Implementing bacteriophages into dosage forms is a significant step for the practical application of phage therapy. While designing a dosage form, bacteriophages as active ingredients may be exposed to excipients, guaranteeing microbial quality. However, only a few antimicrobial preservatives have been studied regarding their interaction with bacteriophages during long-term storage. Here, the stability of the staphylococcal Kayvirus and pseudomonal Pbunavirus with twelve commonly used preservatives was monitored for thirteen weeks to assess the risk of destabilisation of phage suspensions by excipients. The effectiveness of preservatives on the test bacteria, yeast and mould was determined using a microdilution method and the phage lytic activity by plaque enumeration. The antimicrobial activity of preservatives with bacteriophages was confirmed, except benzalkonium chloride and chlorhexidine digluconate, which showed precipitation and were classified as incompatible. A complete loss of phage potency in both tested phages occurred with diazolidinyl urea and in Kayvirus with benzalkonium chloride. For both phages, a slight decrease in titer, by one order of magnitude, was observed with m-cresol, sodium propionate, sodium benzoate, and phenylethyl alcohol. For Kayvirus, thimerosal, parabens, and mono propylene glycol and for Pbunavirus, phenoxyethanol also met the criteria. The decrease by two or more orders was determined for the remaining cases. This study helps select antimicrobial preservatives for optimizing dosage formulations with the therapeutically applicable bacteriophages.
- Klíčová slova
- Antimicrobial preservative, Bacteriophages, Dosage forms formulations, Excipients, Kayvirus, Pbunavirus, Phage therapy, Pseudomonas aeruginosa, Staphylococcus aureus,
- MeSH
- antiinfekční látky * farmakologie chemie MeSH
- bakteriofágy * účinky léků MeSH
- benzalkoniové sloučeniny farmakologie chemie MeSH
- fágová terapie metody MeSH
- konzervační prostředky farmaceutické * chemie farmakologie MeSH
- mikrobiální testy citlivosti MeSH
- pomocné látky chemie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antiinfekční látky * MeSH
- benzalkoniové sloučeniny MeSH
- konzervační prostředky farmaceutické * MeSH
- pomocné látky MeSH
Recent advances in genome editing techniques based on CRISPR-Cas have opened up new possibilities in bacteriophage engineering and, thus, enabled key developments in medicine, nanotechnology, and synthetic biology. Although staphylococcal phage genomes have already been edited, the modification of their structural proteins has not yet been reported. Here, the structure of Staphylococcus phage 812h1 of the Kayvirus genus was modified by inserting a poly histidine tag into an exposed loop of the tail sheath protein. A two-strain editing strategy was applied, utilizing homologous recombination followed by CRISPR-Cas10-assisted counter-selection of the recombinant phages. The His-tagged phage particles can be recognized by specific antibodies, enabling the modified bacteriophages to be employed in numerous techniques. The attachment of the engineered phage to bacteria was visualized by fluorescence microscopy, and its functionality was confirmed using biolayer interferometry biosensing, enzyme-linked immunosorbent assay, and flow cytometry, demonstrating that the genetic modification did not impair its biological activity.
