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Objectives To characterize an MDR bla IMP-4-harbouring plasmid from Enterobacter cloacae EC62 of swine origin in China. Methods Plasmid pIMP-4-EC62 from E. cloacae EC62 was transferred by conjugation via filter mating into Escherichia coli J53. Plasmid DNA was extracted from an E. coli J53 transconjugant and sequenced using single-molecule real-time (SMRT) technology. MIC values for both the isolate EC62 and the transconjugant were determined using the broth microdilution and agar dilution methods. Plasmid stability in both the isolate EC62 and the transconjugant was assessed through a series of passages on antibiotic-free media. Results Plasmid pIMP-4-EC62 is 314351 bp in length, encodes 369 predicted proteins and harbours a novel class 1 integron carrying bla IMP-4 and a group II intron. The bla IMP-4-bearing plasmid belongs to the IncHI2/ST1 incompatibility group. Sequence analysis showed that pIMP-4-EC62 carries four MDR regions and several gene clusters encoding heavy metal resistance. Plasmid pIMP-4-EC62 was stably maintained in both the E. cloacae EC62 isolate and the transconjugant E. coli J53-pIMP-4-EC62 in the absence of selective pressure. Analysis of the evolutionary relatedness of selected IncHI2 plasmids indicates that ST1-type plasmids are key carriers of carbapenemase genes among IncHI2 plasmids. Conclusions pIMP-4-EC62 represents the first fully sequenced IncHI2-type bla IMP-4-harbouring plasmid from E. cloacae in China. Co-location of bla IMP-4 with other resistance genes on an MDR plasmid is likely to further accelerate the dissemination of bla IMP-4 by co-selection among bacteria from humans, animals and the environment under the selective pressure of other antimicrobial agents, heavy metals and disinfectants. [ABSTRACT FROM AUTHOR]

Sortase A (SrtA) has long been recognized as an ideal drug target for therapeutic agents against Gram-positive pathogens. However, the SrtA of Streptococcus suis (Ss-SrtA), an important zoonotic agent, has not been studied. In this study, the enzymatic properties of Ss-SrtA were investigated, and inhibition of Ss-SrtA by natural products was evaluated. Ss-SrtA was expressed and purified. The purified recombinant Ss-SrtA had maximal activity at pH 6.0–7.5, 45°C, and showed a Km of 6.7 μM for the hydrolysis of substrate abz-LPATG-dnp. Different from Staphylococcus aureus SrtA (Sa-SrtA) which is stimulated by Ca2+, Ss-SrtA was observed to be Ca2+ independent. Structural analysis showed that salt bridges formed between K111 and D180 in Ss-SrtA replaced the function of Ca2+ in Sa-SrtA to stabilize the substrate-binding cleft. Site-directed mutagenesis identified H126, C192 and R200 as the key residues of Ss-SrtA active site. To discover potential inhibitors, the percent inhibition of sortase activity by natural products was measured. Among these selected natural products, acteoside, isoquercitrin and baicalin were discovered as novel SrtA inhibitors, with IC50 values of 36.3 ± 1.3 μM, 100.0 ± 1.3 μM and 85.4 ± 1.5 μM, respectively. The inhibitory effects of these three natural products were further confirmed on endogenous Sa-SrtA. Using a previously established S. aureus model with a fluorescent-labeled Sa-SrtA substrate, acteoside, isoquercitrin, and baicalin showed 86%, 28% and 45% inhibition on endogenous Sa-SrtA activity, respectively. Overall, these findings shed new light on enzymatic properties, Ca2+-independent catalytic mechanism and potential inhibitors of Ss-SrtA. [ABSTRACT FROM AUTHOR]

Sortase A ( Srt A), a transpeptidase, anchors surface proteins with an LPXTG-motif sorting signal to the cell envelope. To determine the role of Srt A in the pathogenesis of Staphylococcus aureus, we constructed a mutant strain, ∆ Srt A, by genetic techniques and identified its functions in a S. aureus-induced mastitis mouse model. The histological and myeloperoxidase ( MPO) level results showed that the ∆ Srt A strain attenuated the inflammatory reaction in the mammary tissue of mice compared with wild-type S. aureus challenge. Additionally, the ELISA results showed that the ∆ Srt A strain impaired the induction of pro-inflammatory cytokines such as tumor necrosis factor-α ( TNF-α), interleukin-1β ( IL-1β) and interleukin-6 ( IL-6), and the Western blot results showed that the mutant strain blocked the activation of nuclear factor-κ B ( NF-κ B) and mitogen-activated protein kinases ( MAPKs) by attenuating the degradation and phosphorylation of signaling pathway molecules such as Iκ Bα, p65 and p38. These results suggest that Srt A is a key virulence factor in the pathogenesis of S. aureus-induced mastitis in mice. It appears that the srt A mutant affected the attachment of S. aureus to host cells, thus attenuating the activation of the NF-κ B and MAPK signaling pathways, which regulated the expression of pro-inflammatory cytokines and decreased the susceptibility to mastitis. [ABSTRACT FROM AUTHOR]

