Search Results (15)

The widespread use of antibiotics has led to the emergence of a large number of drug-resistant bacteria, accelerating the dissemination and spread of antibiotic resistance genes (ARGs) in the environment. Bacterial biofilms, serving as reservoirs of ARGs, pose potential risks to environmental health that should not be ignored. Studies on the presence and transfer of ARGs in biofilms have been conducted both domestically and internationally. This article summarises the research progress on ARGs in various environments and analyses the mechanisms and factors influencing the dissemination and transfer of ARGs in microplastics, activated sludge, and pipe wall biofilms, with a particular focus on phage-mediated ARG transfer. We also discuss current research gaps in this field to provide references for future biofilm management and health risk control of ARGs. Full article

Clavibacter is a phytopathogenic genus that causes severe diseases in economically important crops, yet the role of prophages in its evolution, pathogenicity, and adaptation remains poorly understood. In this study, we used PHASTER, Prophage Hunter, and VirSorter2 to identify prophage-like sequences in publicly available Clavibacter genomes. Prophage predictions were checked by hand to make them more accurate. We identified 353 prophages, predominantly in chromosomes, with some detected phage-plasmids. Most prophages exhibited traits of advanced domestication, such as an unimodal genome length distribution, reduced numbers of integrases, and minimal transposable elements, suggesting long-term interactions with their bacterial hosts. Comparative genomic analyses uncovered high genetic diversity, with distinct prophage clusters showing species-specific and interspecies conservation patterns. Functional annotation revealed prophage-encoded genes were involved in sugar metabolism, heavy metal resistance, virulence factors, and antibiotic resistance, highlighting their contribution to host fitness and environmental adaptation. Defense system analyses revealed that, despite lacking CRISPR-Cas, Clavibacter genomes harbor diverse antiviral systems, including PD-Lambda-1, AbiE, and MMB_gp29_gp30, some encoded within prophages. These findings underscore the pervasive presence of prophages in Clavibacter and their role in shaping bacterial adaptability and evolution. Full article

Erysipelas is a significant problem in the waterfowl farming in Poland, and information on the characteristics of the Erysipelothrix rhusiopathiae strains causing this disease is limited. In this study, we determined the serotypes, antimicrobial susceptibility, and potential mechanisms of resistance gene transfer in E. rhusiopathiae isolates (n = 60) from domestic geese and ducks. We also developed a multiplex PCR for the detection of resistance genes. The antimicrobial susceptibility of the isolates was assessed using the broth microdilution method. Resistance genes, integrative conjugative element (ICE)-specific genes, phage-specific genes, and serotype determinants were detected by PCR. Multilocus sequence typing (MLST) was performed for selected resistant strains. The comparative analyses included 260 E. rhusiopathiae strains whose whole genome sequences (WGSs) are publicly available. E. rhusiopathiae isolates represented 7 serotypes, among which serotypes 5 (38.3%) and 1b (28.3%) were the most common. All strains were susceptible to β-lactams, and the vast majority of them were resistant to tetracycline (85%) and enrofloxacin (80%). The percentages of isolates resistant to other antimicrobials used ranged from 3.3% to 16.7%. Ten isolates (16.7%) were found to be multidrug resistant (MDR). The genotypic resistance profiles of the E. rhusiopathiae strains corresponded to their phenotypic resistance, and the amplification patterns obtained using the 10-plex PCR developed in this study were fully consistent with the results of single PCRs. The most prevalent resistance gene was tetM. In enrofloxacin-resistant strains, nonsynonymous mutations in the gyrA and parC genes were identified. The presence of ICE-specific genes was confirmed in resistant strains, and in MDR isolates of serotype 8 that represented sequence type (ST) 113, prophage DNA (Javan630-like) linked to the lsaE gene was additionally detected. The results indicate that β-lactam antibiotics should be the first choice for the treatment of waterfowl erysipelas in Poland. ICEs, including a transposon from the Tn916/Tn1545 family, and bacteriophages are most likely responsible for the transfer of resistance genes in E. rhusiopathiae. Full article