- Klíčová slova
- Bacteriophage, Biosensing Techniques, CRISPR-Cas10, Herelleviridae, Poly histidine Tag, Staphylococcus aureus,
- MeSH
- biosenzitivní techniky * metody MeSH
- CRISPR-Cas systémy * genetika MeSH
- editace genu metody MeSH
- stafylokokové bakteriofágy * genetika MeSH
- Staphylococcus virologie MeSH
- Publikační typ
- časopisecké články MeSH
Macrococci are usually found as commensals on the skin and mucosa of animals and have been isolated from mammal-derived fermented foods; however, they can also act as opportunistic pathogens. Here, we used whole-genome sequencing, comparative genomics, extensive biotyping, MALDI-TOF mass spectrometry, and chemotaxonomy to characterize Macrococcus sp. strains isolated from livestock and human-related specimens. Based on the results of polyphasic taxonomy, we propose the species Macrococcus psychrotolerans sp. nov. (type strain NRL/St 95/376T = CCM 8659T = DSM 111350T) belonging to the Macrococcus caseolyticus phylogenetic clade. It grows at 4°C, and the core genome of the isolates contains suspected genes contributing to low-temperature tolerance. Variable genetic elements include prophages, chromosomal islands, a composite staphylococcal cassette chromosome island, and many plasmids that affect the overall genome expansion and adaptation to specific ecological settings of the studied isolates. Large plasmids carrying the methicillin resistance gene mecB were identified in M. psychrotolerans sp. nov. strains and confirmed as self-transmissible to Staphylococcus aureus in vitro. In addition to plasmids with circular topology, a 150-kb-long linear plasmid with 14.1-kb-long inverted terminal repeats, harboring many IS elements and putative genes for a type IV secretion system was revealed. The described strains were isolated from human clinical material, food-producing animals, meat, and a wooden cheese board and have the potential to proliferate at refrigerator temperatures. Their presence in the food chain and human infections indicates that attention needs to be paid to this potential novel opportunistic pathogen.IMPORTANCEThe study offers insights into the phenotypic and genomic features of a novel species of the genus Macrococcus that occurs in livestock, food, and humans. The large number of diverse mobile genetic elements contributes to the adaptation of macrococci to various environments. The ability of the described microorganisms to grow at refrigerator temperatures, enabled by genes that are predicted to contribute to low-temperature tolerance, raises food safety concerns. Confirmed in vitro conjugative transfer of plasmid-borne mecB gene to S. aureus poses a significant risk of spread of broad β-lactam resistance. In addition, the intergeneric plasmid transfer to S. aureus is indicative of horizontal gene transfer events that may be more frequent than generally accepted. Determining a complete sequence and gene content of linear megaplasmid with exceptional topology for the Staphylococcaceae family suggests its possible role in shuttling adaptive traits through an exchange of genetic information.
- Klíčová slova
- Gram-positive cocci, cephalosporin resistance, cold temperature tolerance, conjugation, food safety, linear plasmid, methicillin resistance,
- MeSH
- antibakteriální látky * farmakologie MeSH
- bakteriální proteiny * genetika metabolismus MeSH
- beta-laktamová rezistence * genetika MeSH
- dobytek mikrobiologie MeSH
- Enterococcaceae * genetika izolace a purifikace účinky léků MeSH
- fylogeneze MeSH
- genom bakteriální MeSH
- lidé MeSH
- plazmidy MeSH
- přenos genů horizontální MeSH
- sekvenování celého genomu MeSH
- stafylokokové infekce mikrobiologie MeSH
- Staphylococcus aureus * genetika účinky léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antibakteriální látky * MeSH
- bakteriální proteiny * MeSH
UNLABELLED: Microviruses are single-stranded DNA viruses infecting bacteria, characterized by T = 1 shells made of single jelly-roll capsid proteins. To understand how microviruses infect their host cells, we have isolated and studied an unusually large microvirus, Ebor. Ebor belongs to the proposed "Tainavirinae" subfamily of Microviridae and infects the model Alphaproteobacterium Rhodobacter capsulatus. Using cryogenic electron microscopy, we show that the enlarged capsid of Ebor is the result of an extended C-terminus of the major capsid protein. The extra packaging space accommodates genes encoding a lytic enzyme and putative methylase, both absent in microviruses with shorter genomes. The capsid is decorated with protrusions at its 3-fold axes, which we show to recognize lipopolysaccharides on the host surface. Cryogenic electron tomography shows that during infection, Ebor attaches to the host cell via five such protrusions. This attachment brings a single pentameric capsomer into close contact with the cell membrane, creating a special vertex through which the genome is ejected. Both subtomogram averaging and single particle analysis identified two intermediates of capsid opening, showing that the interacting penton opens from its center via the separation of individual capsomer subunits. Structural comparison with the model Bullavirinae phage phiX174 suggests that this genome delivery mechanism may be widely present across Microviridae. IMPORTANCE: Tailless Microviridae bacteriophages are major components of the global virosphere. Notably, microviruses are prominent members of the mammalian gut virome, and certain compositions have been linked to serious health disorders; however, a molecular understanding of how they initiate infection of their host remains poorly characterized. We demonstrate that trimeric protrusions located at the corners of a single microvirus capsomer mediate host cell attachment. This interaction triggers opening of the capsomer, driven by separation of subunits from its center, much like flower petals open during blooming. This extensive opening explains how the genome translocation apparatus, along with the genome itself, is able to exit the capsid. "Penton blooming" likely represents a conserved mechanism shared by diverse viruses possessing similar capsid architectures.
