interaction with dendritic cells that are either infected (direct priming), or that have acquired virus proteins but remain uninfected (cross priming). To investigate CD8+ T cell priming pathways for VACV, we engineered the virus to express CPXV12 and CPXV203, two inhibitors of antigen presentation encoded by cowpox virus. These intracellular proteins would be expected to block direct but not cross priming. The inhibitors had diverse impacts on the size of anti-VACV CD8+ T cell responses across epitopes and by different infection routes in mice, superficially suggesting variable use of direct and cross priming. However, when we then tested a form of antigen that requires direct priming, we found surprisingly that CD8+ T cell responses were not diminished by co-expression with CPXV12 and CPXV203. We then directly quantified the impact of CPXV12 and CPXV203 on viral antigen presentation using mass spectrometry, which revealed strong, but incomplete inhibition of antigen presentation by the CPXV proteins. Therefore, direct priming of CD8+ T cells by poxviruses is robust enough to withstand highly potent viral inhibitors of antigen presentation. This is a reminder of the limits of viral immune evasion and shows that viral inhibitors of antigen presentation cannot be assumed to dissect cleanly direct and cross priming of anti-viral CD8+ T cells.ImportanceCD8+ T cells are key to anti-viral immunity, so it is important to understand how they are activated. Many viruses have proteins that protect infected cells from T cell attack by interfering with the process that allows virus infection to be recognised by CD8+ T cells. It is thought that these proteins would also stop infected cells from activating T cells in the first place. However, we show here that this is not the case for two very powerful inhibitory proteins from cowpox virus. This demonstrates the flexibility and robustness of immune processes that turn on the immune responses required to fight infection.

Community-acquired pneumonia: aetiology, antibiotic resistance and prospects of phage therapy.

Bacteria are the most common aetiological agents of community-acquired pneumonia (CAP) and use a variety of mechanisms to evade the host immune system. With the emerging antibiotic resistance, CAP-causing bacteria have now become resistant to most antibiotics. Consequently, significant morbimortality is attributed to CAP despite their varying rates depending on the clinical setting in which the patients being treated. Therefore, there is a pressing need for a safe and effective alternative or supplement to conventional antibiotics. Bacteriophages could be a ray of hope as they are specific in killing their host bacteria. Several bacteriophages had been identified that can efficiently parasitize bacteria related to CAP infection and have shown a promising protective effect. Thus, bacteriophages have shown immense possibilities against CAP inflicted by multidrug-resistant bacteria. This review provides an overview of common antibiotic-resistant CAP bacteria with a comprehensive summarization of the promising bacteriophage candidates for prospective phage therapy.

Bioremediation of malachite green dye by two bacterial strains isolated from textile effluents.

Globally, water pollution from the textile industries is an alarming issue. Malachite Green dye of the triphenylmethane group is an extensively used dye in the fabric industries that is emitted through textile wastewater. This study aimed to isolate and characterize potential Malachite Green (MG) dye degrading bacteria from textile effluents. Different growth and culture parameters such as temperature, pH and dye concentration were optimized to perform the dye-degradation assay using different concentrations of MG dye in the mineral salt medium. A photo-electric-colorimeter was used to measure the decolorizing activity of bacteria at different time intervals after aerobic incubation. Two potential bacterial strains of Enterobacter spp. CV-S1 (accession no: MH450229) and Enterobacter spp. CM-S1 (accession no: MH447289) were isolated from textile effluents exhibiting potential MG dye decoloring efficiency. Further, the RAPD analysis and 16S rRNA sequencing confirmed the genetic differences of the isolated strains. Enterobacter sp CV-S1 and Enterobacter sp CM-S1 can completely decolor MG dye up to 15 mg/L under shaking condition without any requirement of sole carbon source. Thus, these two bacteria have the potency to be utilized in the textile wastewater treatment plant.

Efficacy and potential of phage therapy against multidrug resistant Shigella spp.

Shigella-infected bacillary dysentery or commonly known as Shigellosis is a leading cause of morbidity and mortality worldwide. The gradual emergence of multidrug resistant Shigella spp. has triggered the search for alternatives to conventional antibiotics. Phage therapy could be one such suitable alternative, given its proven long term safety profile as well as the rapid expansion of phage therapy research. To be successful, phage therapy will need an adequate regulatory framework, effective strategies, the proper selection of appropriate phages, early solutions to overcome phage therapy limitations, the implementation of safety protocols, and finally improved public awareness. To achieve all these criteria and successfully apply phage therapy against multidrug resistant shigellosis, a comprehensive study is required. In fact, a variety of phage-based approaches and products including single phages, phage cocktails, mutated phages, genetically engineered phages, and combinations of phages with antibiotics have already been carried out to test the applications of phage therapy against multidrug resistant Shigella. This review provides a broad survey of phage treatments from past to present, focusing on the history, applications, limitations and effective solutions related to, as well as the prospects for, the use of phage therapy against multidrug resistant Shigella spp. and other multidrug resistant bacterial pathogens.