Goose erysipelas is a serious problem in waterfowl breeding in Poland. However, knowledge of the characteristics of Erysipelothrix rhusiopathiae strains causing this disease is limited. In this study, the antimicrobial susceptibility and serotypes of four E. rhusiopathiae strains from domestic geese were determined, and their whole-genome sequences (WGSs) were analyzed to detect resistance genes, integrative and conjugative elements (ICEs), and prophage DNA. Sequence type and the presence of resistance genes and transposons were compared with 363 publicly available E. rhusiopathiae strains, as well as 13 strains of other Erysipelothrix species. Four strains tested represented serotypes 2 and 5 and the MLST groups ST 4, 32, 242, and 243. Their assembled circular genomes ranged from 1.8 to 1.9 kb with a GC content of 36–37%; a small plasmid was detected in strain 1023. Strains 1023 and 267 were multidrug-resistant. The resistance genes detected in the genome of strain 1023 were erm47, tetM, and lsaE-lnuB-ant(6)-Ia-spw cluster, while strain 267 contained the tetM and ermB genes. Mutations in the gyrA gene were detected in both strains. The tetM gene was embedded in a Tn916-like transposon, which in strain 1023, together with the other resistance genes, was located on a large integrative and conjugative-like element of 130 kb designated as ICEEr1023. A minor integrative element of 74 kb was identified in strain 1012 (ICEEr1012). This work contributes to knowledge about the characteristics of E. rhusiopathiae bacteria and, for the first time, reveals the occurrence of erm47 and ermB resistance genes in strains of this species. Phage infection appears to be responsible for the introduction of the ermB gene into the genome of strain 267, while ICEs most likely play a key role in the spread of the other resistance genes identified in E. rhusiopathiae. Full article

Numerous treatment modalities have been employed over the years to eradicate bacterial communities in industrial wastewater. Oxidizing agents and chemical additives, such as ozone, permanganate, glutaraldehyde, and chlorine, are effective in treating microbial contaminants that are typically found in domestic wastewater. However, the chemical complexity of water produced from fracking requires novel approaches, because the microbes have developed mechanisms to overcome typical disinfectants. In this work, we test the effectiveness of bacteriophages for the eradication of two model bacteria from produced water: Pseudomonas aeruginosa and Bacillus megaterium. These bacteria were grown in low salinity produced water and exposed to their corresponding phage. Overall, the total inactivation of the P. aeruginosa population was achieved, as well as the inactivation of B. megaterium. These promising results provide a potentially useful tool for bacterial elimination in overall PW treatment, at an industrial scale. Particularly, since phage treatment is a rapid and cost-effective alternative. Moreover, these results fall within the objectives proposed as part of the sustainable development goals adopted worldwide. Full article

Staphylococcus aureus is a versatile pathogen that does not only occur in humans but also in various wild and domestic animals, including several avian species. When characterizing S. aureus isolates from waterfowl, isolates were identified as atypical CC133 by DNA microarray analysis. They differed from previously sequenced CC133 strains in the presence of the collagen adhesin gene cna; some also showed a different capsule type and a deviant spa type. Thus, they were subjected to whole-genome sequencing. This revealed multiple insertions of large regions of DNA from other S. aureus lineages into a CC133-derived backbone genome. Three distinct strains were identified based on the size and extent of these inserts. One strain comprised two small inserts of foreign DNA up- and downstream of oriC; one of about 7000 nt or 0.25% originated from CC692 and the other, at ca. 38,000 nt or 1.3% slightly larger one was of CC522 provenance. The second strain carried a larger CC692 insert (nearly 257,000 nt or 10% of the strain’s genome), and its CC522-derived insert was also larger, at about 53,500 nt or 2% of the genome). The third strain carried an identical CC692-derived region (in which the same mutations were observed as in the second strain), but it had a considerably larger CC522-like insertion of about 167,000 nt or 5.9% of the genome. Both isolates of the first, and two out of four isolates of the second strain also harbored a hemolysin-beta-integrating prophage carrying “bird-specific” virulence factors, ornithine cyclodeaminase D0K6J8 and a putative protease D0K6J9. Furthermore, isolates had two different variants of SCC elements that lacked mecA/mecC genes. These findings highlight the role of horizontal gene transfer in the evolution of S. aureus facilitated by SCC elements, by phages, and by a yet undescribed mechanism for large-scale exchange of core genomic DNA. Full article

