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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 -
Cell Reports Jun 2024Klebsiella pneumoniae carbapenemase (KPC) poses a major public health risk. Understanding its transmission dynamics requires examining the epidemiological features of...
Klebsiella pneumoniae carbapenemase (KPC) poses a major public health risk. Understanding its transmission dynamics requires examining the epidemiological features of related plasmids. Our study compiled 15,660 bla-positive isolates globally over the past two decades. We found extensive diversity in the genetic background of KPC, with 23 Tn4401-related and 341 non-Tn4401 variants across 163 plasmid types in 14 genera. Intra-K. pneumoniae and cross-genus KPC transmission patterns varied across four distinct periods. In the initial periods, plasmids with narrow host ranges gradually established a survival advantage. In later periods, broad-host-range plasmids became crucial for cross-genera transmission. In total, 61 intra-K. pneumoniae and 66 cross-genus transmission units have been detected. Furthermore, phylogenetic reconstruction dated the origin of KPC transmission back to 1991 and revealed frequent exchanges across countries. Our research highlights the frequent and transient spread events of KPC mediated by plasmids across multiple genera and offers theoretical support for high-risk plasmid monitoring.
PubMed: 38923465
DOI: 10.1016/j.celrep.2024.114351 -
Veterinary Sciences Jun 2024Raw milk and dairy products can serve as potential vectors for transmissible bacterial, viral and protozoal diseases, alongside harboring antimicrobial-resistance genes....
Raw milk and dairy products can serve as potential vectors for transmissible bacterial, viral and protozoal diseases, alongside harboring antimicrobial-resistance genes. This study monitors the changes in the antimicrobial-resistance gene pool in raw milk and cheese, from farm to consumer, utilizing next-generation sequencing. Five parallel sampling runs were conducted to assess the resistance gene pool, as well as phage or plasmid carriage and potential mobility. In terms of taxonomic composition, in raw milk the Firmicutes phylum made up 41%, while the Proteobacteria phylum accounted for 58%. In fresh cheese, this ratio shifted to 93% Firmicutes and 7% Proteobacteria. In matured cheese, the composition was 79% Firmicutes and 21% Proteobacteria. In total, 112 antimicrobial-resistance genes were identified. While a notable reduction in the resistance gene pool was observed in the freshly made raw cheese compared to the raw milk samples, a significant growth in the resistance gene pool occurred after one month of maturation, surpassing the initial gene frequency. Notably, the presence of extended-spectrum beta-lactamase (ESBL) genes, such as (100% coverage, 99.3% identity) and (97.1% coverage, 96.2% identity), raised concerns; these genes have a major public health relevance. In total, nineteen such genes belonging to nine gene families (, , , , , , , , ) have been identified. The largest number of resistance genes were identified against fluoroquinolone drugs, which determined efflux pumps predominantly. Our findings underscore the importance of monitoring gene pool variations throughout the product pathway and the potential for horizontal gene transfer in raw products. We advocate the adoption of a new approach to food safety investigations, incorporating next-generation sequencing techniques.
PubMed: 38922012
DOI: 10.3390/vetsci11060265 -
Pathogens (Basel, Switzerland) Jun 2024Sternal bursitis, a common inflammatory condition in poultry, poses significant challenges to both animal welfare and public health. This study aimed to investigate the...
Sternal bursitis, a common inflammatory condition in poultry, poses significant challenges to both animal welfare and public health. This study aimed to investigate the prevalence, antimicrobial resistance, and genetic characteristics of isolates associated with sternal bursitis in chickens. Ninety-eight samples were collected from affected chickens, and 24 isolates were identified. Antimicrobial susceptibility testing revealed resistance to multiple agents, with a notable prevalence of aminoglycoside resistance genes. Whole genome sequencing elucidated the genetic diversity and virulence profiles of the isolates, highlighting the predominance of clonal complex 5 (CC5) strains. Additionally, biofilm formation assays demonstrated moderate biofilm production capacity among the isolates. These findings underscore the importance of vigilant monitoring and targeted interventions to mitigate the impact of sternal bursitis in poultry production systems.
PubMed: 38921816
DOI: 10.3390/pathogens13060519 -
Pathogens (Basel, Switzerland) Jun 2024The bacterial agent of Lyme disease, , exists in an enzootic cycle by adapting to dissimilar mammalian and tick environments. The genetic elements necessary for host and...
The bacterial agent of Lyme disease, , exists in an enzootic cycle by adapting to dissimilar mammalian and tick environments. The genetic elements necessary for host and vector adaptation are spread across a bacterial genome comprised of a linear chromosome and essential linear and circular plasmids. The promoter trap system, In Vivo Expression Technology (IVET), has been used to identify promoters of that are transcriptionally active specifically during infection of a murine host. However, an observed infection bottleneck effect in mice prevented the application of this system to study promoters induced in a tick environment. In this study, we adapted a membrane-based in vitro feeding system as a novel method to infect the spp. vector with . Once adapted, we performed IVET screens as a proof of principle via an infected bloodmeal on the system. The screen yielded promoters that are induced during tick infection and verified relative expression levels using qRT-PCR. The results of our study demonstrate the potential of our developed in vitro tick feeding system and IVET systems to gain insight into the adaptive gene expression of the Lyme disease bacteria to the tick vector.