Antimicrobial peptides from different plant sources: Isolation, characterisation, and purification.

Antimicrobial peptides (AMPs), the self-defence products of organisms, are extensively distributed in plants. They can be classified into several groups, including thionins, defensins, snakins, lipid transfer proteins, glycine-rich proteins, cyclotides and hevein-type proteins. AMPs can be extracted and isolated from different plants and plant organs such as stems, roots, seeds, flowers and leaves. They perform various physiological defensive mechanisms to eliminate viruses, bacteria, fungi and parasites, and so could be used as therapeutic and preservative agents. Research on AMPs has sought to obtain more detailed and reliable information regarding the selection of suitable plant sources and the use of appropriate isolation and purification techniques, as well as examining the mode of action of these peptides. Well-established AMP purification techniques currently used include salt precipitation methods, absorption-desorption, a combination of ion-exchange and reversed-phase C18 solid phase extraction, reversed-phase high-performance liquid chromatography (RP-HPLC), and the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) method. Beyond these traditional methods, this review aims to highlight new and different approaches to the selection, characterisation, isolation, purification, mode of action and bioactivity assessment of a range of AMPs collected from plant sources. The information gathered will be helpful in the search for novel AMPs distributed in the plant kingdom, as well as providing future directions for the further investigation of AMPs for possible use on humans.

Biodegradation of Crystal Violet dye by bacteria isolated from textile industry effluents.

Industrial effluent containing textile dyes is regarded as a major environmental concern in the present world. Crystal Violet is one of the vital textile dyes of the triphenylmethane group; it is widely used in textile industry and known for its mutagenic and mitotic poisoning nature. Bioremediation, especially through bacteria, is becoming an emerging and important sector in effluent treatment. This study aimed to isolate and identify Crystal Violet degrading bacteria from industrial effluents with potential use in bioremediation. The decolorizing activity of the bacteria was measured using a photo electric colorimeter after aerobic incubation in different time intervals of the isolates. Environmental parameters such as pH, temperature, initial dye concentration and inoculum size were optimized using mineral salt medium containing different concentration of Crystal Violet dye. Complete decolorizing efficiency was observed in a mineral salt medium containing up to 150 mg/l of Crystal Violet dye by 10% (v/v) inoculums of Enterobacter sp. CV-S1 tested under 72 h of shaking incubation at temperature 35 °C and pH 6.5. Newly identified bacteria Enterobacter sp. CV-S1, confirmed by 16S ribosomal RNA sequencing, was found as a potential bioremediation biocatalyst in the aerobic degradation/de-colorization of Crystal Violet dye. The efficiency of degrading triphenylmethane dye by this isolate, minus the supply of extra carbon or nitrogen sources in the media, highlights the significance of larger-scale treatment of textile effluent.

Shigella flexneri serotype 1c derived from serotype 1a by acquisition of gtrIC gene cluster via a bacteriophage.

BACKGROUND: Shigella spp. are the primary causative agents of bacillary dysentery. Since its emergence in the late 1980s, the S. flexneri serotype 1c remains poorly understood, particularly with regard to its origin and genetic evolution. This article provides a molecular insight into this novel serotype and the gtrIC gene cluster that determines its unique immune recognition. RESULTS: A PCR of the gtrIC cluster showed that serotype 1c isolates from different geographical origins were genetically conserved. An analysis of sequences flanking the gtrIC cluster revealed remnants of a prophage genome, in particular integrase and tRNA(Pro) genes. Meanwhile, Southern blot analyses on serotype 1c, 1a and 1b strains indicated that all the tested serotype 1c strains may have had a common origin that has since remained distinct from the closely related 1a and 1b serotypes. The identification of prophage genes upstream of the gtrIC cluster is consistent with the notion of bacteriophage-mediated integration of the gtrIC cluster into a pre-existing serotype. CONCLUSIONS: This is the first study to show that serotype 1c isolates from different geographical origins share an identical pattern of genetic arrangement, suggesting that serotype 1c strains may have originated from a single parental strain. Analysis of the sequence around the gtrIC cluster revealed a new site for the integration of the serotype converting phages of S. flexneri. Understanding the origin of new pathogenic serotypes and the molecular basis of serotype conversion in S. flexneri would provide information for developing cross-reactive Shigella vaccines.