Antimicrobial resistance is increasing despite new treatments being employed, so novel strategies are required to ensure that bacterial infections remain treatable. Bacteriophages (phages; bacteria viruses) have the potential to be used as natural antimicrobial methods to control bacterial pathogens such as Salmonella spp. A Salmonella phage, Wara, was isolated from environmental water samples at the Subaé River Basin, Salvador de Bahia, Brazil. The basin has environmental impacts in its main watercourses arising from the dumping of domestic and industrial effluents and agricultural and anthropological activities. The phage genome sequence was determined by Oxford Nanopore Technologies (ONT) MinION and Illumina HiSeq sequencing, and assembly was carried out by Racon (MinION) and Unicycler (Illumina, Illumina + MinION). The genome was annotated and compared to other Salmonella phages using various bioinformatics approaches. MinION DNA sequencing combined with Racon assembly gave the best complete genome sequence. Phylogenetic analysis revealed that Wara is a member of the Tequintavirus genus. A lack of lysogeny genes, antimicrobial resistance, and virulence genes indicated that Wara has therapeutic and biocontrol potential against Salmonella species in healthcare and agriculture. Full article

Canine diarrhea is a common intestinal illness that is usually caused by viruses, bacteria, and parasites, and canine diarrhea may induce morbidity and mortality of domestic dogs if treated improperly. Recently, viral metagenomics was applied to investigate the signatures of the enteric virome in mammals. In this research, the characteristics of the gut virome in healthy dogs and dogs with diarrhea were analyzed and compared using viral metagenomics. The alpha diversity analysis indicated that the richness and diversity of the gut virome in the dogs with diarrhea were much higher than the healthy dogs, while the beta diversity analysis revealed that the gut virome of the two groups was quite different. At the family level, the predominant viruses in the canine gut virome were certified to be Microviridae, Parvoviridae, Siphoviridae, Inoviridae, Podoviridae, Myoviridae, and others. At the genus level, the predominant viruses in the canine gut virome were certified to be Protoparvovirus, Inovirus, Chlamydiamicrovirus, Lambdavirus, Dependoparvovirus, Lightbulbvirus, Kostyavirus, Punavirus, Lederbergvirus, Fibrovirus, Peduovirus, and others. However, the viral communities between the two groups differed significantly. The unique viral taxa identified in the healthy dogs group were Chlamydiamicrovirus and Lightbulbvirus, while the unique viral taxa identified in the dogs with diarrhea group were Inovirus, Protoparvovirus, Lambdavirus, Dependoparvovirus, Kostyavirus, Punavirus, and other viruses. Phylogenetic analysis based on the near-complete genome sequences showed that the CPV strains collected in this study together with other CPV Chinese isolates clustered into a separate branch, while the identified CAV-2 strain D5-8081 and AAV-5 strain AAV-D5 were both the first near-complete genome sequences in China. Moreover, the predicted bacterial hosts of phages were certified to be Campylobacter, Escherichia, Salmonella, Pseudomonas, Acinetobacter, Moraxella, Mediterraneibacter, and other commensal microbiota. In conclusion, the enteric virome of the healthy dogs group and the dogs with diarrhea group was investigated and compared using viral metagenomics, and the viral communities might influence canine health and disease by interacting with the commensal gut microbiome. Full article

