Staphylococcus spp., especially Staphylococcus aureus (S. aureus), is an important pathogen in hospital-acquired infection and food poisoning. Here, we developed a multienzyme isothermal rapid amplification combined with duplex quantitative PCR (duplex MIRA-qPCR) method, which can simultaneously detect the S. aureus species-specific conserved gene FMN-bgsfp and the Staphylococcus genus-specific conserved gene tuf. This assay enabled the amplification of DNA within 20 min at a constant temperature of 39 °C. Specificity analysis indicated that all nine common Staphylococcus species were positive and non-Staphylococcus spp. were negative for tuf gene, whereas S. aureus was positive, non-aureus Staphylococci species and non-Staphylococcus spp. were negative for FMN-bgsfp gene, suggesting that duplex MIRA-qPCR exhibited high specificity. Meanwhile, the sensitivity was tested and the limit of detection (LoD) was 3 × 10² CFU/mL. The coefficient variation values ranged from 0.13% to 2.09%, indicating that the assay had good repeatability. Furthermore, all the nine common Staphylococcus species (including S. aureus) could be detected from four kinds of simulated samples and the LoD of S. aureus was 8.56 × 10³ CFU/mL. In conclusion, the duplex MIRA-qPCR has advantages of stronger specificity, lower detection threshold, shorter detection time, and simpler operation, which is an effective tool to detect S. aureus and non-aureus Staphylococci spp. infections rapidly.

A rising incidence of clinical infections has been caused by Kluyvera, a significant opportunistic pathogen. Meanwhile, Kluyvera acts as an important reservoir of bla_CTX-Ms, which are the dominant genes of class A extended-spectrum β-lactamases (ESBLs). In this work, 60 strains of Kluyvera were subjected to phylogenetic relationship reconstruction, antimicrobial susceptibility testing, and antibiotic resistance genes prediction. All mature bla_CTX-Ms were gathered to perform subgroup reclassification. The findings demonstrate that Kluyvera has a large gene pool with significant genetic flexibility. Notably, 25% of strains showed simultaneous detection of ESBLs and carbapenem resistance genes. The genotypes of fourteen novel bla_CTX-Ms were identified. A new subgroup classification approach for bla_CTX-Ms was defined by using 20 amino acid site variants, which could split bla_CTX-Ms into 10 subgroups. The results of the subgroup division were consistent with the phylogenetic clustering. More significantly, we proposed a novel bla_CTX-M subgroup, KLUS, that is chromosomally encoded in K. sichuanensis and the new species put forward in this study, showing amino acid differences from the currently known sequences. Cloning and transformation tests demonstrated that the recipient bacteria had a robust phenotype of cefotaxime resistance. Closely related Kluyvera species had bla_CTX-Ms in the same subgroup. Our research lays the groundwork for a deeper comprehension of Kluyvera and emphasizes how important a bla_CTX-M reservoir it is. We provide an update on bla_CTX-M subgroups reclassification from the aspects of phylogenetic relationship, amino acid differences, and the new subgroup KLUS, which needs to be strengthen monitored due to its strong resistance phenotype to cefotaxime.

To understand the characteristics of Vibrio isolates in aquatic environments in China and their public health significance, this study investigated water samples in six cities in China in 2020. A total of 88 sampling locations were included and Vibrio isolates were identified in 81 of them. A total of 143 Vibrio isolates belonging to 16 species were selected for characterization. The population structure of Vibrio species showed great differences among the six cities, indicating regional specificity. The presence of virulence genes was examined for the isolates (n = 78) of five pathogenic Vibrio species. All isolates except one (n = 77) contained at least one virulence gene and isolates belonging to the same species showed very similar virulence gene profiles. Then, 26 isolates from 12 species were examined by multilocus sequence typing and were assigned to 25 STs, of which 24 STs were new. Also, the presence of antibiotic-resistant genes was investigated for all 143 isolates and only three isolates were found to contain genes from aminoglycosides, phenicols, beta-lactams or the tetracycline family. Our results provide valuable insights into the Vibrio community in Chinese aquatic environments and can be applied as guidance for the environmental surveillance of the risk of Vibrio isolates.