Structural and functional divergence of the newly identified GtrIc from its Gtr family of conserved Shigella flexneri serotype-converting glucosyltransferases.

Glucosyltransferases (Gtrs) and O-acetyltransferase (Oac) are integral membrane proteins embedded within the cytoplasmic membrane of Shigella flexneri. Gtrs and Oac are responsible for unidirectional host serotype conversion by altering the epitopic properties of the bacterial surface lipopolysaccharide (LPS) O-antigen. In this study, we present the membrane topology of a recently recognized Gtr, GtrIc, which is known to mediate S. flenxeri serotype switching from 1a to 1c. The GtrIc topology is shown to deviate from those typically seen in S. flexneri Gtrs. GtrIc has 11 hydrophilic loops, 10 transmembrane helices, a double intramembrane dipping loop 5, and a cytoplasmic N- and C-terminus. Along with a unique membrane topology, the identification of non-critical Gtr-conserved peptide motifs within large periplasmic loops (N-terminal D/ExD/E and C-terminal KK), which have previously been proven essential for the activity of other Gtrs, challenge current opinions of a similar mechanism for enzyme function between members of the S. flexneri Gtr family.

A novel glucosyltransferase involved in O-antigen modification of Shigella flexneri serotype 1c.

The O antigen of serotype 1c differs from the unmodified O antigen of serotype Y by the addition of a disaccharide (two glucosyl groups) to the tetrasaccharide repeating unit. It was shown here that addition of the first glucosyl group is mediated by the previously characterized gtrI cluster, which is found within a cryptic prophage at the proA locus in the bacterial chromosome. Transposon mutagenesis was performed to disrupt the gene responsible for addition of the second glucosyl group, causing reversion to serotype 1a. Colony immunoblotting was used to identify the desired revertants, and subsequent sequencing, cloning, and functional expression successfully identified the gene encoding serotype 1c-specific O-antigen modification. This gene (designated gtrIC) was present as part of a three-gene cluster, similar to other S. flexneri glucosyltransferase genes. Relative to the other S. flexneri gtr clusters, the gtrIC cluster is more distantly related and appears to have arrived in S. flexneri from outside the species. Analysis of surrounding sequence suggests that the gtrIC cluster arrived via a novel bacteriophage that was subsequently rendered nonfunctional by a series of insertion events.

Peptide mimotopes of complex carbohydrates in Salmonella enterica serovar typhi which react with both carbohydrate-specific monoclonal antibody and polyclonal sera from typhoid patients.

Polyclonal sera from typhoid patients and a monoclonal antibody, mAb ATVi, which recognizes the capsular polysaccharide Vi antigen (ViCPS), were used to select for peptides that mimic the ViCPS by using a phage-displayed random 12-mer peptide library. Two major common mimotopes selected from the library carried the amino acid sequences TSHHDSHGLHRV and ENHSPVNIAHKL. Enzyme-linked immunosorbent assays (ELISAs) showed that these peptides carry mimotopes to ViCPS. Phage clones that contained the 12-mer peptides were also tested against pooled/individual typhoid patients' sera and found to have 3 to 5 times higher binding compared to normal sera. By using Phage-ELISA assays, the derived synthetic peptides, TSHHDSHGLHRV and ENHSPVNIAHKL, were tested against a monoclonal antibody mAb ATVi and over 2-fold difference in binding was found between these peptides and a control unrelated peptide, CTLTTKLYC. Inhibition of the mAb's binding to ViCPS indicated that the synthetic peptides successfully competed with the capsular polysaccharide for antibody binding.

Mimotopes of the Vi antigen of Salmonella enterica serovar typhi identified from phage display peptide library.

The capsular polysaccharide Vi antigen (ViCPS) is an essential virulence factor and also a protective antigen of Salmonella enterica serovar Typhi. A random 12-mer phage-displayed peptide library was used to identify mimotopes (epitope analogues) of this antigen by panning against a ViCPS-specific monoclonal antibody (MAb) ATVi. Approximately 75% of the phage clones selected in the fourth round carried the peptide sequence TSHHDSHGLHRV, and the rest of the clones harbored ENHSPVNIAHKL and other related sequences. These two sequences were also obtained in a similar panning process by using pooled sera from patients with a confirmed diagnosis of typhoid fever, suggesting they mimic immunodominant epitopes of ViCPS antigens. Binding of MAb ATVi to the mimotopes was specifically blocked by ViCPS, indicating that they interact with the same binding site (paratope) of the MAb. Data and reagents generated in this study have important implications for the development of peptide-base diagnostic tests and peptide vaccines and may also provide a better understanding of the pathogenesis of typhoid fever.