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Antibiotics (Basel, Switzerland) Jun 2024The aim of this study is to investigate the occurrence of plasmid mediated quinolone resistance (PMQR) determinants in isolates collected from broilers, laying hens and...
The aim of this study is to investigate the occurrence of plasmid mediated quinolone resistance (PMQR) determinants in isolates collected from broilers, laying hens and poultry farm environments. One hundred and thirty-nine isolates were isolated from broilers (n = 41), laying hens (n = 53), eggs (n = 4) and the environment (n = 41) of 23 poultry farms located in northeastern of Tunisia. Antimicrobial susceptibility testing was performed on all isolates according to the recommendation of the European Committee on Antimicrobial Susceptibility Testing guidelines. The detection of PMQR genes: , , , , , , and (6) gene was performed using polymerase chain reaction (PCR) and specific primers. (6')-Ib amplicons were further analyzed by digestion with to identify the (6')- variant. Mutations in GyrA and the occurrence of RE-CmeABC efflux pump were determined by mismatch amplification mutation assay (MAMA) PCR and PCR, respectively. In addition, eleven isolates were selected to determine their clonal lineage by MLST. The 139 isolates were resistant to ciprofloxacin, and 86 (61.8%) were resistant to nalidixic acid. High rates of resistance were also observed toward erythromycin (100%), azithromycin (96.4%), tetracycline (100%), chloramphenicol (98.56%), ampicillin (66.1%), amoxicillin-clavulanic acid (55.39%), and kanamycin (57.55%). However, moderate resistance rates were observed for gentamicin (9.35%) and streptomycin (22.3%). All quinolone-resistant isolates harbored the Thr-86-Ile amino acid substitution in GyrA, and the RE-CmeABC efflux pump was detected in 40.28% of isolates. Interestingly, the , , , and (6')-- were detected in 57.7%, 61.15%, 21.58%, and 10% of isolates, respectively. The eleven isolates studied by MLST belonged to a new sequence type ST13450. This study described for the first time the occurrence of PMQR genes in isolates in Tunisia and globally.
PubMed: 38927193
DOI: 10.3390/antibiotics13060527 -
Antibiotics (Basel, Switzerland) Jun 2024In recent years, global concern over increasing multidrug resistance (MDR) among various serotypes has grown significantly. However, reports on MDR Paratyphi B remain...
BACKGROUND
In recent years, global concern over increasing multidrug resistance (MDR) among various serotypes has grown significantly. However, reports on MDR Paratyphi B remain scarce, let alone the extensively drug-resistant (XDR) strains.
METHODS
In this retrospective study, we investigated the isolates of Paratyphi B in Jiangsu Province over the past decade and carried out antimicrobial susceptibility tests, then the strains were sequenced and bioinformatics analyses were performed.
RESULTS
27 Paratyphi B strains were identified, of which the predominant STs were ST42 (11), ST86 (10), and ST2814 (5). Among these strains, we uncovered four concerning XDR Paratyphi B ST2814 strains (4/5) which were previously unreported. These alarmingly resistant isolates showed resistance to all three major antibiotic classes for treatment and even the last resort treatment tigecycline. Bioinformatics analysis revealed high similarity between the plasmids harbored by these XDR strains and diverse serotypes and from China and neighboring regions. Notably, these four plasmids carried the gene responsible for multiple antibiotic resistance by regulating the AcrAB-TolC pump, predominantly originating from China. Additionally, a distinct MDR ST42(1/11) strain with an ICE on chromosome was also identified. Furthermore, phylogenetic analysis of global ST42/ST2814 isolates highlighted the regional specificity of these strains, with Jiangsu isolates clustering together with domestic isolates and XDR ST2814 forming a distinct branch, suggesting adaptation to local antibiotic pressures.
CONCLUSIONS
This research underscores the pressing need for closely monitoring the MDR/XDR Paratyphi B, particularly the emerging ST2814 strains in Jiangsu Province, to effectively curb its spread and protect public health. Moreover, surveillance should be strengthened across different ecological niches and genera to track resistance genes and horizontal gene transfer elements under the concept of "ONE HEALTH".
PubMed: 38927185
DOI: 10.3390/antibiotics13060519 -
Antibiotics (Basel, Switzerland) May 2024Carbapenemase-producing (CP-KP) represents a global threat to public health, with limited antimicrobial therapeutic options. In this study, we analyzed a...
BACKGROUND
Carbapenemase-producing (CP-KP) represents a global threat to public health, with limited antimicrobial therapeutic options. In this study, we analyzed a ceftazidime/avibactam (CAZ-AVI)-resistant isolate obtained from a patient previously exposed to CAZ-AVI expressing a novel carbapenemase (KPC)-3 variant.