Vibrio fluvialis is an emerging foodborne pathogenic bacterium that can cause severe cholera-like diarrhea and various extraintestinal infections, posing challenges to public health and food safety worldwide. The arginine deiminase (ADI) pathway plays an important role in bacterial environmental adaptation and pathogenicity. However, the biological functions and regulatory mechanisms of the pathway in V. fluvialis remain unclear. In this study, we demonstrate that L-arginine upregulates the expression of the ADI gene cluster and promotes the growth of V. fluvialis. The ADI gene cluster, which we proved to be comprised of two operons, arcD and arcACB, significantly enhances the survival of V. fluvialis in acidic environments both in vitro (in culture medium and in macrophage) and in vivo (in mice). The mRNA level and reporter gene fusion analyses revealed that ArgR, a transcriptional factor, is necessary for the activation of both arcD and arcACB transcriptions. Bioinformatic analysis predicted the existence of multiple potential ArgR binding sites at the arcD and arcACB promoter regions that were further confirmed by electrophoretic mobility shift assay, DNase I footprinting, or point mutation analyses. Together, our study provides insights into the important role of the ArgR-ADI pathway in the survival of V. fluvialis under acidic conditions and the detailed molecular mechanism. These findings will deepen our understanding of how environmental changes and gene expression interact to facilitate bacterial adaptations and virulence.

CTXΦ is a lysogenic filamentous phage that carries the genes encoding cholera toxin (ctxAB), the main virulence factor of Vibrio cholerae. The toxigenic conversion of environmental V. cholerae strains through CTXΦ lysogenic infection is crucial for the emergence of new pathogenic clones. A special allelic form of CTXΦ, called pre-CTXΦ, is a precursor of CTXΦ and without ctxAB. Different members of the pre-CTXΦ and CTXΦ families are distinguished by the sequence of the transcriptional repressor-coding gene rstR. Multiple rstR alleles can coexist within a single strain, demonstrating the diverse structure and complex genomic integration patterns of CTXΦ/pre-CTXΦ prophage on the chromosome. Exploration of the diversity and co-integration patterns of CTXΦ/pre-CTXΦ prophages in V. cholerae can help to understand the evolution of this phage family. In this study, 21 V. cholerae strains, which were shown to carry the CTXΦ/pre-CTXΦ prophages as opposed to typical CTX^ETΦ-RS1 structure, were selected from approximately 1000 strains with diverse genomes. We identified two CTXΦ members and six pre-CTXΦ members with distinct rstR alleles, revealing complex chromosomal DNA integration patterns and arrangements of different prophages in these strains. Promoter activity assays showed that the transcriptional repressor RstR protected against CTXΦ superinfection by preventing the replication and integration of CTXΦ/pre-CTXΦ phages containing the same rstR allele, supporting the co-integration of the diverse CTXΦ/pre-CTXΦ members observed. The numbers and types of prophages and their co-integration arrangements in serogroup O139 strains were more complex than those in serogroup O1 strains. Also, these CTXΦ/pre-CTXΦ members were shown to present the bloom period of the CTXΦ/pre-CTXΦ family during wave 2 of the seventh cholera pandemic. Together, these analyses deepen our comprehension of the genetic variation of CTXΦ and pre-CTXΦ and provide insights into the evolution of the CTXΦ/pre-CTXΦ family in the seventh cholera pandemic.

Plague, caused by Yersinia pestis, remains a significant public health concern, yet the determinants of many toxin-associated proteins encoded by this pathogen remain poorly understood. Here, we characterized two previously unexplored Rearrangement hotspot (Rhs) proteins, YPO3609 and YPO3615, using a combination of bioinformatic analyses, bacterial toxicity assays, DNA degradation assays, SOS reporter assays, and RT-qPCR analysis. The C-terminal domains of YPO3609 and YPO3615 confer nuclease toxicity, with YPO3609 containing a WHH motif of the HNH nuclease superfamily and YPO3615 harboring a restriction endonuclease-like aspartate dyad. Heterologous expression of these C-terminal toxin domains in Escherichia coli inhibited growth, caused DNA degradation, elicited SOS-related responses, and induced cellular filamentation. Co-expression experiments identified YPO3610 and YPO3616 as the cognate immunity proteins of YPO3609 and YPO3615, respectively, while site-directed mutagenesis further showed that H411 and D1442/D1447 contribute to the nuclease-associated activities of the two toxins. Homologs of both toxin domains were broadly distributed in Pseudomonadota, with the C-terminal domain of YPO3609 showing a wider distribution. These findings characterize two functional Rhs toxin–immunity modules encoded by Y. pestis, and provide new insights into Rhs-associated nuclease toxins and their related homologs.