First Characterization of Acinetobacter baumannii-Specific Filamentous Phages.

Filamentous bacteriophages belonging to the order Tubulavirales, family Inoviridae, significantly affect the properties of Gram-negative bacteria, but filamentous phages of many important pathogens have not been described so far. The aim of this study was to examine A. baumannii filamentous phages for the first time and to determine their effect on bacterial virulence. The filamentous phages were detected in 15.3% of A. baumannii strains as individual prophages in the genome or as tandem repeats, and a slightly higher percentage was detected in the culture collection (23.8%). The phylogenetic analyses revealed 12 new genera within the Inoviridae family. Bacteriophages that were selected and isolated showed structural and genomic characteristics of the family and were unable to form plaques. Upon host infection, these phages did not significantly affect bacterial twitching motility and capsule production but significantly affected growth kinetics, reduced biofilm formation, and increased antibiotic sensitivity. One of the possible mechanisms of reduced resistance to antibiotics is the observed decreased expression of efflux pumps after infection with filamentous phages.

A Video Mosaicing-Based Sensing Method for Chicken Behavior Recognition on Edge Computing Devices.

Chicken behavior recognition is crucial for a number of reasons, including promoting animal welfare, ensuring the early detection of health issues, optimizing farm management practices, and contributing to more sustainable and ethical poultry farming. In this paper, we introduce a technique for recognizing chicken behavior on edge computing devices based on video sensing mosaicing. Our method combines video sensing mosaicing with deep learning to accurately identify specific chicken behaviors from videos. It attains remarkable accuracy, achieving 79.61% with MobileNetV2 for chickens demonstrating three types of behavior. These findings underscore the efficacy and promise of our approach in chicken behavior recognition on edge computing devices, making it adaptable for diverse applications. The ongoing exploration and identification of various behavioral patterns will contribute to a more comprehensive understanding of chicken behavior, enhancing the scope and accuracy of behavior analysis within diverse contexts.

Antibacterial Potential of Non-Tailed Icosahedral Phages Alone and in Combination with Antibiotics.

Non-tailed icosahedral phages belonging to families Fiersviridae (phages MS2 and Qbeta), Tectiviridae (PRD1) and Microviridae (phiX174) have not been considered in detail so far as potential antibacterial agents. The aim of the study was to examine various aspects of the applicability of these phages as antibacterial agents. Antibacterial potential of four phages was investigated via bacterial growth and biofilm formation inhibition, lytic spectra determination, and phage safety examination. The phage phiX174 was combined with different classes of antibiotics to evaluate potential synergistic interactions. In addition, the incidence of phiX174-insensitive mutants was analyzed. The results showed that only phiX174 out of four phages tested against their corresponding hosts inhibited bacterial growth for > 90% at different multiplicity of infection and that only this phage considerably prevented biofilm formation. Although all phages show the absence of potentially undesirable genes, they also have extremely narrow lytic spectra. The synergism was determined between phage phiX174 and ceftazidime, ceftriaxone, ciprofloxacin, macrolides, and chloramphenicol. It was shown that the simultaneous application of agents is more effective than successive treatment, where one agent is applied first. The analysis of the appearance of phiX174 bacteriophage-insensitive mutants showed that mutations occur with a frequency of 10-3. The examined non-tailed phages have a limited potential for use as antibacterial agents, primarily due to a very narrow lytic spectrum and the high frequency of resistant mutants appearance, but Microviridae can be considered in the future as biocontrol agents against susceptible strains of E. coli in combinations with conventional antimicrobial agents.

Do Ganoderma Species Represent Novel Sources of Phenolic Based Antimicrobial Agents?