The metagenomic whole genome shotgun sequencing (mWGS) approach was used to detect signatures of the rhizosphere microbiomes of Dipterygium glaucum and surrounding bulk soil microbiomes, and to detect differential microbial responses due to watering. Preliminary results reflect the reliability of the experiment and the rationality of grouping microbiomes. Based on the abundance of non-redundant genes, bacterial genomes showed the highest level, followed by Archaeal and Eukaryotic genomes, then, the least abundant viruses. Overall results indicate that most members of bacteria have a higher abundance/relative abundance (AB/RA) pattern in the rhizosphere towards plant growth promotion, while members of eukaryota have a higher pattern in bulk soil, most likely acting as pathogens. The results also indicate the contribution of mycorrhiza (genus Rhizophagus) in mediating complex mutualistic associations between soil microbes (either beneficial or harmful) and plant roots. Some of these symbiotic relationships involve microbes of different domains responding differentially to plant root exudates. Among these are included the bacterial genus Burkholderia and eukaryotic genus Trichoderma, which have antagonistic activities against the eukaryotic genus Fusarium. Another example involves Ochrobactrum phage POA1180, its bacterial host and plant roots. One of the major challenges in plant nutrition involves other microbes that manipulate nitrogen levels in the soil. Among these are the microbes that perform contraversal actions of nitrogen fixation (the methanogen Euryarchaeota) and ammonia oxidation (Crenarchaeota). The net nitrogen level in the soil is originally based on the AB/RA of these microbes and partially on the environmental condition. Watering seems to influence the AB/RA of a large number of soil microbes, where drought-sensitive microbes (members of phyla Acidobacteria and Gemmatimonadetes) showed an increased AB/RA pattern after watering, while others (Burkholderia and Trichoderma) seem to be among microbes assisting plants to withstand abiotic stresses. This study sheds light on the efficient use of mWGS in the taxonomic assignment of soil microbes and in their response to watering. It also provides new avenues for improving biotic and abiotic resistance in domestic plant germplasm via the manipulation of soil microbes. Full article

Peste des petits ruminants virus (PPRV) causes a highly devastating disease, peste des petits ruminants (PPR) of sheep and goats, that threatens food security, small ruminant production, and the conservation of wild small ruminants in many developing countries, especially in Africa. Robust serological and molecular diagnostic tools are available to detect PPRV infection, but they were mainly developed for domestic sheep and goats. The presence of a wide host range for PPRV does present serological diagnostic challenges. New innovative diagnostic tools are needed to detect PPRV in atypical hosts (e.g., Camelidae, Suidae, and Bovinae), in wildlife ecosystems and in complex field situations. Interestingly, single-domain antigen binding fragments (nanobodies) derived from heavy-chain-only camelid antibodies have emerged as a new hope in the development of accurate, rapid, and cost-effective diagnostic tools in veterinary and biomedical fields that are suitable for low-income countries. The main objective of this study was to construct an immune nanobody library to retrieve PPRV-reactive nanobodies that enable the development of diagnostic and therapeutic nanobodies in the future. Here, a strategy was developed whereby an alpaca (Vicugna pacos) was immunized with a live attenuated vaccine strain (PPRV/N/75/1) to raise an affinity-matured immune response in the heavy-chain-only antibody classes. The nanobody gene repertoire was engineered in pMECS-GG phagemid, whereby a ccdB gene (encoding a lethal protein) was substituted by the nanobody gene. An immune nanobody library with approximately sixty-four million independent transformants was constructed, of which 100% contained an insert with the proper size of nanobody gene. Following phage display and biopanning, nine nanobodies that specifically recognise completely inactivated PPRV were identified on enzyme-linked immunosorbent assay. They showed superb potency in rapidly identifying PPRV, which is likely to open a new perspective in the diagnosis and possible treatment of PPR infection. Full article

To overcome the obstacle of antimicrobial resistance, researchers are investigating the use of phage therapy as an alternative and/or supplementation to antibiotics to treat and prevent infections both in humans and in animals. In the first part of this review, we describe the unique biological characteristics of bacteriophages and the crucial aspects influencing the success of phage therapy. However, despite their efficacy and safety, there is still no specific legislation that regulates their use. In the second part of this review, we describe the comprehensive research done in the past and recent years to address the use of phage therapy for the treatment and prevention of bacterial disease affecting domestic animals as an alternative to antibiotic treatments. While in farm animals, phage therapy efficacy perspectives have been widely studied in vitro and in vivo, especially for zoonoses and diseases linked to economic losses (such as mastitis), in pets, studies are still few and rather recent. Full article

Outer membrane vesicles (OMVs) are nanostructures mostly produced by blebbing of the outer membrane in Gram negative bacteria. They contain biologically active proteins and perform a variety of processes. OMV production is also a typical response to events inducing stress in the bacterial envelope. In these cases, hypervesiculation is regarded as a strategy to avoid the dangerous accumulation of undesired products within the periplasm. Several housekeeping genes influence the biogenesis of OMVs, including those correlated with peptidoglycan and cell wall dynamics. In this work, we have investigated the relationship between OMV production and the lysis module of the E. coli DLP12 cryptic prophage. This module is an operon encoding a holin, an endolysin and two spannins, and is known to be involved in cell wall maintenance. We find that deleting the lysis module increases OMV production, suggesting that during evolution this operon has been domesticated to regulate vesiculation, likely through the elimination of non-recyclable peptidoglycan fragments. We also show that the expression of the lysis module is negatively regulated by environmental stress stimuli as high osmolarity, low pH and low temperature. Our data further highlight how defective prophages finely contribute to bacterial host fitness. Full article