- Klíčová slova
- Microviridae, Rhodobacter, electron microscopy, structural biology, virion structure,
- MeSH
- elektronová kryomikroskopie MeSH
- genom virový * MeSH
- kapsida ultrastruktura MeSH
- Microviridae * genetika fyziologie ultrastruktura MeSH
- virové plášťové proteiny genetika metabolismus chemie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- virové plášťové proteiny MeSH
Antibiotic-resistant strains of Staphylococcus aureus pose a significant threat in healthcare, demanding urgent therapeutic solutions. Combining bacteriophages with conventional antibiotics, an innovative approach termed phage-antibiotic synergy, presents a promising treatment avenue. However, to enable new treatment strategies, there is a pressing need for methods to assess their efficacy reliably and rapidly. Here, we introduce a novel approach for real-time monitoring of pathogen lysis dynamics employing the piezoelectric quartz crystal microbalance (QCM) with dissipation (QCM-D) technique. The sensor, a QCM chip modified with the bacterium S. aureus RN4220 ΔtarM, was utilized to monitor the activity of the enzyme lysostaphin and the phage P68 as model lytic agents. Unlike conventional QCM solely measuring resonance frequency changes, our study demonstrates that dissipation monitoring enables differentiation of bacterial growth and lysis caused by cell-attached lytic agents. Compared to reference turbidimetry measurements, our results reveal distinct alterations in the growth curve of the bacteria adhered to the sensor, characterized by a delayed lag phase. Furthermore, the dissipation signal analysis facilitated the precise real-time monitoring of phage-mediated lysis. Finally, the QCM-D biosensor was employed to evaluate the synergistic effect of subinhibitory concentrations of the antibiotic amoxicillin with the bacteriophage P68, enabling monitoring of the lysis of P68-resistant wild-type strain S. aureus RN4220. Our findings suggest that this synergy also impedes the formation of bacterial aggregates, the precursors of biofilm formation. Overall, this method brings new insights into phage-antibiotic synergy, underpinning it as a promising strategy against antibiotic-resistant bacterial strains with broad implications for treatment and prevention.
- Klíčová slova
- Staphylococcus aureus, Antimicrobial treatment, Multidrug-resistant bacteria, Phage therapy, Phage-antibiotic synergy, Piezoelectric biosensor,
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriofágy fyziologie MeSH
- bakteriolýza * účinky léků MeSH
- biosenzitivní techniky * metody přístrojové vybavení MeSH
- lysostafin farmakologie MeSH
- mikrorovnovážné techniky křemenného krystalu * metody MeSH
- Staphylococcus aureus * účinky léků virologie růst a vývoj MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antibakteriální látky MeSH
- lysostafin MeSH
Single-stranded DNA bacteriophages of the Microviridae family are major components of the global virosphere. Microviruses are highly abundant in aquatic ecosystems and are prominent members of the mammalian gut microbiome, where their diversity has been linked to various chronic health disorders. Despite the clear importance of microviruses, little is known about the molecular mechanism of host infection. Here, we have characterized an exceptionally large microvirus, Ebor, and provide crucial insights into long-standing mechanistic questions. Cryogenic electron microscopy of Ebor revealed a capsid with trimeric protrusions that recognise lipopolysaccharides on the host surface. Cryogenic electron tomography of the host cell colonized with virus particles demonstrated that the virus initially attaches to the cell via five such protrusions, located at the corners of a single pentamer. This interaction triggers a stargate mechanism of capsid opening along the 5-fold symmetry axis, enabling delivery of the virus genome. Despite variations in specific virus-host interactions among different Microviridae family viruses, structural data indicate that the stargate mechanism of infection is universally employed by all members of the family. Startlingly, our data reveal a mechanistic link for the opening of relatively small capsids made out of a single jelly-roll fold with the structurally unrelated giant viruses.