Ganoderma species have been recognized as potential antimicrobial (AM) agents and have been used in traditional Chinese medicine (TCM) for a long time. The aim of this study is to examine the AM potential of autochthonous Ganoderma species (G. applanatum, G. lucidum, G. pfeifferi and G. resinaceum) from Serbia. The extraction of fungal material was prepared in different solvents (ethanol-EtOH, water-H2O, chloroform-CHCl3). Antibacterial activity (ABA) was determined using disk-diffusion, agar-well diffusion, and micro-dilution method, while for antifungal properties disk-diffusion and pour plate method were applied. Antiviral activity was tested on model DNA virus LK3 and determined by plaque assay. Statistical PCA analysis was applied for detection of correlation effects of phenolics and AM activities, while LC-MS/MS was performed for phenolics quantification. G. resinaceum CHCl3 extract expressed the most potent ABA against P. aeruginosa (MIC = 6.25 mg/mL), probably due to presence of flavonoids and 2,5-dihydroxybenzoic acid. Among H2O extracts, the highest ABA was determined for G. pfeifferi against both E. coli and S. aureus (21 and 19 mm, respectively). EtOH extracts of G. pfeifferi and G. resinaceum were the most effective against A. niger (23.8 and 20.15 mm, respectively), with special impact of phenolic acids and flavonoid isorhamnetin, while C. albicans showed the lowest susceptibility. The most potent antiviral inhibitor was G. lucidum (70.73% growth inhibition) due to the high amount of phenolic acids. To the best of our knowledge, this is the first report of a methodical AM profile of G. pfeifferi and G. resinaceum from the Balkan region including PCA analysis.

Abolishment of morphology-based taxa and change to binomial species names: 2022 taxonomy update of the ICTV bacterial viruses subcommittee.

This article summarises the activities of the Bacterial Viruses Subcommittee of the International Committee on Taxonomy of Viruses for the period of March 2021-March 2022. We provide an overview of the new taxa proposed in 2021, approved by the Executive Committee, and ratified by vote in 2022. Significant changes to the taxonomy of bacterial viruses were introduced: the paraphyletic morphological families Podoviridae, Siphoviridae, and Myoviridae as well as the order Caudovirales were abolished, and a binomial system of nomenclature for species was established. In addition, one order, 22 families, 30 subfamilies, 321 genera, and 862 species were newly created, promoted, or moved.

Detection and Molecular Characterization of Rotavirus Infections in Children and Adults with Gastroenteritis from Vojvodina, Serbia.

Rotaviruses (RV) are the leading cause of gastroenteritis in infants, young children, and adults, responsible for serious disease burden. In the period 2012-2018, a cross-sectional study was conducted using stool samples collected from patients with acute gastroenteritis from Vojvodina, Serbia. We described age and gender distribution, as well as seasonal patterns of RV prevalence. Out of 1853 included stool samples, RV was detected in 29%. Hospitalized children between 1-2 years old were especially affected by RV infection (45%). The highest prevalence of infection was observed during the colder, winter/spring months. We compared sequenced representative G and P genotypes circulating in Serbia with vaccine strains and determined their genetic similarity. Genotype combination G2P[4] was the most prevalent (34.6%), followed by G2P[8] (24.1%) and G1P[8] (21.1%). Given that several epitopes were conserved, neutralization motifs among circulating strains can be characterized as sufficiently matching vaccine strains Rotarix™ and RotaTeq™, but existing antigenic disparities should not be overlooked. The present results contribute to a better insight into the prevalence of rotavirus infection in our region and point out the need for epidemiological surveillance of rotaviruses before the introduction of vaccines. These data can help formulate future vaccine strategies in Serbia.

An Optimized Checkerboard Method for Phage-Antibiotic Synergy Detection.

