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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.
Topics: Plasmids; beta-Lactamases; Klebsiella pneumoniae; Phylogeny; Bacterial Proteins; Humans; Klebsiella Infections
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 -
Current Issues in Molecular Biology May 2024A single nucleotide variant in mitochondrial DNA (mtDNA) 1555A>G is associated with drug-induced hearing loss. For the 1555A>G mutation site, 1555A wild-type and 1555G...
A single nucleotide variant in mitochondrial DNA (mtDNA) 1555A>G is associated with drug-induced hearing loss. For the 1555A>G mutation site, 1555A wild-type and 1555G mutant-type plasmids were constructed, respectively. In this study, a PCR method based on the TaqMan amplification refractory mutation system was proposed to detect mtDNA 1555A>G. A common upstream primer, a common TaqMan probe, and two downstream allele-specific primers with mismatched bases were designed. One-step amplification and detection of the wild-type and mutant type at the 1555 site were realized for the deafness-related gene through two reactions. Based on this detection method, the minimum detection limit of the wild-type and mutant type detection systems for plasmids was 50 copies/μL. The minimum sensitivity for the detection of nucleic acids in real dried blood spot (DBS) samples was 0.1 ng/μL. In the normal DBS DNA sample, the detection limit of the mutation abundance reached 0.78%. The specificity of the detection method was 100%, and the coefficient of variation was less than 3.36%. This approach was validated using clinical DNA extracted from 113 DBS samples of newborns. Additionally, it showed 100% agreement with bi-directional Sanger sequencing. It can be used as an optional method for the clinical detection of deafness-related genes.
PubMed: 38920998
DOI: 10.3390/cimb46060326 -
Infection and Immunity Jun 2024Cold shock proteins (Csp) are pivotal nucleic acid binding proteins known for their crucial roles in the physiology and virulence of various bacterial pathogens...
Cold shock proteins (Csp) are pivotal nucleic acid binding proteins known for their crucial roles in the physiology and virulence of various bacterial pathogens affecting plant, insect, and mammalian hosts. However, their significance in bacterial pathogens of teleost fish remains unexplored. subsp. (hereafter ) is a psychrotrophic pathogen and the causative agent of furunculosis in marine and freshwater fish. Four genes (, and ) have been identified in the genome of J223 (wild type). Here, we evaluated the role of DNA binding proteins, CspB and CspD, in physiology and virulence in lumpfish (). Δ, Δ, and the double ΔΔ mutants were constructed and characterized. Δ and ΔΔ mutants showed a faster growth at 28°C, and reduced virulence in lumpfish. Δ showed a slower growth at 28°C, biofilm formation, lower survival in low temperatures and freezing conditions (-20°C, 0°C, and 4°C), deficient in lipopolysaccharide synthesis, and low virulence in lumpfish. Additionally, ΔΔ mutants showed less survival in the presence of bile compared to the wild type. Transcriptome analysis revealed that 200, 37, and 921 genes were differentially expressed in Δ, Δ, and ΔΔ respectively. In Δ and ΔΔ virulence genes in the chromosome and virulence plasmid were downregulated. Our analysis indicates that CspB and CspD mostly act as a transcriptional activator, influencing cell division (e.g., ), virulence factors (e.g., ), and ultimately virulence.
PubMed: 38920386
DOI: 10.1128/iai.00011-24 -
BMC Veterinary Research Jun 2024Acinetobacter lwoffii (A. lwoffii) is a Gram-negative bacteria common in the environment, and it is the normal flora in human respiratory and digestive tracts. The...
BACKGROUND
Acinetobacter lwoffii (A. lwoffii) is a Gram-negative bacteria common in the environment, and it is the normal flora in human respiratory and digestive tracts. The bacteria is a zoonotic and opportunistic pathogen that causes various infections, including nosocomial infections. The aim of this study was to identify A. lwoffii strains isolated from bovine milk with subclinical mastitis in China and get a better understanding of its antimicrobial susceptibility and resistance profile. This is the first study to analyze the drug resistance spectrum and corresponding mechanisms of A. lwoffii isolated in raw milk.
RESULTS
Four A. lwoffii strains were isolated by PCR method. Genetic evolution analysis using the neighbor-joining method showed that the four strains had a high homology with Acinetobacter lwoffii. The strains were resistant to several antibiotics and carried 17 drug-resistance genes across them. Specifically, among 23 antibiotics, the strains were completely susceptible to 6 antibiotics, including doxycycline, erythromycin, polymyxin, clindamycin, imipenem, and meropenem. In addition, the strains showed variable resistance patterns. A total of 17 resistance genes, including plasmid-mediated resistance genes, were detected across the four strains. These genes mediated resistance to 5 classes of antimicrobials, including beta-lactam, aminoglycosides, fluoroquinolones, tetracycline, sulfonamides, and chloramphenicol.
CONCLUSION
These findings indicated that multi-drug resistant Acinetobacter lwoffii strains exist in raw milk of bovine with subclinical mastitis. Acinetobacter lwoffii are widespread in natural environmental samples, including water, soil, bathtub, soap box, skin, pharynx, conjunctiva, saliva, gastrointestinal tract, and vaginal secretions. The strains carry resistance genes in mobile genetic elements to enhance the spread of these genes. Therefore, more attention should be paid to epidemiological surveillance and drug resistant A. lwoffii.
Topics: Animals; Cattle; Mastitis, Bovine; Female; Acinetobacter; Milk; China; Anti-Bacterial Agents; Microbial Sensitivity Tests; Acinetobacter Infections; Drug Resistance, Bacterial; Drug Resistance, Multiple, Bacterial
PubMed: 38918815
DOI: 10.1186/s12917-024-04119-3