Objectives: To characterize an MDR blaIMP-4-harbouring plasmid from Enterobacter cloacae EC62 of swine origin in China.Methods: Plasmid pIMP-4-EC62 from E. cloacae EC62 was transferred by conjugation via filter mating into Escherichia coli J53. Plasmid DNA was extracted from an E. coli J53 transconjugant and sequenced using single-molecule real-time (SMRT) technology. MIC values for both the isolate EC62 and the transconjugant were determined using the broth microdilution and agar dilution methods. Plasmid stability in both the isolate EC62 and the transconjugant was assessed through a series of passages on antibiotic-free media.Results: Plasmid pIMP-4-EC62 is 314351 bp in length, encodes 369 predicted proteins and harbours a novel class 1 integron carrying blaIMP-4 and a group II intron. The blaIMP-4-bearing plasmid belongs to the IncHI2/ST1 incompatibility group. Sequence analysis showed that pIMP-4-EC62 carries four MDR regions and several gene clusters encoding heavy metal resistance. Plasmid pIMP-4-EC62 was stably maintained in both the E. cloacae EC62 isolate and the transconjugant E. coli J53-pIMP-4-EC62 in the absence of selective pressure. Analysis of the evolutionary relatedness of selected IncHI2 plasmids indicates that ST1-type plasmids are key carriers of carbapenemase genes among IncHI2 plasmids.Conclusions: pIMP-4-EC62 represents the first fully sequenced IncHI2-type blaIMP-4-harbouring plasmid from E. cloacae in China. Co-location of blaIMP-4 with other resistance genes on an MDR plasmid is likely to further accelerate the dissemination of blaIMP-4 by co-selection among bacteria from humans, animals and the environment under the selective pressure of other antimicrobial agents, heavy metals and disinfectants. [ABSTRACT FROM AUTHOR]

Flavobacterium psychrophilum is the causative agent of rainbow trout fry syndrome and bacterial cold-water disease in salmonid fish worldwide. As an important fish pathogen, F. psychrophilum is frequently exposed to multiple invading genetic elements in natural environments. Endonuclease Cas9 provides bacteria with adaptive interference against invading genetic elements. Previous studies revealed that several F. psychrophilum strains harbored a type II-C Cas9 called Fp1Cas9, but little is known about the potential role of this endonuclease against invading genetic elements. In this work, we identified a gene encoding a novel type II-C Cas9 called Fp2Cas9 from F. psychrophilum strain CN46. Through bacterial RNA sequencing, we demonstrated active transcription of both Fp2Cas9 and pre-crRNAs in strain CN46. Bioinformatics analysis further revealed that the transcription of Fp2Cas9 and pre-crRNAs was driven by a newly integrated promoter sequence and a promoter element embedded within each CRISPR repeat, respectively. To formally demonstrate that Fp2Cas9 and associated crRNAs yielded functional interference in strain CN46, a plasmid interference assay was performed, resulting in adaptive immunity to target DNA sequences in Flavobacterium bacteriophages. Phylogenetic analysis demonstrated that Fp2Cas9 was present only in several F. psychrophilum isolates. Phylogenetic analysis revealed that this novel endonuclease was probably acquired through horizontal gene transfer from the CRISPR-Cas9 system in an unidentified Flavobacterium species. Comparative genomics analysis further showed that the Fp2Cas9 was integrated into the type II-C CRISPR-Cas locus in strain CN38 instead of the original Fp1Cas9. Taken together, our results shed light on the origin and evolution of Fp2Cas9 gene and demonstrated that this novel endonuclease provided adaptive interference against bacteriophage infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Chen, Wang, Lu and Li.)

Sortase A (SrtA) is a cysteine transpeptidase and virulence factor from Staphylococcus aureus (S. aureus) that catalyses the attachment and display of surface proteins on the cell wall, thereby mediating bacterial adhesion to host tissues, host-cell entry and evasion of the immune response. As a result, SrtA has become an important target in the development of therapies for S. aureus infections. In this study, we used the new reference strain S. aureus Newman D2C to investigate the role of SrtA in a murine model of bloodstream infection, when the impact of coagulase and haemolysin is excluded. The results suggested that deletion of SrtA reduced the bacterial burden on the heart, liver and kidneys by blunting the host proinflammatory cytokine response at an early point in infection. Kidneys, but not heart or liver, formed abscesses on the sixth day following non-lethal infection, and this effect was diminished by SrtA mutation. These findings indicate that SrtA is a determining virulence factor in lethality and formation of renal abscesses in mice followed by S. aureus bloodstream infection. We have thus established a convenient in vitro and mouse model for developing SrtA-targeted therapeutic strategies., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)