Phage-antibiotic synergy is a promising therapeutic strategy, but there is no reliable method for synergism estimation. Although the time-kill curve assay is a gold standard, the method is not appropriate for fast and extensive screening of the synergy. The aim is to optimize the checkerboard method to determine phage-chemical agent interactions, to check its applicability by the time-kill curve method, and to examine whether the synergy can be obtained with both simultaneous and successive applications of these agents. In addition, the aim is to determine interactions of the Pseudomonas phage JG024 with ciprofloxacin, gentamicin, or ceftriaxone, as well as the Staphylococcus phage MSA6 and SES43300 with ciprofloxacin, gentamicin, and oxacillin. The results show that the optimized checkerboard method is reliable and that results correspond to those obtained by the time-kill curve. The synergy is detected with the phage JG024 and ciprofloxacin or ceftriaxone against Pseudomonas aeruginosa, and the phage SES43300 with ciprofloxacin against MRSA. The synergy was obtained after simultaneous applications, and in the case of P. aeruginosa, after application of the second agent with delay of one hour, indicating that simultaneous application is the best mode of synergy exploitation for therapy. The checkerboard method can be used for thorough clinical studies on synergy and in the future for personalized therapy when infections are caused by multiple resistant bacteria.

Filamentous Pseudomonas Phage Pf4 in the Context of Therapy-Inducibility, Infectivity, Lysogenic Conversion, and Potential Application.

More than 20% of all Pseudomonas aeruginosa are infected with Pf4-related filamentous phage and although their role in virulence of P. aeruginosa strain PAO1 is well documented, its properties related to therapy are not elucidated in detail. The aim of this study was to determine how phage and antibiotic therapy induce Pf4, whether the released virions can infect other strains and how the phage influences the phenotype of new hosts. The subinhibitory concentrations of ciprofloxacin and mitomycin C increased Pf4 production for more than 50% during the first and sixth hour of exposure, respectively, while mutants appearing after infection with obligatory lytic phage at low MOI produced Pf4 more than four times after 12-24 h of treatment. This indicates that production of Pf4 is enhanced during therapy with these agents. The released virions can infect new P. aeruginosa strains, as confirmed for models UCBPP-PA14 (PA14) and LESB58, existing both episomally and in a form of a prophage, as confirmed by PCR, RFLP, and sequencing. The differences in properties of Pf4-infected, and uninfected PA14 and LESB58 strains were obvious, as infection with Pf4 significantly decreased cell autoaggregation, pyoverdine, and pyocyanin production, while significantly increased swimming motility and biofilm production in both strains. In addition, in strain PA14, Pf4 increased cell surface hydrophobicity and small colony variants' appearance, but also decreased twitching and swarming motility. This indicates that released Pf4 during therapy can infect new strains and cause lysogenic conversion. The infection with Pf4 increased LESB58 sensitivity to ciprofloxacin, gentamicin, ceftazidime, tetracycline, and streptomycin, and PA14 to ciprofloxacin and ceftazidime. Moreover, the Pf4-infected LESB58 was re-sensitized to ceftazidime and tetracycline, with changes from resistant to intermediate resistant and sensitive, respectively. The obtained results open a new field in phage therapy-treatment with selected filamentous phages in order to re-sensitize pathogenic bacteria to certain antibiotics. However, this approach should be considered with precautions, taking into account potential lysogenic conversion.

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.

Phages from Genus Bruynoghevirus and Phage Therapy: Pseudomonas Phage Delta Case.

The applicability and safety of bacteriophage Delta as a potential anti-Pseudomonas aeruginosa agent belonging to genus Bruynoghevirus (family Podoviridae) was characterised. Phage Delta belongs to the species Pseudomonas virus PaP3, which has been described as a temperate, with cos sites at the end of the genome. The phage Delta possesses a genome of 45,970 bp that encodes tRNA for proline (Pro), aspartic acid (Asp) and asparagine (Asn) and does not encode any known protein involved in lysogeny formation or persistence. Analysis showed that phage Delta has 182 bp direct terminal repeats at the end of genome and lysogeny was confirmed, neither upon infection at low nor at high multiplicity of infection (MOI). The turbid plaques that appear on certain host lawns can result from bacteriophage insensitive mutants that occur with higher frequency (10-4). In silico analysis showed that the genome of Delta phage does not encode any known bacterial toxin or virulence factor, determinants of antibiotic resistance and known human allergens. Based on the broad host range and high lytic activity against planktonic and biofilm cells, phage Delta represents a promising candidate for phage therapy.