- Publikační typ
- časopisecké články MeSH
- preprinty MeSH
To broaden the substrate scope of microbial cell factories towards renewable substrates, rational genetic interventions are often combined with adaptive laboratory evolution (ALE). However, comprehensive studies enabling a holistic understanding of adaptation processes primed by rational metabolic engineering remain scarce. The industrial workhorse Pseudomonas putida was engineered to utilize the non-native sugar D-xylose, but its assimilation into the bacterial biochemical network via the exogenous xylose isomerase pathway remained unresolved. Here, we elucidate the xylose metabolism and establish a foundation for further engineering followed by ALE. First, native glycolysis is derepressed by deleting the local transcriptional regulator gene hexR. We then enhance the pentose phosphate pathway by implanting exogenous transketolase and transaldolase into two lag-shortened strains and allow ALE to finetune the rewired metabolism. Subsequent multilevel analysis and reverse engineering provide detailed insights into the parallel paths of bacterial adaptation to the non-native carbon source, highlighting the enhanced expression of transaldolase and xylose isomerase along with derepressed glycolysis as key events during the process.
- MeSH
- metabolické inženýrství MeSH
- pentózofosfátový cyklus MeSH
- Pseudomonas putida * genetika MeSH
- transaldolasa genetika MeSH
- xylosa * metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- transaldolasa MeSH
- xylosa * MeSH
Novel species of coagulase-negative staphylococci, which could serve as reservoirs of virulence and antimicrobial resistance factors for opportunistic pathogens from the genus Staphylococcus, are recognized in human and animal specimens due to advances in diagnostic techniques. Here, we used whole-genome sequencing, extensive biotyping, MALDI-TOF mass spectrometry, and chemotaxonomy to characterize five coagulase-negative strains from the Staphylococcus haemolyticus phylogenetic clade obtained from human ear swabs, wounds, and bile. Based on the results of polyphasic taxonomy, we propose the species Staphylococcus brunensis sp. nov. (type strain NRL/St 16/872T = CCM 9024T = LMG 31872T = DSM 111349T). The genomic analysis revealed numerous variable genomic elements, including staphylococcal cassette chromosome (SCC), prophages, plasmids, and a unique 18.8 kb-long genomic island SbCIccrDE integrated into the ribosomal protein L7 serine acetyltransferase gene rimL. SbCIccrDE has a cassette chromosome recombinase (ccr) gene complex with a typical structure found in SCCs. Based on nucleotide and amino acid identity to other known ccr genes and the distinct integration site that differs from the canonical methyltransferase gene rlmH exploited by SCCs, we classified the ccr genes as novel variants, ccrDE. The comparative genomic analysis of SbCIccrDE with related islands shows that they can accumulate virulence and antimicrobial resistance factors creating novel resistance elements, which reflects the evolution of SCC. The spread of these resistance islands into established pathogens such as Staphylococcus aureus would pose a great threat to the healthcare system. IMPORTANCE The coagulase-negative staphylococci are important opportunistic human pathogens, which cause bloodstream and foreign body infections, mainly in immunocompromised patients. The mobile elements, primarily the staphylococcal cassette chromosome mec, which confers resistance to methicillin, are the key to the successful dissemination of staphylococci into healthcare and community settings. Here, we present a novel species of the Staphylococcus genus isolated from human clinical material. The detailed analysis of its genome revealed a previously undescribed genomic island, which is closely related to the staphylococcal cassette chromosome and has the potential to accumulate and spread virulence and resistance determinants. The island harbors a set of conserved genes required for its mobilization, which we recognized as novel cassette chromosome recombinase genes ccrDE. Similar islands were revealed not only in the genomes of coagulase-negative staphylococci but also in S. aureus. The comparative genomic study contributes substantially to the understanding of the evolution and pathogenesis of staphylococci.