Burkholderia cenocepacia, is a Gram-negative opportunistic pathogen that belongs to Burkholderia cepacia complex (BCC) group. BCC representatives carry various pathogenicity factors and can infect humans and plants. Phages as bacterial viruses play a significant role in biodiversity and ecological balance in the environment. Specifically, horizontal gene transfer (HGT) and lysogenic conversion (temperate phages) influence microbial diversification and fitness. In this study, we describe the prevalence and gene content of prophages in 16 fully sequenced B. cenocepacia genomes stored in NCBI database. The analysis was conducted in silico by manual and automatic approaches. Sixty-three potential prophage regions were found and classified as intact, incomplete, questionable, and artifacts. The regions were investigated for the presence of known virulence factors, resulting in the location of sixteen potential pathogenicity mechanisms, including toxin–antitoxin systems (TA), Major Facilitator Superfamily (MFS) transporters and responsible for drug resistance. Investigation of the region’s closest neighborhood highlighted three groups of genes with the highest occurrence—tRNA-Arg, dehydrogenase family proteins, and ABC transporter substrate-binding proteins. Searches for antiphage systems such as BacteRiophage EXclusion (BREX) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) in the analyzed strains suggested 10 sequence sets of CRISPR elements. Our results suggest that intact B. cenocepacia prophages may provide an evolutionary advantage to the bacterium, while domesticated prophages may help to maintain important genes. Full article

Fusarium verticillioides is the primary causal agent of Fusarium ear and kernel rot in maize, producing fumonisin mycotoxins that are toxic to humans and domestic animals. Rapid detection and monitoring of fumonisin-producing fungi are pivotally important for the prevention of mycotoxins from entering into food/feed products. Chicken-derived single-chain variable fragments (scFvs) against cell wall-bound proteins from F. verticillioides were isolated from an immunocompetent phage display library. Comparative phage enzyme-linked immunosorbant assays (ELISAs) and sequencing analyses identified four different scFv antibodies with high sensitivity. Soluble antibody ELISAs identified two highly sensitive scFv antibodies, FvCA3 and FvCA4, with the latter being slightly more sensitive. Three-dimensional modeling revealed that the FvCA4 may hold a better overall structure with CDRH3, CDRL1 and CDRL3 centered in the core region of antibody surface compared with that of other scFvs. Immunofluorescence labeling revealed that the binding of FvCA4 antibody was localized to the cell walls of conidiospores and hyphae of F. verticillioides, confirming the specificity of this antibody for a surface target. This scFv antibody was able to detect the fungal mycelium as low as 10⁻² μg/mL and contaminating mycelium at a quantity of 10⁻² mg/g maize. This is the first report that scFv antibodies derived from phage display have a wide application for rapid and accurate detection and monitoring of fumonisin-producing pathogens in agricultural samples. Full article

Fusarium mycotoxins directly accumulated in grains during the infection of wheat and other cereal crops by Fusarium head blight (FHB) pathogens are detrimental to humans and domesticated animals. Prevention of the mycotoxins via the development of FHB-resistant varieties has been a challenge due to the scarcity of natural resistance against FHB pathogens. Various antibodies specific to Fusarium fungi and mycotoxins are widely used in immunoassays and antibody-mediated resistance in planta against Fusarium pathogens has been demonstrated. Antibodies fused to antifungal proteins have been shown to confer a very significantly enhanced Fusarium resistance in transgenic plants. Thus, antibody fusions hold great promise as an effective tool for the prevention of mycotoxin contaminations in cereal grains. This review highlights the utilization of protective antibodies derived from phage display to increase endogenous resistance of wheat to FHB pathogens and consequently to reduce mycotoxins in field. The role played by Fusarium-specific antibody in the resistance is also discussed. Full article