Are Bordetella bronchiseptica Siphoviruses (Genus Vojvodinavirus) Appropriate for Phage Therapy-Bacterial Allies or Foes?

Bordetella bronchiseptica is a respiratory animal pathogen that shows growing resistance to commonly used antibiotics, which has necessitated the examination of new antimicrobials, including bacteriophages. In this study, we examined the previously isolated and partially characterized B. bronchiseptica siphoviruses of the genus Vojvodinavirus (LK3, CN1, CN2, FP1 and MW2) for their ability to inhibit bacterial growth and biofilm, and we examined other therapeutically important properties through genomic analysis and lysogeny experiments. The phages inhibited bacterial growth at a low multiplicity of infection (MOI = 0.001) of up to 85% and at MOI = 1 for >99%. Similarly, depending on the phages and MOIs, biofilm formation inhibition ranged from 65 to 95%. The removal of biofilm by the phages was less efficient but still considerably high (40-75%). Complete genomic sequencing of Bordetella phage LK3 (59,831 bp; G + C 64.01%; 79 ORFs) showed integrase and repressor protein presence, indicating phage potential to lysogenize bacteria. Lysogeny experiments confirmed the presence of phage DNA in bacterial DNA upon infection using PCR, which showed that the LK3 phage forms more or less stable lysogens depending on the bacterial host. Bacterial infection with the LK3 phage enhanced biofilm production, sheep blood hemolysis, flagellar motility, and beta-lactam resistance. The examined phages showed considerable anti-B. bronchiseptica activity, but they are inappropriate for therapy because of their temperate nature and lysogenic conversion of the host bacterium.

A comprehensive method for determining cellular uptake of purine nucleoside phosphorylase and adenylosuccinate synthetase inhibitors by H. pylori.

Due to the growing number of Helicobacter pylori strains resistant to currently available antibiotics, there is an urgent need to design new drugs utilizing different molecular mechanisms than those that have been used up to now. Enzymes of the purine salvage pathway are possible targets of such new antibiotics because H. pylori is not able to synthetize purine nucleotides de novo. The bacterium's recovery of purines and purine nucleotides from the environment is the only source of these essential DNA and RNA building blocks. We have identified formycins and hadacidin as potent inhibitors of purine nucleoside phosphorylase (PNP) and adenylosuccinate synthetase (AdSS) from H. pylori - two key enzymes of the purine salvage pathway. However, we have found that these compounds are not effective in H. pylori cell cultures. To address this issue, we have developed a universal comprehensive method for assessing H. pylori cell penetration by drug candidates, with three alternative detection assays. These include liquid chromatography tandem mass spectrometry, UV absorption, and inhibition of the target enzyme by the tested compound. Using this approach, we have shown that cellular uptake by H. pylori of formycins and hadacidin is very poor, which reveals why their in vitro inhibition of PNP and AdSS and their effect on H. pylori cell cultures are so different. The cell penetration assessment method developed here will be extremely useful for validating the cellular uptake of other drug candidates, facilitating the design of new potent therapeutic agents against H. pylori. KEY POINTS: • A method for assessing H. pylori cells penetration by drug candidates is described. • Three alternative detection assays that complement each other can be used. • The method may be adapted for other bacteria as well.

Bacterial Viruses Subcommittee and Archaeal Viruses Subcommittee of the ICTV: update of taxonomy changes in 2021.

In this article, we - the Bacterial Viruses Subcommittee and the Archaeal Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV) - summarise the results of our activities for the period March 2020 - March 2021. We report the division of the former Bacterial and Archaeal Viruses Subcommittee in two separate Subcommittees, welcome new members, a new Subcommittee Chair and Vice Chair, and give an overview of the new taxa that were proposed in 2020, approved by the Executive Committee and ratified by vote in 2021. In particular, a new realm, three orders, 15 families, 31 subfamilies, 734 genera and 1845 species were newly created or redefined (moved/promoted).