METHODS
Antimicrobial susceptibility testing was performed using reference broth microdilution. Whole-genome sequencing (WGS) was performed using Illumina and Nanopore Technologies. Short- and long-reads were combined with Unicycler. Assemblies were investigated for multilocus sequence typing (MLST), antimicrobial resistance genes, porins, and plasmids.
RESULTS
The . isolate (KP_RM_1) was resistant to CAZ-AVI, expanded-spectrum cephalosporins, amikacin, ertapenem, and cefiderocol (FDC) but was susceptible to tigecycline, colistin, trimethoprim/sulfamethoxazole, meropenem-vaborbactam, and imipenem-relebactam. WGS revealed that the KP_RM_1 genome is composed of a single chromosome of 5 Mbp and five circular plasmids. Further analysis showed the presence of novel located on a 72 kb plasmid. KPC-216 differs from KPC-3 by a Lysin (K) insertion at position 168 (+K168).
CONCLUSIONS
We report the identification of a new KPC-3 variant associated with CAZ-AVI resistance. The KPC variants associated with CAZ-AVI resistance should be determined to promptly inform clinicians and start the appropriate antimicrobial therapy.
PubMed: 38927174
DOI: 10.3390/antibiotics13060507 -
Biomolecules Jun 2024MicroRNAs (miRNAs) are highly conserved endogenous single-stranded non-coding RNA molecules that play a crucial role in regulating gene expression to maintain normal...
Downregulation of miR-1388 Regulates the Expression of Antiviral Genes via Tumor Necrosis Factor Receptor ()-Associated Factor 3 Targeting Following poly(I:C) Stimulation in Silver Carp ().
MicroRNAs (miRNAs) are highly conserved endogenous single-stranded non-coding RNA molecules that play a crucial role in regulating gene expression to maintain normal physiological functions in fish. Nevertheless, the specific physiological role of miRNAs in lower vertebrates, particularly in comparison to mammals, remains elusive. Additionally, the mechanisms underlying the control of antiviral responses triggered by viral stimulation in fish are still not fully understood. In this study, we investigated the regulatory impact of miR-1388 on the signaling pathway mediated by IFN regulatory factor 3 (). Our findings revealed that following stimulation with the viral analog poly(I:C), the expression of miR-1388 was significantly upregulated in primary immune tissues and macrophages. Through a dual luciferase reporter assay, we corroborated a direct targeting relationship between miR-1388 and tumor necrosis factor receptor ()-associated factor 3 (). Furthermore, our study demonstrated a distinct negative post-transcriptional correlation between miR-1388 and . We observed a significant negative post-transcriptional regulatory association between miR-1388 and the levels of antiviral genes following poly(I:C) stimulation. Utilizing reporter plasmids, we elucidated the role of miR-1388 in the antiviral signaling pathway activated by . By intervening with siRNA-, we validated that miR-1388 regulates the expression of antiviral genes and the production of type I interferons () through its interaction with . Collectively, our experiments highlight the regulatory influence of miR-1388 on the -mediated signaling pathway by targeting post poly(I:C) stimulation. These findings provide compelling evidence for enhancing our understanding of the mechanisms through which fish miRNAs participate in immune responses.
Topics: Animals; MicroRNAs; Poly I-C; Carps; TNF Receptor-Associated Factor 3; Down-Regulation; Interferon Regulatory Factor-3; Gene Expression Regulation; Fish Proteins; Signal Transduction
PubMed: 38927097
DOI: 10.3390/biom14060694 -
Biomolecules Jun 2024Clickable nucleosides, most often 5-ethynyl-2'-deoxyuridine (EtU), are widely used in studies of DNA replication in living cells and in DNA functionalization for...
Clickable nucleosides, most often 5-ethynyl-2'-deoxyuridine (EtU), are widely used in studies of DNA replication in living cells and in DNA functionalization for bionanotechology applications. Although clickable dNTPs are easily incorporated by DNA polymerases into the growing chain, afterwards they might become targets for DNA repair systems or interfere with faithful nucleotide insertion. Little is known about the possibility and mechanisms of these post-synthetic events. Here, we investigated the repair and (mis)coding properties of EtU and two bulkier clickable pyrimidine nucleosides, 5-(octa-1,7-diyn-1-yl)-U (C8-AlkU) and 5-(octa-1,7-diyn-1-yl)-C (C8-AlkC). In vitro, EtU and C8-AlkU, but not C8-AlkC, were excised by SMUG1 and MBD4, two DNA glycosylases from the base excision repair pathway. However, when placed into a plasmid encoding a fluorescent reporter inactivated by repair in human cells, EtU and C8-AlkU persisted for much longer than uracil or its poorly repairable phosphorothioate-flanked derivative. DNA polymerases from four different structural families preferentially bypassed EtU, C8-AlkU and C8-AlkC in an error-free manner, but a certain degree of misincorporation was also observed, especially evident for DNA polymerase β. Overall, clickable pyrimidine nucleotides could undergo repair and be a source of mutations, but the frequency of such events in the cell is unlikely to be considerable.