Both temperate and obligately lytic phages have crucial roles in the biology of staphylococci. While superinfection exclusion among closely related temperate phages is a well-characterized phenomenon, the interactions between temperate and lytic phages in staphylococci are not understood. Here, we present a resistance mechanism toward lytic phages of the genus Kayvirus, mediated by the membrane-anchored protein designated PdpSau encoded by Staphylococcus aureus prophages, mostly of the Sa2 integrase type. The prophage accessory gene pdpSau is strongly linked to the lytic genes for holin and ami2-type amidase and typically replaces genes for the toxin Panton-Valentine leukocidin (PVL). The predicted PdpSau protein structure shows the presence of a membrane-binding α-helix in its N-terminal part and a cytoplasmic positively charged C terminus. We demonstrated that the mechanism of action of PdpSau does not prevent the infecting kayvirus from adsorbing onto the host cell and delivering its genome into the cell, but phage DNA replication is halted. Changes in the cell membrane polarity and permeability were observed from 10 min after the infection, which led to prophage-activated cell death. Furthermore, we describe a mechanism of overcoming this resistance in a host-range Kayvirus mutant, which was selected on an S. aureus strain harboring prophage 53 encoding PdpSau, and in which a chimeric gene product emerged via adaptive laboratory evolution. This first case of staphylococcal interfamily phage-phage competition is analogous to some other abortive infection defense systems and to systems based on membrane-destructive proteins. IMPORTANCE Prophages play an important role in virulence, pathogenesis, and host preference, as well as in horizontal gene transfer in staphylococci. In contrast, broad-host-range lytic staphylococcal kayviruses lyse most S. aureus strains, and scientists worldwide have come to believe that the use of such phages will be successful for treating and preventing bacterial diseases. The effectiveness of phage therapy is complicated by bacterial resistance, whose mechanisms related to therapeutic staphylococcal phages are not understood in detail. In this work, we describe a resistance mechanism targeting kayviruses that is encoded by a prophage. We conclude that the defense mechanism belongs to a broader group of abortive infections, which is characterized by suicidal behavior of infected cells that are unable to produce phage progeny, thus ensuring the survival of the host population. Since the majority of staphylococcal strains are lysogenic, our findings are relevant for the advancement of phage therapy.
- Klíčová slova
- Kayvirus, Staphylococcus aureus, abortive infection, bacteriophage evolution, bacteriophage therapy, bacteriophages, cell death, lysogeny, phage resistance, phage therapy,
- MeSH
- lidé MeSH
- lyzogenie MeSH
- membránové proteiny genetika MeSH
- profágy * genetika MeSH
- stafylokokové bakteriofágy genetika MeSH
- stafylokokové infekce * mikrobiologie MeSH
- Staphylococcus aureus genetika MeSH
- Staphylococcus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- membránové proteiny MeSH
Staphylococci from the Staphylococcus intermedius-Staphylococcus hyicus species group include numerous animal pathogens and are an important reservoir of virulence and antimicrobial resistance determinants. Due to their pathogenic potential, they are possible causative agents of zoonoses in humans; therefore, it is important to address the properties of these strains. Here we used a polyphasic taxonomic approach to characterize the coagulase-negative staphylococcal strain NRL/St 03/464T, isolated from the nostrils of a healthy laboratory rat during a microbiological screening of laboratory animals. The 16S rRNA sequence, MALDI-TOF mass spectrometry and positive urea hydrolysis and beta-glucuronidase tests clearly distinguished it from closely related Staphylococcus spp. All analyses have consistently shown that the closest relative is Staphylococcus chromogenes; however, values of digital DNA-DNA hybridization <35.3% and an average nucleotide identity <81.4% confirmed that the analyzed strain is a distinct Staphylococcus species. Whole-genome sequencing and expert annotation of the genome revealed the presence of novel variable genetic elements, including two plasmids named pSR9025A and pSR9025B, prophages, genomic islands and a composite transposon that may confer selective advantages to other bacteria and enhance their survival. Based on phenotypic, phylogenetic and genomic data obtained in this study, the strain NRL/St 03/464T (= CCM 9025T = LMG 31873T = DSM 111348T) represents a novel species with the suggested name Staphylococcus ratti sp. nov.
- Klíčová slova
- Hyicus-Intermedius species group, Staphylococcus, genomic island, laboratory rat, taxonomy, variable genetic element, whole genome sequencing,
- Publikační typ
- časopisecké články MeSH