ICTV Virus Taxonomy Profile: Inoviridae.

Members of the family Inoviridae are non-enveloped flexible filamentous bacteriophages (600-2500×6-10 nm) with supercoiled, circular, positive-sense, single-stranded DNA genomes of 5.5-10.6 kb, encoding 7-15 proteins. They absorb to the pili of Gram-negative bacteria and replicate their DNA by a rolling-circle mechanism with progeny released from cells by extrusion without killing the host. Phage DNA can persist extra-chromosomally or integrate into the bacterial genome. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Inoviridae, which is available at ictv.global/report/inoviridae.

Preisach Elasto-Plastic Model for Mild Steel Hysteretic Behavior-Experimental and Theoretical Considerations.

The Preisach model already successfully implemented for axial and bending cyclic loading is applied for modeling of the plateau problem for mild steel. It is shown that after the first cycle plateau disappears an extension of the existing Preisach model is needed. Heat dissipation and locked-in energy is calculated due to plastic deformation using the Preisach model. Theoretical results are verified by experiments performed on mild steel S275. The comparison of theoretical and experimental results is evident, showing the capability of the Presicah model in predicting behavior of structures under cyclic loading in the elastoplastic region. The purpose of this paper is to establish a theoretical background for embedded sensors like regenerated fiber Bragg gratings (RFBG) for measurement of strains and temperature in real structures. In addition, the present paper brings a theoretical base for application of nested split-ring resonator (NSRR) probes in measurements of plastic strain in real structures.

ICTV Virus Taxonomy Profile: Plectroviridae.

Members of the family Plectroviridae produce particles that are non-enveloped rigid rods (70-280×10-16 nm). The supercoiled, circular, single-stranded DNA genome of about 4.5-8.3 kb, encodes 4-13 proteins. Viruses of this family infect cell wall-less bacteria, adsorbing to the bacterial surface, replicating their DNA by a rolling-circle mechanism or transposition, and releasing progeny from cells by extrusion, without killing the host. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Plectroviridae which is available at ictv.global/report/plectroviridae.

Optimized Method for Pseudomonas aeruginosa Integrative Filamentous Bacteriophage Propagation.

Filamentous bacteriophages frequently infect Pseudomonas aeruginosa and alter its phenotypic traits, including virulence factors. The first step in examination of these phages is to obtain suspensions with high virus titer, but as there are no methods for integrative filamentous phage multiplication, the aim was to design, describe, and compare two methods for this purpose. As models, three strains of Pseudomonas aeruginosa, containing (pro)phages Pf4, Pf5, and PfLES were used (PAO1, UCBPP-PA14, and LESB58, respectively). Method 1 comprised propagation of phages in 6 L of bacterial culture for 48 h, and method 2 applied 600 mL culture and incubation for 6 days with centrifugation and addition of new medium and inoculum at 2-day intervals. In method 1, phages were propagated by culture agitation, followed by centrifugation and filtration (0.45 and 0.22 µm), and in method 2, cultures were agitated and centrifuged several times to remove bacteria without filtration. Regardless of the propagation method, supernatants were subjected to concentration by PEG8000 and CsCl equilibrium density gradient centrifugation, and phage bands were removed after ultracentrifugation and dialyzed. In the obtained suspensions, phage titer was determined, and concentration of isolated ssDNA from virions was measured. When propagation method 2 was compared with method 1, the phage bands in CsCl were much thicker, phage number was 3.5-7.4 logs greater, and concentration of ssDNA was 7.6-22.4 times higher. When phage count was monitored from days 2 to 6, virion numbers increased for 1.8-5.6 logs, depending on phage. We also observed that filamentous phage plaques faded after 8 h of incubation when the double layer agar spot method was applied, whereas the plaques were visible for 24 h on single-layer agar. Finally, for the first time, we confirmed existence of replicative form and virions of PfLES (pro)phage as well as its ability to produce plaques. Similarly, for the first time, we confirmed plaque production of Pf5 (pro)phage present in P. aeruginosa strain UCBPP-PA14. The described method 2 has many advantages and can be further improved and adopted for filamentous phages of other hosts.