Topics: DNA Repair; Humans; Pyrimidine Nucleotides; Click Chemistry; DNA-Directed DNA Polymerase; Deoxyuridine; DNA; DNA Replication; Uracil-DNA Glycosidase
PubMed: 38927084
DOI: 10.3390/biom14060681 -
Microbial Cell Factories Jun 2024Currently, industrial fermentation of Botrytis cinerea is a significant source of abscisic acid (ABA). The crucial role of ABA in plants and its wide range of...
BACKGROUND
Currently, industrial fermentation of Botrytis cinerea is a significant source of abscisic acid (ABA). The crucial role of ABA in plants and its wide range of applications in agricultural production have resulted in the constant discovery of new derivatives and analogues. While modifying the ABA synthesis pathway of existing strains to produce ABA derivatives is a viable option, it is hindered by the limited synthesis capacity of these strains, which hinders further development and application.
RESULTS
In this study, we knocked out the bcaba4 gene of B. cinerea TB-31 to obtain the 1',4'-trans-ABA-diol producing strain ZX2. We then studied the fermentation broth of the batch-fed fermentation of the ZX2 strain using metabolomic analysis. The results showed significant accumulation of 3-hydroxy-3-methylglutaric acid, mevalonic acid, and mevalonolactone during the fermentation process, indicating potential rate-limiting steps in the 1',4'-trans-ABA-diol synthesis pathway. This may be hindering the flow of the synthetic pathway. Additionally, analysis of the transcript levels of terpene synthesis pathway genes in this strain revealed a correlation between the bchmgr, bcerg12, and bcaba1-3 genes and 1',4'-trans-ABA-diol synthesis. To further increase the yield of 1',4'-trans-ABA-diol, we constructed a pCBg418 plasmid suitable for the Agrobacterium tumefaciens-mediated transformation (ATMT) system and transformed it to obtain a single-gene overexpression strain. We found that overexpression of bchmgr, bcerg12, bcaba1, bcaba2, and bcaba3 genes increased the yield of 1',4'-trans-ABA-diol. The highest yielding ZX2 A3 strain was eventually screened, which produced a 1',4'-trans-ABA-diol concentration of 7.96 mg/g DCW (54.4 mg/L) in 144 h of shake flask fermentation. This represents a 2.1-fold increase compared to the ZX2 strain.
CONCLUSIONS
We utilized metabolic engineering techniques to alter the ABA-synthesizing strain B. cinerea, resulting in the creation of the mutant strain ZX2, which has the ability to produce 1',4'-trans-ABA-diol. By overexpressing the crucial genes involved in the 1',4'-trans-ABA-diol synthesis pathway in ZX2, we observed a substantial increase in the production of 1',4'-trans-ABA-diol.
Topics: Botrytis; Abscisic Acid; Fermentation; Metabolic Engineering; Fungal Proteins
PubMed: 38926702
DOI: 10.1186/s12934-024-02460-8 -
Nature Communications Jun 2024Phage predation is generally assumed to reduce microbial proliferation while not contributing to the spread of antibiotic resistance. However, this assumption does not...
Phage predation is generally assumed to reduce microbial proliferation while not contributing to the spread of antibiotic resistance. However, this assumption does not consider the effect of phage predation on the spatial organization of different microbial populations. Here, we show that phage predation can increase the spread of plasmid-encoded antibiotic resistance during surface-associated microbial growth by reshaping spatial organization. Using two strains of the bacterium Escherichia coli, we demonstrate that phage predation slows the spatial segregation of the strains during growth. This increases the number of cell-cell contacts and the extent of conjugation-mediated plasmid transfer between them. The underlying mechanism is that phage predation shifts the location of fastest growth from the biomass periphery to the interior where cells are densely packed and aligned closer to parallel with each other. This creates straighter interfaces between the strains that are less likely to merge together during growth, consequently slowing the spatial segregation of the strains and enhancing plasmid transfer between them. Our results have implications for the design and application of phage therapy and reveal a mechanism for how microbial functions that are deleterious to human and environmental health can proliferate in the absence of positive selection.
Topics: Plasmids; Escherichia coli; Bacteriophages; Drug Resistance, Bacterial; Anti-Bacterial Agents; Conjugation, Genetic
PubMed: 38926498
DOI: 10.1038/s41467-024-49840-7 -
Scientific Reports Jun 2024Ultra-high dose rate (UHDR) irradiation has been shown to have a sparing effect on healthy tissue, an effect known as 'FLASH'. This effect has been studied across...