PubMed: 38921785
DOI: 10.3390/pathogens13060487 -
Pathogens (Basel, Switzerland) May 2024is the main pathogen of chicken necrotic enteritis (NE) causing huge economic losses in the poultry industry. Although dietary secondary bile acid deoxycholic acid...
is the main pathogen of chicken necrotic enteritis (NE) causing huge economic losses in the poultry industry. Although dietary secondary bile acid deoxycholic acid (DCA) reduced chicken NE, the accumulation of conjugated tauro-DCA (TDCA) raised concerns regarding DCA efficacy. In this study, we aimed to deconjugate TDCA by bile salt hydrolase (BSH) to increase DCA efficacy against the NE pathogen . Assays were conducted to evaluate the inhibition of growth, hydrogen sulfide (HS) production, and virulence gene expression by TDCA and DCA. BSH activity and sequence alignment were conducted to select the gene for cloning. The gene from was PCR-amplified and cloned into plasmids pET-28a (pET-BSH) and pDR111 (pDR-BSH) for expressing the BSH protein in BL21 and 168 (BSH), respectively. His-tag-purified BSH from BL21 cells was evaluated by SDS-PAGE, Coomassie blue staining, and a Western blot (WB) assays. Secretory BSH from was analyzed by a Dot-Blot. -BSH was evaluated for the inhibition of growth. growth reached 7.8 log10 CFU/mL after 24 h culture. growth was at 8 vs. 7.4, 7.8 vs. 2.6 and 6 vs. 0 log10 CFU/mL in 0.2, 0.5, and 1 mM TDCA vs. DCA, respectively. Compared to TDCA, DCA reduced HS production and the virulence gene expression of , , , and . BSH activity was observed in and under anaerobe but not under 10% CO air. After the sequence alignment of from ten bacteria, from was selected, cloned into pET-BSH, and sequenced at 951 bp. After pET-BSH was transformed in BL21, BSH expression was assessed around 35 kDa using Coomassie staining and verified for His-tag using WB. After the subcloned and amylase signal peptide sequence was inserted into pDR-BSH, was transformed and named -BSH. The transformation was evaluated using PCR with around 3 kb and BSH around 5 kb. Secretory BSH expressed from -BSH was determined for His-tag using Dot-Blot. Importantly, growth was reduced greater than 59% log10 CFU/mL in the -BSH media precultured with 1 vs. 0 mM TDCA. In conclusion, TDCA was less potent than DCA against virulence, and recombinant secretory BSH from -BSH reduced growth, suggesting a new potential intervention against the pathogen-induced chicken NE.
PubMed: 38921762
DOI: 10.3390/pathogens13060464 -
Current Issues in Molecular Biology Jun 2024We produced a recombinant eel luteinizing hormone (rec-eel LH) analog with high potency in Chinese hamster ovary DG44 (CHO DG44) cells. The tethered eel LH mutant...
We produced a recombinant eel luteinizing hormone (rec-eel LH) analog with high potency in Chinese hamster ovary DG44 (CHO DG44) cells. The tethered eel LH mutant (LH-M), which had a linker comprising the equine chorionic gonadotropin (eLH/CG) β-subunit carboxyl-terminal peptide (CTP) region (amino acids 115 to 149), was inserted between the β-subunit and α-subunit of wild-type tethered eel LH (LH-wt). Monoclonal cells transfected with the tethered eel LH-wt and eel LH-M plasmids were isolated from five to nine clones of CHO DG44 cells, respectively. The secreted quantities abruptly increased on day 3, with peak levels of 5000-7500 ng/mL on day 9. The molecular weight of tethered rec-eel LH-wt was 32-36 kDa, while that of tethered rec-eel LH-M increased to approximately 38-44 kDa, indicating the detection of two bands. Treatment with the peptide N-glycanase F decreased the molecular weight by approximately 8 kDa. The oligosaccharides at the eCG β-subunit O-linked glycosylation sites were appropriately modified post-translation. The EC value and maximal responsiveness of eel LH-M increased by approximately 2.90- and 1.29-fold, respectively, indicating that the mutant exhibited more potent biological activity than eel LH-wt. Phosphorylated extracellular regulated kinase (pERK1/2) activation resulted in a sharp peak 5 min after agonist treatment, with a rapid decrease thereafter. These results indicate that the new tethered rec-eel LH analog had more potent activity in cAMP response than the tethered eel LH-wt in vitro. Taken together, this new eel LH analog can be produced in large quantities using a stable CHO DG44 cell system.
PubMed: 38921034
DOI: 10.3390/cimb46060363 -
Current Issues in Molecular Biology Jun 2024Uropathogenic (UPEC) is the main cause of urinary tract infections (UTIs) and carries virulence and resistance factors often found in mobilizable genetic elements, such...
Uropathogenic (UPEC) is the main cause of urinary tract infections (UTIs) and carries virulence and resistance factors often found in mobilizable genetic elements, such as plasmids or pathogenicity islands (PAIs). UPEC is part of the extraintestinal pathogenic (ExPEC), but hybrid strains possessing both diarrheagenic (DEC) and ExPEC traits, termed "hypervirulent", present a significant health threat. This study assessed the prevalence of UPEC PAIs, ExPEC sequence types (ST), DEC genes, carbapenemase and extended-spectrum β-lactamase (ESBL) phenotypes, resistance genotypes, and plasmids in 40 clinical isolates of UPEC. Results showed that 72.5% of isolates had PAIs, mainly PAI IV (53%). ESBL phenotypes were found in 65% of β-lactam-resistant isolates, with 100% of carbapenem-resistant isolates producing carbapenemase. The predominant ESBL gene was (60%), and the most common resistance gene in fluoroquinolone and aminoglycoside-resistant isolates was (93%). Plasmids were present in 57% of isolates, and 70% belonged to the ST131 clonal group. Molecular markers for DEC pathotypes were detected in 20 isolates, with 60% classified as hybrid pathotypes. These findings indicate significant pathogenic potential and the presence of hybrid pathotypes in UTI clinical isolates in the Mexican population.
PubMed: 38921024
DOI: 10.3390/cimb46060353