Synergistic activity of bile salts and their derivatives in combination with conventional antimicrobial agents against Acinetobacter baumannii.

ETHNOPHARMACOLOGICAL RELEVANCE: Bile traditionally was used in wound healing, having erodent, antioxidant and antimicrobial potential. Acinetobacter baumannii is a frequent etiological agent of wound infections, exhibiting high level of resistance to conventional antibiotics. AIM OF THE STUDY: To determine the effect of selected bile acid sodium salts and their 3-dehydro (i.e. 3-oxo) derivatives, as well as their combinations with commercial antibiotics against A. baumanniia, to confirm bile ethnopharmacological application in wound healing from aspect of microbiology. MATERIALS AND METHODS: The sensitivity of reference and multidrug resistant (MDR) A. baumannii strains to bile salts, their derivatives and conventional antibiotics were examined by a microtiter plate method. The interaction of bile salts/derivatives and antibiotics was examined by a checkerboard method and time kill curve method. The interaction of bile salts with ciprofloxacin in terms of micelles formation was examined by DOSY NMR technique. RESULTS: The bile salts sodium deoxycholate (Na-DCA) and sodium chenodeoxycholate (Na-CDCA), as well as their derivatives sodium 3-dehydro-deoxycholate (Na-3DH-DCA) and sodium 3-dehydro-chenodeoxycholate (Na-3DH-CDCA), potentiate antibiotic activity and resensitize A. baumannii. The bile salts and their derivatives enhance A. baumannii sensitivity to antibiotics, particularly those that should penetrate cell to exhibit activity. The sodium salts of bile acid derivatives, namely Na-3DH-DCA and Na-3DH-CDCA, showed synergy against both reference and MDR strain in combination with ciprofloxacin or gentamicin, while synergy with gentamicin was obtained in all combinations, regardless of bile salt type and bacterial strains. The synergy with Na-3DH-CDCA was further confirmed by the time-kill curve method, as bacterial number decreased after 12 h. NMR experiment revealed that this bile salt derivative and ciprofloxacin form co-aggregates when bile salts concentration was higher than critical micelle concentrations (CMC), which indicate the possibility that bile salts enhance ciprofloxacin cell penetration by membrane destabilization, contributing to the synergy. CONCLUSION: The synergistic interactions between bile salts or derivatives with ciprofloxacin and particularly gentamicin represent a promising strategy for the treatment of A. baumannii wound infections.

Anti-Acinetobacter baumannii activity of selected phytochemicals alone, in binary combinations and in combinations with conventional antibiotics.

Acinetobacter baumannii is one of the emerging multidrug- and pandrug-resistant pathogens. The aim of the study was to determine anti-A. baumannii activity of selected terpenes, terpenoids and phenylpropanoids alone, in binary combinations, and in combinations with conventional antibiotics using microdilution-checkerboard and time-kill curve method. The most effective were terpenoids carvacrol (7.0-28.0 µg mL-1) and thymol (22.0-76.0 µg mL-1), as well as phenylpropanoid compound eugenol (90.5-304.0 µg mL-1), with the active concentrations were comparable to antibiotics. The binary combinations showed additive or indifferent effects. The combination gentamicin-carvacrol was synergistic only against reference strain (FICI = 0.50), while other combinations were additive. The best bacteriostatic activity showed carvacrol, thymol or eugenol in combination with ciprofloxacin (FICI range 0.11-0.50) against both the reference and multidrug-resistant strains. The synergistic effect was further confirmed by time kill curve method and obtained after only 15 h. The results indicate a new possible therapeutic strategy against multiple resistant A. baumannii.