Ultra-high dose rate (UHDR) irradiation has been shown to have a sparing effect on healthy tissue, an effect known as 'FLASH'. This effect has been studied across several radiation modalities, including photons, protons and clinical energy electrons, however, very little data is available for the effect of FLASH with Very High Energy Electrons (VHEE). pBR322 plasmid DNA was used as a biological model to measure DNA damage in response to Very High Energy Electron (VHEE) irradiation at conventional (0.08 Gy/s), intermediate (96 Gy/s) and ultra-high dose rates (UHDR, (2 × 10 Gy/s) at the CERN Linear Electron Accelerator (CLEAR) user facility. UHDRs were used to determine if the biological FLASH effect could be measured in the plasmid model, within a hydroxyl scavenging environment. Two different concentrations of the hydroxyl radical scavenger Tris were used in the plasmid environment to alter the proportions of indirect damage, and to replicate a cellular scavenging capacity. Indirect damage refers to the interaction of ionising radiation with molecules and species to generate reactive species which can then attack DNA. UHDR irradiated plasmid was shown to have significantly reduced amounts of damage in comparison to conventionally irradiated, where single strand breaks (SSBs) was used as the biological endpoint. This was the case for both hydroxyl scavenging capacities. A reduced electron energy within the VHEE range was also determined to increase the DNA damage to pBR322 plasmid. Results indicate that the pBR322 plasmid model can be successfully used to explore and test the effect of UHDR regimes on DNA damage. This is the first study to report FLASH sparing with VHEE, with induced damage to pBR322 plasmid DNA as the biological endpoint. UHDR irradiated plasmid had reduced amounts of DNA single-strand breaks (SSBs) in comparison with conventional dose rates. The magnitude of the FLASH sparing was a 27% reduction in SSB frequency in a 10 mM Tris environment and a 16% reduction in a 100 mM Tris environment.
Topics: Plasmids; DNA Damage; Electrons; Dose-Response Relationship, Radiation; Humans; Particle Accelerators; DNA Breaks, Single-Stranded
PubMed: 38926450
DOI: 10.1038/s41598-024-65055-8 -
Microbial Biotechnology Jun 2024Ethylene and ethylene oxide are widely used in the chemical industry, and ethylene is also important for its role in fruit ripening. Better sensing systems would assist...
Ethylene and ethylene oxide are widely used in the chemical industry, and ethylene is also important for its role in fruit ripening. Better sensing systems would assist risk management of these chemicals. Here, we characterise the ethylene regulatory system in Mycobacterium strain NBB4 and use these genetic parts to create a biosensor. The regulatory genes etnR1 and etnR2 and cognate promoter P were combined with a fluorescent reporter gene (fuGFP) in a Mycobacterium shuttle vector to create plasmid pUS301-EtnR12P. Cultures of M. smegmatis mc-155(pUS301-EtnR12P) gave a fluorescent signal in response to ethylene oxide with a detection limit of 0.2 μM (9 ppb). By combining the epoxide biosensor cells with another culture expressing the ethylene monooxygenase, the system was converted into an ethylene biosensor. The co-culture was capable of detecting ethylene emission from banana fruit. These are the first examples of whole-cell biosensors for epoxides or aliphatic alkenes. This work also resolves long-standing questions concerning the regulation of ethylene catabolism in bacteria.
Topics: Biosensing Techniques; Ethylenes; Ethylene Oxide; Mycobacterium; Musa; Genes, Reporter; Plasmids
PubMed: 38925606
DOI: 10.1111/1751-7915.14511 -
Journal of Colloid and Interface Science Jun 2024Nucleic acid-based therapies are transforming medicine, but rely on an efficient delivery vehicle such as lipid nanoparticles (LNPs). Concerns exists in the nanomedicine...
Nucleic acid-based therapies are transforming medicine, but rely on an efficient delivery vehicle such as lipid nanoparticles (LNPs). Concerns exists in the nanomedicine field, that a large fraction of the LNPs in the ensemble does not contain any nucleic acid cargo and thus exert no functional effect. Nevertheless, how LNP lipid formulation, the LNP preparation method employed and nucleic acid cargo size correlates with the proportion of empty LNPs remains largely unexplored. Here we employ a well-established single particle based method to study nucleic acid loading heterogeneity in LNPs. We find that only a minor fraction of LNPs are "empty", both for LNPs loaded with siRNA, mRNA and plasmids. For clinically relevant LNPs for mRNA delivery, we never detected more than 16% empty nanoparticles in the ensemble. Thus employing standard LNP lipid-cargo combinations and preparation schemes results in LNPs with the potential to serve their biomedical function.
PubMed: 38925059
DOI: 10.1016/j.jcis.2024.06.158